Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic Analysis of the Role of Heparan Sulfate Proteoglycans†
Identifieur interne : 000828 ( Istex/Corpus ); précédent : 000827; suivant : 000829Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic Analysis of the Role of Heparan Sulfate Proteoglycans†
Auteurs : Gizette V. Sperinde ; Matthew A. NugentSource :
- Biochemistry [ 0006-2960 ] ; 2000.
Abstract
The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.
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DOI: 10.1021/bi992243d
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic Analysis of the Role of Heparan Sulfate Proteoglycans†</title><author><name sortKey="Sperinde, Gizette V" sort="Sperinde, Gizette V" uniqKey="Sperinde G" first="Gizette V." last="Sperinde">Gizette V. Sperinde</name><affiliation><mods:affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</mods:affiliation></affiliation></author><author><name sortKey="Nugent, Matthew A" sort="Nugent, Matthew A" uniqKey="Nugent M" first="Matthew A." last="Nugent">Matthew A. Nugent</name><affiliation><mods:affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School ofMedicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339;E-mail nugent@biochem.bumc.bu.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:CF0F213D60C325EE792FA567983A500DA0EECBC5</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1021/bi992243d</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000828</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000828</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic
Analysis of the Role of Heparan Sulfate Proteoglycans<ref type="bib" target="#bi992243dAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Sperinde, Gizette V" sort="Sperinde, Gizette V" uniqKey="Sperinde G" first="Gizette V." last="Sperinde">Gizette V. Sperinde</name><affiliation><mods:affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</mods:affiliation></affiliation></author><author><name sortKey="Nugent, Matthew A" sort="Nugent, Matthew A" uniqKey="Nugent M" first="Matthew A." last="Nugent">Matthew A. Nugent</name><affiliation><mods:affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School ofMedicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339;E-mail nugent@biochem.bumc.bu.edu.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published" when="2000-03-10">2000</date><date when="2000-04-04">2000</date><biblScope unit="vol">39</biblScope><biblScope unit="issue">13</biblScope><biblScope unit="page" from="3788">3788</biblScope><biblScope unit="page" to="3796">3796</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>vsmc</json:string><json:string>hspg</json:string><json:string>intracellular</json:string><json:string>receptor</json:string><json:string>native vsmc</json:string><json:string>rate constant</json:string><json:string>chloroquine</json:string><json:string>biol</json:string><json:string>biochemistry</json:string><json:string>native cell</json:string><json:string>internalization</json:string><json:string>time point</json:string><json:string>quantitated</json:string><json:string>cytoplasmic</json:string><json:string>heparan</json:string><json:string>lysosomal</json:string><json:string>datum</json:string><json:string>hspgdeficient</json:string><json:string>sulfate</json:string><json:string>solubilized</json:string><json:string>chem</json:string><json:string>intracellular processing</json:string><json:string>laemmli sample buffer</json:string><json:string>laemmli</json:string><json:string>chlorate</json:string><json:string>cell surface</json:string><json:string>growth factor</json:string><json:string>triplicate determination</json:string><json:string>degradation</json:string><json:string>differential equation</json:string><json:string>cell surface receptor</json:string><json:string>heparan sulfate</json:string><json:string>intracellular degradation</json:string><json:string>hspgdeficient cell</json:string><json:string>chloroquine treatment</json:string><json:string>cell biol</json:string><json:string>fragment</json:string><json:string>excretion</json:string><json:string>final concentration</json:string><json:string>heparan sulfate proteoglycans</json:string><json:string>experimental datum</json:string><json:string>sodium chlorate</json:string><json:string>intracellular level</json:string><json:string>vascular smooth muscle cell</json:string><json:string>fibroblast growth factor</json:string><json:string>molecular weight</json:string><json:string>kinetic analysis</json:string><json:string>present study</json:string><json:string>cytoplasmic fraction</json:string><json:string>band corresponding</json:string><json:string>life technology</json:string><json:string>hspgdeficient vsmc</json:string><json:string>apparent rate constant</json:string><json:string>pulse chase experiment</json:string><json:string>time course</json:string><json:string>nuclear fraction</json:string><json:string>fragment formation</json:string><json:string>excretion reaction</json:string><json:string>computer simulation</json:string><json:string>acid phosphatase quantitation</json:string><json:string>radioactivity</json:string></teeft></keywords><author><json:item><name>SPERINDE Gizette V.</name><affiliations><json:string>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</json:string></affiliations></json:item><json:item><name>NUGENT Matthew A.</name><affiliations><json:string>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</json:string><json:string>Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School ofMedicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339;E-mail nugent@biochem.bumc.bu.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-9DV1S2MW-W</arkIstex><language><json:string>unknown</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.</abstract><qualityIndicators><score>10</score><pdfWordCount>6412</pdfWordCount><pdfCharCount>39817</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>9</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>712</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>254</abstractWordCount><abstractCharCount>1711</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic Analysis of the Role of Heparan Sulfate Proteoglycans†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>39</volume><issue>13</issue><pages><first>3788</first><last>3796</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-9DV1S2MW-W</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences medicales</json:string></inist></categories><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1021/bi992243d</json:string></doi><id>CF0F213D60C325EE792FA567983A500DA0EECBC5</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic
Analysis of the Role of Heparan Sulfate Proteoglycans†</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2000 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="e-published" when="2000-03-10">2000</date><date when="2000-04-04">2000</date><date type="Copyright" when="2000">2000</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main" xml:lang="en">Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic
Analysis of the Role of Heparan Sulfate Proteoglycans<ref type="bib" target="#bi992243dAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Sperinde</surname><forename type="first">Gizette V.</forename></persName><affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118
</affiliation></author><author xml:id="author-0001" role="corresp"><persName><surname>Nugent</surname><forename type="first">Matthew A.</forename></persName><affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118
</affiliation><affiliation role="corresp"> Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School of Medicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339; E-mail nugent@biochem.bumc.bu.edu.</affiliation></author><idno type="istex">CF0F213D60C325EE792FA567983A500DA0EECBC5</idno><idno type="ark">ark:/67375/TPS-9DV1S2MW-W</idno><idno type="DOI">10.1021/bi992243d</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published" when="2000-03-10">2000</date><date when="2000-04-04">2000</date><biblScope unit="vol">39</biblScope><biblScope unit="issue">13</biblScope><biblScope unit="page" from="3788">3788</biblScope><biblScope unit="page" to="3796">3796</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><abstract><p>The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG)
has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized
FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells
(VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment
with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow
for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low
molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that
the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and
processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the
greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further
suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored
in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and
prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG,
FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to
mediate prolonged or unique biological responses through intracellular processes.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="zxx"></language></langUsage></profileDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi992243d</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic
Analysis of the Role of Heparan Sulfate Proteoglycans<xref rid="bi992243dAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Sperinde</surname><given-names>Gizette V.</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Nugent</surname><given-names>Matthew A.</given-names></name><xref rid="bi992243dAF1">*</xref></contrib><aff>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118
</aff></contrib-group><author-notes><corresp id="bi992243dAF1">
Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School of
Medicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339;
E-mail nugent@biochem.bumc.bu.edu.</corresp></author-notes><pub-date pub-type="epub"><day>10</day><month>03</month><year>2000</year></pub-date><pub-date pub-type="ppub"><day>04</day><month>04</month><year>2000</year></pub-date><volume>39</volume><issue>13</issue><fpage>3788</fpage><lpage>3796</lpage><history><date date-type="received"><day>27</day><month>09</month><year>1999</year></date><date date-type="rev-recd"><day>05</day><month>01</month><year>2000</year></date><date date-type="asap"><day>10</day><month>03</month><year>2000</year></date><date date-type="issue-pub"><day>04</day><month>04</month><year>2000</year></date></history><permissions><copyright-statement>Copyright © 2000 American Chemical Society</copyright-statement><copyright-year>2000</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG)
has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized
FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells
(VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment
with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow
for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low
molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that
the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and
processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the
greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further
suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored
in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and
prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG,
FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to
mediate prolonged or unique biological responses through intracellular processes.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi992243d</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi992243dAF2"><label>†</label><p>
This work was supported in part by Grants R01HL56200 and
P01HL46902 from the National Institutes of Health and by Departmental grants from the Massachusetts Lions Eye Research Fund and
Research to Prevent Blindness, Inc.</p></notes></front><body><sec id="d7e112"><title></title><p>Fibroblast growth factor 2 (FGF-2)<xref rid="bi992243db00001" ref-type="bibr"></xref> belongs to a family
of at least 18 related polypeptides (<italic toggle="yes"><xref rid="bi992243db00001" ref-type="bibr"></xref></italic>). At the cell surface,
FGF-2 binds to tyrosine kinase receptors (FGFR1−4) as well
as heparan sulfate proteoglycans (HSPG) (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00002" ref-type="bibr"></xref>−<xref rid="bi992243db00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00004" ref-type="bibr"></xref></named-content></italic>). The biological role of the FGFRs has been characterized extensively
(<italic toggle="yes"><xref rid="bi992243db00003" ref-type="bibr"></xref></italic>). The interaction of FGF-2 with FGFR has been shown
to result in the activation of several intracellular signaling
cascades, which include activation of the mitogen-activated
protein kinases and phospholipase Cγ, leading to cell
proliferation, migration, and differentiation (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00001" ref-type="bibr"></xref>, <xref rid="bi992243db00003" ref-type="bibr"></xref></named-content></italic>). FGF-2
binding to HSPG has been shown to modulate FGF-2 activity
at a number of levels. It has been shown that the interaction
of FGF-2 with HSPG increases the binding affinity of FGF-2
for FGFR, leading to increased cellular response (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00005" ref-type="bibr"></xref>−<xref rid="bi992243db00006" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00007" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00008" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00009" ref-type="bibr"></xref></named-content></italic>).
Recent evidence suggests a direct involvement of HSPG in
signaling, such as the FGF-2-mediated dephosphorylation of
syndecan 4 (<italic toggle="yes"><xref rid="bi992243db00010" ref-type="bibr"></xref></italic>).
</p><p>There have been numerous reports implicating the involvement of internalized FGF-2 or its receptors in mediating
intracellular biological function (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00007" ref-type="bibr"></xref>, <xref rid="bi992243db00011" ref-type="bibr"></xref></named-content></italic>). Upon binding, FGF-2/FGFR complexes are internalized. The complexes are
subsequently directed to endosomes and lysosomes, where
degradation of ligand, receptor, or the entire complex occurs
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00012" ref-type="bibr"></xref>, <xref rid="bi992243db00013" ref-type="bibr"></xref></named-content></italic>). Several studies have demonstrated that this process
is inhibited by the addition of lysosomal inhibitors in various
cell types, including venular endothelial cells (<italic toggle="yes"><xref rid="bi992243db00014" ref-type="bibr"></xref></italic>), chinese
hamster lung fibroblasts (<italic toggle="yes"><xref rid="bi992243db00015" ref-type="bibr"></xref></italic>), and bovine aortic endothelial
cells (<italic toggle="yes"><xref rid="bi992243db00016" ref-type="bibr"></xref></italic>). However, there have been indications that FGF-2, its receptors, and HSPG may also be directed to other
intracellular destinations (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00017" ref-type="bibr"></xref>−<xref rid="bi992243db00018" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00019" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00020" ref-type="bibr"></xref></named-content></italic>). Indeed, there have been
reports of nuclear localization of FGF-2 in a variety of cell
types (<italic toggle="yes">11, 14, 21</italic>). Nuclear accumulation of FGF-2 has been
shown to be cell-cycle-dependent, occurring in the G1−S
transition (<italic toggle="yes"><xref rid="bi992243db00016" ref-type="bibr"></xref></italic>). FGFR have also been shown to be localized
to the nucleus and perinuclear region, where it is suggested
that they might continue to signal (<italic toggle="yes">20, 22, 23</italic>). The role of
HSPG in modulating the intracellular fate of FGF-2 and its
receptors is poorly understood. HSPG are constitutively
internalized and degraded, through both lysosomal as well
as nonlysosomal pathways in rat ovarian granulosa cells (<italic toggle="yes"><xref rid="bi992243db00024" ref-type="bibr"></xref></italic>).
Heparan sulfate has also been found localized in the nuclei
of hepatocytes (<italic toggle="yes">21, 25, 26</italic>). We have recently shown that
HSPG inhibit the accumulation of FGF-2 degradation
products in the medium of vascular smooth muscle cells
(VSMC) (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>).
</p><p>In the present study, the role of HSPG in modulating the
intracellular degradation of FGF-2 was investigated in
VSMC. VSMC were chosen for these studies for their
sensitive regulation by FGF-2, heparin, and HSPG in vivo
and in vitro (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00027" ref-type="bibr"></xref>−<xref rid="bi992243db00028" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00029" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00030" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00031" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi992243db00032" ref-type="bibr"></xref></named-content></italic>). VSMC were rendered HSPG-deficient
by treatment with sodium chlorate. The consequences of the
presence and absence of HSPG on the intracellular degradation of FGF-2 was analyzed. The intracellular processing of
FGF-2 occurred in stages. First, low molecular weight
(LMW) fragments (4−10 kDa) were detected in both native
and HSPG-deficient VSMC. However, the LMW fragments
accumulated to much higher levels in native cells as
compared with HSPG-deficient VSMC. LMW fragments of
FGF-2 constituted the greatest fraction of internalized growth
factor in native VSMC. Pulse−chase experiments showed a
product/precursor relationship between 18 kDa FGF-2 and
the low molecular weight (LMW) intermediates. The half-life of these LMW intermediates was significantly greater
in native VSMC (8−18 h) as compared with HSPG-deficient
VSMC (2−4 h). Inhibition of lysosomal degradation by
chloroquine treatment greatly reduced the intracellular
concentrations of FGF-2 in native VSMC but had no effect
on HSPG-deficient VSMC. There are distinct differences in
FGF-2 processing in the presence and absence of HSPG in
VSMC. It is possible that the regulated processing of FGF-2
by HSPG has important biological consequences.
</p></sec><sec id="d7e213"><title>Materials and Methods</title><p><italic toggle="yes">Materials.</italic> Human recombinant FGF-2 was from Scios-Nova, Inc. (Mountain View, CA).
</p><p><sup>125</sup>I-FGF-2 was prepared by a modification of the Bolton−Hunter procedure (<italic toggle="yes"><xref rid="bi992243db00006" ref-type="bibr"></xref></italic>). This method has been demonstrated
to produce active <sup>125</sup>I-FGF-2 as determined by its ability to
stimulate DNA synthesis in quiescent Balb/c3T3 cells. The
specific activity ranged from 80 to 90 μCi/μg. [<sup>35</sup>S]Sulfate
and <sup>125</sup>I-Bolton−Hunter reagent were from DuPont NEN
(Boston, MA). Sodium chlorate (NaClO<sub>3</sub>) was obtained from
Fluka (Ronkonkoma, NY). <italic toggle="yes">p</italic>-Nitrophenyl phosphate (PNP),
Nonidet P-40, and other reagent-grade chemicals were from
Sigma (St. Louis, MO).
</p><p><italic toggle="yes">Cell Culture.</italic> Vascular smooth muscle cells (VSMC) were
used between passages 4 and 8. VSMC, isolated as described
(<italic toggle="yes"><xref rid="bi992243db00033" ref-type="bibr"></xref></italic>), were from Coriell Cell Repositories (Camden, NJ).
Cells were maintained in 75 cm<sup>2</sup> vented culture flasks
(Costar, Cambridge, MA) in Dulbecco's modified Eagle's
medium (DMEM, low glucose, Life Technologies, Inc.),
supplemented with 20% fetal bovine serum (FBS) (Life
Technologies, Inc.), penicillin (100 units/mL), streptomycin
(100 μg/mL), and glutamine (2 mM). Cell number was
determined with a Coulter counter, or relative cell numbers
were determined by acid phosphatase quantitation (<italic toggle="yes"><xref rid="bi992243db00034" ref-type="bibr"></xref></italic>).
Briefly, cells were incubated in 0.1M sodium acetate (pH
5.5), 0.1% Triton X-100, and 10 mM <italic toggle="yes">p</italic>-nitrophenyl phosphate (Sigma 104 phosphatase substrate) for 45 min at 37
°C. The reaction was stopped by the addition of 1 N NaOH
and absorbance was determined at 410 nm with a Shimadzu
UV−vis spectrophotometer<bold>.</bold></p><p><italic toggle="yes">Chlorate Treatment.</italic> Chlorate treatment was performed as
described previously (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>) to generate HSPG-deficient VSMC.
To obtain similar cell numbers for native and chlorate-treated
VSMC at the end of the chlorate treatment procedure,
chlorate-treated cells were plated at a higher initial seeding
density. VSMC were plated at 25 000/cm<sup>2</sup> for native and at
37 500/cm<sup>2</sup> for chlorate-treated cells in low-glucose DMEM,
0.5% dialyzed calf serum (DCS) (Sigma), and glutamine (2
mM). Following cell attachment, approximately (∼4 h), cells
were treated with or without sodium chlorate (75 mM final
concentration) for 48 h at 37 °C.
</p><p><italic toggle="yes">Cellular Fractionation.</italic> Cell fractionation was performed
as described previously (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>). Briefly, cells were washed three
times with ice-cold binding buffer (DMEM, 25 mM HEPES,
and 0.05% gelatin) and cell-surface-bound <sup>125</sup>I-FGF-2 was
removed by sequential extraction with a high-salt buffer [20
mM HEPES and 1 M NaCl, pH 7.4; 0.5 mL/well for 5 s;
followed by a wash with phosphate-buffered saline (PBS)]
and high salt/acid buffer (10 mM sodium acetate and 1 M
NaCl, pH 5, for 5 min; followed by a wash with PBS). Cells
were trypsinized with 0.5 mL of 0.01% trypsin and 0.53 mM
EDTA (Life Technologies, Inc.) per well. Trypsinization was
stopped by the addition of 50 μL/well calf serum (Hyclone,
UT). Cell suspensions were centrifuged for 30 s at 10000<italic toggle="yes">g</italic>.
Cell pellets were resuspended by briefly vortexing in 200
μL of homogenization buffer (10 mM HEPES, pH 7.9, 10
mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and
0.5 mM PMSF) and incubated on ice for 15 min (<italic toggle="yes"><xref rid="bi992243db00020" ref-type="bibr"></xref></italic>). After
this incubation, 12.5 μL of a 10% Nonidet P-40 solution
was added to each tube and the samples were vortexed
vigorously for 10 s. This was followed by another centrifugation, 30 s at 10000<italic toggle="yes">g</italic>. The supernatant was collected as the
cytoplasmic fraction. The crude nuclear pellet was washed
two additional times by repeating the procedure outlined
above. The nuclear pellet was then resuspended in 100 μL
of homogenization buffer. Radioactivity in these fractions
was quantitated by counting in a Packard Model 5650 γ
counter. Boiling Laemmli sample buffer was added to the
fractions obtained for electrophoretic analysis. Virtually full
recovery of the cytoplasmic fraction was observed, based
on acid phosphatase quantitation in the cytoplasmic fraction
as compared with nonfractionated, whole cells. Based on
recovery and quantitation of the lysosomal contaminant, acid
phosphatase, in the nuclear fraction (<italic toggle="yes"><xref rid="bi992243db00034" ref-type="bibr"></xref></italic>), less than 0.5%
lysosomal contamination was found in the nuclear fraction
(<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">TCA Precipitation.</italic> Samples were subjected to TCA
precipitation to separate incorporated radioactivity from
unincorporated or small fragments. Briefly, 5 μL of sample
was combined with 5 μL of BSA solution (10 mg/mL), 10
μL of KI (10 mM), and 70 μL of NaCl (150 mM). Then
12.5 μL of 100% TCA (ice cold) was added to the final
mixture to precipitate proteins. The precipitate was collected
by centrifugation at 10000<italic toggle="yes">g</italic> for 10 min. Radioactivity in the
supernatant and pellet was quantitated with a γ counter.
</p><p><italic toggle="yes">Gel Electrophoresis.</italic> Cell layers were solubilized in boiling
Laemmli sample buffer. Samples were normalized to relative
cell numbers on the basis of acid phosphatase quantitation
and subjected to SDS−polyacrylamide gel electrophoresis
(PAGE) (16% running gel, 5% stacking gel) (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>). Bio-Rad
prestained standards were used for molecular mass calibration. <sup>125</sup>I-labeled protein bands were visualized through
exposure of the gel onto a phosphorimager SF phosphor plate
(Molecular Dynamics).
</p><p><italic toggle="yes">Rate Constants.</italic> Numerical analysis was based on first-order reactions. Each step in the FGF-2 binding, internalization, and processing pathway was represented as a chemical
reaction, where product formation was dependent on the first-order concentration of the precursor pool. All models were
fitted to experimental data and assumed uniform binding sites
for FGF-2 on the cell surface, for both the high- and low-affinity receptors. Cooperative and competitive interactions
were assumed to be negligible. Nonspecific interactions were
low and assumed to be negligible at the concentration ranges
of the experiments (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>). In each case, a series of equations
were derived as a model to describe the specific experiment
and these were solved simultaneously and compared with
experimental data, to yield kinetic rate constants. The terms
are as follows: [F] is the concentration of soluble FGF-2 in
the medium at any time, [R] is the concentration of free
FGFR remaining at any time, [F·R] is the concentration of
FGF-2/FGFR complexes at any time, [H] is the concentration
of free HSPG sites present at any time, [F·H] is the
concentration of FGF-2/HSPG complexes, [Fe] is the concentration of internalized FGF-2 (18 and 16 kDa), [F(LMW)]
is the concentration of processed low molecular fragments
of FGF-2, [F(deg)] is the concentration of TCA-soluble
byproducts of FGF-2 secreted into the medium, <italic toggle="yes">k</italic><sub>onR</sub> is the
rate constant for the forward reaction of FGF-2 binding to
FGFR, <italic toggle="yes">k</italic><sub>offR</sub> is the rate constant for the dissociation of the
FGF-2/FGFR complex, <italic toggle="yes">k</italic><sub>onH</sub> is the rate constant for the
forward reaction of FGF-2 binding to HSPG, <italic toggle="yes">k</italic><sub>offH</sub> is the rate
constant for the dissociation of the FGF-2/HSPG complex,
<italic toggle="yes">k</italic><sub>eR</sub> is the rate constant for the internalization of FGF-2/FGFR
complex, <italic toggle="yes">k</italic><sub>eH</sub> is the rate constant for the internalization of
FGF-2/HSPG complex, <italic toggle="yes">k</italic><sub>deg</sub> is the rate constant for the
degradation of FGF-2 into TCA-soluble fragments detected
in the medium, and <italic toggle="yes">k</italic><sub>LMW</sub> is the rate constant for the
processing of internalized FGF-2 into low molecular weight
fragments.
</p><p><italic toggle="yes">Endocytic Reactions.</italic> Endocytosis was simulated on the
basis of reactions 1−4. At relatively short time scales, the
assumption that internalized FGF-2 is virtually completely
in the form of high molecular weight FGF-2 still holds. It
was assumed that HSPG and receptors internalize FGF-2
independently. Downstream reactions were accounted for by
the inclusion of <italic toggle="yes">k</italic><sub>d</sub>, which represents the sum of the rate
constants of the subsequent reactions.<xref rid="bi992243de00001"></xref><disp-formula content-type="pre-labeled" id="bi992243de00001"><!--%@md;sys;6q@%Reaction 1%@hm;8@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%F%@fnx;];vis;full@% + %@fn;[;vis;full;auto@%R%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;↔;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%onR%@sbx@%%@sbx@%%@au@%%@ex@%%@ital@%k%@rsf@%%@sb@%offR%@sbx@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%Reaction 2%@ah;8@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%F%@fnx;];vis;full@% + %@fn;[;vis;full;auto@%H%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;↔;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%onH%@sbx@%%@sbx@%%@au@%%@ex@%%@ital@%k%@rsf@%%@sb@%offH%@sbx@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%Reaction 3%@ah;8@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;→;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%eR%@sbx@%%@sbx@%%@au@%%@ex@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%Reaction 4%@ah;8@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;→;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%eH%@sbx@%%@sbx@%%@au@%%@ex@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi992243de00001.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
The above reactions were described by a series of first-order
differential equations, shown below:<xref rid="bi992243de00005"></xref><disp-formula content-type="pre-labeled" id="bi992243de00005"><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%onR%@sbx@%%@fn;[;vis;full;auto@%F%@fnx;];vis;full@%%@fn;[;vis;full;auto@%R%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%offR%@sbx@%%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%eR%@sbx@%%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@%%@id;reqid;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%onH%@sbx@%%@fn;[;vis;full;auto@%F%@fnx;];vis;full@%%@fn;[;vis;full;auto@%H%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%offH%@sbx@%%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%eH%@sbx@%%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@%%@id;reqid;2@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%eR%@sbx@%%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@% + %@ital@%k%@rsf@%%@sb@%eH%@sbx@%%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%deg%@sbx@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@id;reqid;3@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = − %@fr@%d%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% − %@fr@%d%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@%%@id;reqid;4@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi992243de00005.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
To determine postinternalization events, all upstream reactions were evaluated simultaneously. Reactions 5 and 6 were
included with reactions 1−4 to simulate the binding,
internalization, and processing of FGF-2:<xref rid="bi992243de00009"></xref><disp-formula content-type="pre-labeled" id="bi992243de00009"><!--%@md;sys;6q@%Reaction 5%@hm;9@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;→;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%LMW%@sbx@%%@sbx@%%@au@%%@ex@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%F%@fn;(;vis;full;unlock@%LMW%@fnx;);vis;full@%%@fnx;];vis;full@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%Reaction 6%@ah;9@%%@bp@%%@/mh;%lnwidth?18q:0@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@% %@mspx;$-$90@%%@aw;→;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%deg%@sbx@%%@sbx@%%@au@%%@ex@%%@exx@%%@awx@% %@fn;[;vis;full;auto@%F%@fn;(;vis;full;unlock@%deg%@fnx;);vis;full@%%@fnx;];vis;full@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi992243de00009.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
The above reactions were described by eqs 1 and 2 and the
following processing reactions:<xref rid="bi992243de00011"></xref><disp-formula content-type="pre-labeled" id="bi992243de00011"><!--%@md;sys;6q@%%@mpos;l@%%@fr@%d%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%eR%@sbx@%%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@% + %@ital@%k%@rsf@%%@sb@%eH%@sbx@%%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%LMW%@sbx@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@% − %@ital@%k%@rsf@%%@sb@%deg%@sbx@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@id;reqid;5@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F%@fn;(;vis;full;unlock@%LMW%@fnx;);vis;full@%%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%LMW%@sbx@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@id;reqid;6@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F deg%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = %@ital@%k%@rsf@%%@sb@%deg%@sbx@%%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@id;reqid;7@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><!--%@md;sys;6q@%%@fr@%d%@fn;[;vis;full;auto@%F%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% = − %@fr@%d%@fn;[;vis;full;auto@%F·R%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% − %@fr@%d%@fn;[;vis;full;auto@%F·H%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% − %@fr@%d%@fn;[;vis;full;auto@%Fe%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% − %@fr@%d%@fn;[;vis;full;auto@%F%@fn;(;vis;full;unlock@%LMW%@fnx;);vis;full@%%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@% −[S_EL2;quad]\
%@fr@%d%@fn;[;vis;full;auto@%F%@fn;(;vis;full;unlock@%deg%@fnx;);vis;full@%%@fnx;];vis;full@%%@fd@%d%@ital@%t%@rsf@%%@frx@%%@id;reqid;8@%%@lp;&3q;1@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi992243de00011.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
An iterative process was used, in a computer simulation using
FORTRAN on a 100 MHz Think Pad 560 (IBM), which
numerically integrated the model equations, given a set of
initial reaction rate constants, by a fourth-order Runge-Kutta
step method. The values derived through the numerical
integrator were then compared to experimental data and the
difference (χ<sup>2</sup>) was minimized with every iteration. The
iterative procedure continued until theoretical values were
obtained that minimize χ<sup>2</sup>. This was done by reguessing for
the values of the rate constants through the Levenberg−Marquardt method (<italic toggle="yes"><xref rid="bi992243db00035" ref-type="bibr"></xref></italic>) and reintegrating the equations. The
iteration stopped when the value of χ<sup>2</sup> was within 0.1% of
the previous value.
</p></sec><sec id="d7e443"><title>Results</title><p><italic toggle="yes">LMW Fragments of FGF-2 Accumulate in Native VSMC.
</italic>Endocytosis of a growth factor/receptor complex generally
leads to the degradation of the complex. Degradation of either
ligand or receptor has been regarded as a shut-off mechanism
of the signaling process. However, FGF-2 has a relatively
long intracellular half-life (<italic toggle="yes"><xref rid="bi992243db00036" ref-type="bibr"></xref></italic>) as compared with non-heparin-binding mitogens (<italic toggle="yes"><xref rid="bi992243db00037" ref-type="bibr"></xref></italic>), suggesting that intracellular
degradation of FGF-2 is subject to regulation. Therefore, the
role of HSPG in modulating FGF-2 catabolism was investigated. Secreted, processed byproducts of FGF-2 degradation
had previously been shown to accumulate in the medium
with slower kinetics in native VSMC as compared with
HSPG-deficient cells (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>), suggesting that HSPG inhibit
intracellular degradation of FGF-2.
</p><p>The intracellular state of FGF-2 was analyzed at different
stages of processing in native and HSPG-deficient VSMC.
Cells were exposed to <sup>125</sup>I-FGF-2 at 37 °C to allow for uptake
and processing. At each time point, cell-surface-associated
<sup>125</sup>I-FGF-2 was removed (high ionic strength, low pH wash)
and the cells were solubilized and subjected to SDS−PAGE
analysis. At the earliest time points, the majority of the <sup>125</sup>I-FGF-2 was composed of 18 and 16 kDa forms. With
increasing incubation times a group of LMW fragments of
FGF-2 formed in both native and HSPG-deficient VSMC
(Figure <xref rid="bi992243df00001"></xref>). Only native VSMC retained and accumulated
significant amounts of the LMW fragments, suggesting that
HSPG-deficient cells were able to process FGF-2 in a similar
manner but were unable to retain the processed fragments.
The relative intensities of the bands in each lane in Figure <xref rid="bi992243df00001"></xref>
were quantitated and the LMW fragments constituted the
majority of the FGF-2 present in the native cells: greater
than 70% after 16 h and 90% after 24 h (Figure <xref rid="bi992243df00002"></xref>). Within
HSPG-deficient VSMC, the LMW fragments constituted a
maximum of 30% of total intracellular FGF-2 present after
prolonged incubations (24−36 h). These data suggest that
the large differences in the total intracellular levels of FGF-2
in native versus HSPG-deficient VSMC arose largely from
the increased accumulation of LMW fragments within native
cells. The intracellular levels of the 18 and 16 kDa species
of FGF-2 in the HSPG-deficient VSMC were virtually the
same as in the native cells. However, LMW fragments
reached a rapid steady state, followed by a steady decline at
approximately 12 h in HSPG-deficient cells. At the later time
points, native cells retained 10-fold higher concentrations
of LMW fragments, as compared to HSPG-deficient VSMC.
<fig id="bi992243df00001" position="float" orientation="portrait"><label>1</label><caption><p>Intracellular processing of FGF-2 within native and HSPG-deficient VSMC. Native (A) and HSPG-deficient VSMC (B) were treated with<sup>125</sup>I-FGF-2 (0.28 nM) for the indicated times
at 37 °C. At each time point, cell-surface-bound FGF-2 was
removed, and cells were solubilized with boiling Laemmli sample
buffer and applied to an SDS−16% polyacrylamide gel. Bands were
visualized by the phosphorimager and quantitated with Image Quant
software. Similar results were observed in four separate experiments.
</p></caption><graphic xlink:href="bi992243df00001.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi992243df00002" position="float" orientation="portrait"><label>2</label><caption><p>Total composition of 18 kDa, 16 kDa, and LMW fragments within native and HSPG-deficient VSMC.<sup>125</sup>I-FGF-2
was added to native (A) and HSPG-deficient (B) VSMC (0.28 nM)
for the indicated times at 37 °C. At each time point, cell-surface-bound FGF-2 was removed, and cells were solubilized in boiling
Laemmli sample buffer and applied to an SDS−polyacrylamide
gel (Figure <xref rid="bi992243df00001"></xref>). Percentage radioactivity in each band from Figure
<xref rid="bi992243df00001"></xref> was determined by a phosphorimager. <sup>125</sup>I radioactivity was
calculated in bands corresponding to 18 (+), 16 (○), and LMW
(◇) fragments by multiplying the relative composition of each lane
by the total amount of radioactivity per lane, as determined by γ
counting. The results presented are representative of the average
of triplicate determinations. Similar results were observed in three
separate experiments.</p></caption><graphic xlink:href="bi992243df00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">LMW Fragments Are Cleared at a Slower Rate in Native
VSMC. </italic>Pulse chase experiments were performed to isolate
the relevant FGF-2 intracellular processing steps that resulted
in the observed differences in uptake of FGF-2 in native and
HSPG-deficient VSMC. Native and HSPG-deficient VSMC
were exposed to <sup>125</sup>I-FGF-2 for 2 h at 37 °C (pulse). After
this pulse period, medium containing <sup>125</sup>I-FGF-2 and cell-surface-bound <sup>125</sup>I-FGF-2 were removed. Fresh medium was
added back to the cells and the incubation continued for
various times at 37 °C for the chase. At each time point,
medium was collected and the cells were extracted for
analysis of processed FGF-2. LMW fragments were the
primary constituent, after approximately 12 h of chase period
(Figure <xref rid="bi992243df00003"></xref>). After a 2 h exposure to FGF-2, both native and
HSPG-deficient VSMC accumulated similar levels of LMW
fragments, which allowed for a direct comparison of FGF-2
clearance under these conditions. Comparison of internal
FGF-2 immediately after the pulse period to that after 2 h
of chase (<italic toggle="yes">t</italic> = 0 and 2 h in Figure <xref rid="bi992243df00003"></xref>) suggests that the
formation of LMW fragments occurred rapidly. Additionally,
a product/precursor relationship existed between the higher
molecular weight forms of initially internalized, 18 kDa
FGF-2 and the processed fragments that were generated
during prolonged exposures. Total radioactivity in each lane
was quantitated (Figure <xref rid="bi992243df00003"></xref>C). The half-life of the LMW
intermediates was greatly prolonged in native VSMC (∼18
h; apparent first-order rate constant of 0.04 h<sup>-1</sup>) as compared
with HSPG-deficient VSMC (∼4 h; apparent first-order rate
constant of 0.17 h<sup>-1</sup>).
<fig id="bi992243df00003" position="float" orientation="portrait"><label>3</label><caption><p>Intracellular clearance of processed FGF-2 in native and HSPG-deficient VSMC. Native (A) and HSPG-deficient VSMC (B) were treated with<sup>125</sup>I-FGF-2 (0.28 nM) for 2 h at 37 °C (pulse),
to allow for <sup>125</sup>I-FGF-2 internalization. After this time, cells were
washed with binding buffer and high-salt, low-pH buffer (see
Materials and Methods) to remove all soluble and cell-bound <sup>125</sup>I-FGF-2. Medium was added back to the cells together with unlabeled
FGF-2 at a final concentration of 0.28 nM (chase, time = 0 h).
Cells were incubated for the indicated times at 37 °C. Cell layers
were solubilized with boiling Laemmli sample buffer, radioactivity
was quantitated by γ counting (C) in both native (○) and HSPG-deficient VSMC (·) and samples were applied to SDS−PAGE.
The results presented represent the pooled fractions of triplicate
determinations.</p></caption><graphic xlink:href="bi992243df00003.eps" position="float" orientation="portrait"></graphic></fig></p><p>The clearance of FGF-2 comprises a sum of processes that
lead to the eventual excretion of small molecular weight
FGF-2 degradation products. Although TCA soluble processed fragments of FGF-2 comprised the majority of the
cellular excreted FGF-2, experiments were conducted to
evaluate the extent of FGF-2 recycling. When VSMC were
allowed to excrete <sup>125</sup>I-FGF-2 under conditions where a large
excess of unlabeled FGF-2 was included in the medium to
prevent rebinding of recycled intact <sup>125</sup>I-FGF-2, no significant
intact <sup>125</sup>I-FGF-2 was detected in the incubation medium
(data not shown). Therefore, it is unlikely that recycling of
FGF-2 occurs to any significant degree in VSMC under these
experimental conditions.
</p><p><italic toggle="yes">LMW Are Generated with Rapid Kinetics in Native VSMC.
</italic>The differences in FGF-2 processing between native and
HSPG-deficient VSMC may arise at various stages of growth
factor binding, endocytosis, intracellular processing, and
excretion. It has been shown that the presence of HSPG alters
FGF-2 binding to cell surface receptors (<italic toggle="yes">6, 8, 9</italic>). To identify
the steps in the FGF-2 processing pathway that are controlled
by HSPG, a kinetic analysis of FGF-2 endocytosis, intracellular processing, and excretion was performed.
</p><p>To isolate the endocytic rate constant for FGF-2 at 37 °C,
short time scale internalization of FGF-2 was carried out to
avoid the complexities of downstream processing events.
Cells were exposed to <sup>125</sup>I-FGF-2 over a 30 min time course
and the amount of <sup>125</sup>I-FGF-2 in each experimental compartment (medium, cell surface, and intracellular) was measured.
At each time point, the medium was collected and <sup>125</sup>I-FGF-2
content was determined by TCA precipitation. HSPG and
receptor-bound FGF-2 were removed and counted through
high-salt and low-pH extractions sequentially. Internalized
FGF-2 was extraction with 0.5% Triton X-100 in PBS. The
endocytic rate constants were determined by fitting eqs 1−4
to the experimental data (see Materials and Methods) (Table
<xref rid="bi992243dt00001"></xref>). The apparent receptor endocytic rate constant was 3-fold
greater for HSPG-deficient VSMC as compared with native
cells. This may reflect a higher percentage of FGF-2
internalization through receptors in HSPG-deficient VSMC.
<table-wrap id="bi992243dt00001" position="float" orientation="portrait"><label>1</label><caption><p>Kinetic Constants for FGF-2 Internalization in Native VSMC<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">kinetic constants</oasis:entry><oasis:entry namest="2" nameend="2">native VSMC</oasis:entry><oasis:entry namest="3" nameend="3">HSPG-deficient VSMC
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">k</italic><sub>eR</sub> (h<sup>-1</sup>)
</oasis:entry><oasis:entry colname="2">0.72
</oasis:entry><oasis:entry colname="3">2.16
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">k</italic><sub>eH</sub> (h<sup>-1</sup>)
</oasis:entry><oasis:entry colname="2">0.29
</oasis:entry><oasis:entry colname="3">N/A</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Data for the amounts of FGF-2 in the medium, bound to HSPG,
bound to receptor sites, and inside the cell were collected over a 30-min time course and were simultaneously fit to eqs 1−4. The computer
simulation minimized the difference between the model and the data.
Endocytosis rate constants for receptors, <italic toggle="yes">k</italic><sub>eR</sub>, and HSPG, <italic toggle="yes">k</italic><sub>eH</sub>, were
determined.</p></table-wrap-foot></table-wrap></p><p>To identify the relative extent to which slow production
or fast excretion of LMW fragments lead to the differences
observed in the levels of these fragments formed in native
and HSPG-deficient cells (Figure <xref rid="bi992243df00001"></xref>, panels A and B,
respectively), a kinetic analysis was performed in conjunction
with process simulation based on reactions 1−6 and eqs 1−8
(Figures <xref rid="bi992243df00004"></xref> and <xref rid="bi992243df00005"></xref>). Cells were exposed to <sup>125</sup>I-FGF-2 at 37
°C to allow for uptake and processing. At each time point,
cell-surface-associated <sup>125</sup>I-FGF-2 was removed (high ionic
strength, low pH wash) and the cells were solubilized and
subjected to SDS−PAGE analysis. Processed <sup>125</sup>I-FGF-2
fragments excreted into the medium were quantitated by
TCA precipitation. Internalized FGF-2, visualized on gels
as 18 and 16 kDa bands, were grouped as one pool. The
results of the process simulation best fit are shown in Figures
<xref rid="bi992243df00004"></xref> and <xref rid="bi992243df00005"></xref>. The rate constant for LMW fragment formation was
increased by greater than 6-fold within native as compared
with HSPG-deficient VSMC, suggesting that native cells
produce LMW fragments at faster rates (Table <xref rid="bi992243dt00002"></xref>). The rate
constant governing the overall excretion of degraded FGF-2
into the medium was similar within both native and HSPG-deficient cells. This excretion rate did not reflect the much
slower removal of LMW observed during the pulse chase
experiments, indicating that the major pathway of FGF-2
degradation does not include the LMW fragments. Taken
together, these data suggest that a primary contributor of
FGF-2 accumulation within native cells was the increased
production and slow degradation of LMW fragments of FGF-2.
<fig id="bi992243df00004" position="float" orientation="portrait"><label>4</label><caption><p>FGF-2 processing and secretion within native VSMC.<sup>125</sup>I-FGF-2 was added to native VSMC (0.28 nM) for the indicated
times at 37 °C. At each time point, cell-surface-bound FGF-2 was
removed, medium was subject to TCA precipitation, and cells were
solubilized in boiling Laemmli sample buffer and applied to an
SDS−polyacrylamide gel. Radioactivity in each lane was quantitated with a phosphorimager. <sup>125</sup>I radioactivity was calculated in
bands corresponding to 18 + 16 kDa (○) and LMW (◇) (A), and
TCA-soluble (degraded FGF-2) radioactivity was measured in the
medium (B) (□). The results represent the averages of triplicate
determinations. First-order differential equations (eqs 1−8) were
fit to the data to produce the theoretical isotherms (lines shown).</p></caption><graphic xlink:href="bi992243df00004.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi992243df00005" position="float" orientation="portrait"><label>5</label><caption><p>FGF-2 processing and secretion within HSPG-deficient VSMC.<sup>125</sup>I-FGF-2 was added to native VSMC (0.28 nM) for the
indicated times at 37 °C. At each time point, cell-surface-bound
FGF-2 was removed, and cells were solubilized in boiling Laemmli
sample buffer and applied to an SDS−polyacrylamide gel. Radioactivity in each lane was quantitated by using the phosphorimager.
<sup>125</sup>I radioactivity was calculated in bands corresponding to 18 +
16 kDa (○) and LMW (◇) (A), and TCA-soluble (degraded FGF-2) radioactivity was measured in the medium (B). The results are
the average of triplicate determinations. First-order differential
equations (eqs 1−8) were fit to the data to generate the theoretical
isotherms (line shown).</p></caption><graphic xlink:href="bi992243df00005.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi992243dt00002" position="float" orientation="portrait"><label>2</label><caption><p>Kinetic Constants for FGF-2 Processing in Native and HSPG-Deficient VSMC<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">kinetic constants</oasis:entry><oasis:entry namest="2" nameend="2">native VSMC</oasis:entry><oasis:entry namest="3" nameend="3">HSPG-deficient VSMC
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">k</italic><sub>LMW</sub> (h<sup>-1</sup>)
</oasis:entry><oasis:entry colname="2">0.17
</oasis:entry><oasis:entry colname="3">0.026
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"><italic toggle="yes">k</italic><sub>deg</sub> (h<sup>-1</sup>)
</oasis:entry><oasis:entry colname="2">0.45
</oasis:entry><oasis:entry colname="3">0.50</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> The results of the FGF-2 processing studies (Figures <xref rid="bi992243df00004"></xref> and <xref rid="bi992243df00005"></xref>), along
with values for the amounts of FGF-2 bound to receptors and HSPG
on the cell surface, and the endocytosis rate constants (Table <xref rid="bi992243dt00001"></xref>) were
simultaneously fit to eqs 1−8. The computer simulation minimized
the difference between the model and the data.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">LMW Fragment Formation Is Required for FGF-2 Cytoplasmic Retention. </italic>Native VSMC accumulated higher intracellular concentrations of FGF-2 as compared with HSPG-deficient cells (<italic toggle="yes">11</italic>; Figures <xref rid="bi992243df00001"></xref>−5). The differences in
intracellular levels of FGF-2 could be accounted for by the
increased amounts of LMW fragments in native VSMC. If
the formation of LMW fragments of FGF-2 was responsible
for increased accumulation and high retention of FGF-2 in
VSMC, then inhibiting intracellular degradation of FGF-2
might result in a specific reduction in the accumulation of
FGF-2 within native VSMC. To test this hypothesis, both
native and HSPG-deficient VSMC were treated with the
lysosomal inhibitor chloroquine (50 μM) 30 min prior to <sup>125</sup>I-FGF-2 addition at 37 °C. At each time point, cell surface
FGF-2 was removed for quantitation and the cells were
fractionated into cytoplasmic (Figure <xref rid="bi992243df00006"></xref>) and nuclear (Figure
<xref rid="bi992243df00007"></xref>) pools. The cytoplasmic accumulation of FGF-2 was
reduced in native cells treated with chloroquine. Chloroquine
treatment reduced cytoplasmic accumulation of FGF-2 to
levels found in HSPG-deficient cells. Chloroquine addition
did not have a significant impact on FGF-2 accumulation in
HSPG-deficient cells, consistent with the finding that LMW
products do not constitute a high percentage of FGF-2 in
these cells. The nuclear accumulation of FGF-2 in native
and HSPG-deficient VSMC was virtually unaffected by
chloroquine treatment. Taken together, these data suggest
that FGF-2 processing was mediated by lysosomes to a large
extent. The fact that chloroquine reduced FGF-2 levels
specifically in native cells indicated that the processing of
FGF-2 was necessary for cellular retention of this growth
factor. Nuclear targeting of FGF-2, in contrast, appears to
be independent of lysosomal degradation. This is consistent
with our previous data as well as that of others, suggesting
that mainly intact 18 kDa FGF-2 enters the nuclear compartment (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>).
<fig id="bi992243df00006" position="float" orientation="portrait"><label>6</label><caption><p>Effect of chloroquine on<sup>125</sup>I-FGF-2 cytoplasmic uptake
in native and HSPG-deficient VSMC. Native (A) and HSPG-deficient (B) VSMC were treated with (◇) or without (○) 50 μM
chloroquine for 30 min. <sup>125</sup>I-FGF-2 was added at a final concentration of 0.28 nM, and cells were incubated at 37 °C for the indicated
times. At each time point, cell-surface-bound FGF-2 was removed,
and cells were extracted and fractionated into cytoplasmic and
nuclear pools. Radioactivity was quantitated by a γ counter. The
results presented represent the averages ± SE of triplicate determinations.</p></caption><graphic xlink:href="bi992243df00006.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi992243df00007" position="float" orientation="portrait"><label>7</label><caption><p>Effect of chloroquine on<sup>125</sup>I-FGF-2 nuclear uptake in
native and HSPG-deficient VSMC. Native (A) and HSPG-deficient
(B) VSMC were treated with (◇) or without (○) 50 μM chloroquine
for 30 min. <sup>125</sup>I-FGF-2 was added at a final concentration of 0.28
nM, and cells were incubated at 37 °C for the indicated times. At
each time point, cell-surface-bound FGF-2 was removed, and cells
were extracted and fractionated into cytoplasmic and nuclear pools.
Radioactivity was quantitated by a γ counter. The results presented
represent the averages ± SE of triplicate determinations.</p></caption><graphic xlink:href="bi992243df00007.tif" position="float" orientation="portrait"></graphic></fig></p><p>Treatment with chloroquine had no effects on cell number
or FGF-2 binding to the cell surface in either native or
HSPG-deficient VSMC, during the time course of these
experiments (data not shown). SDS−PAGE electrophoresis
confirmed that cellular degradation of FGF-2 was inhibited.
Only 18 kDa FGF-2 was visualized with chloroquine
treatment. Treatment with ammonium chloride or leupeptin,
additional lysosomal inhibitors, showed similar effects on
the intracellular levels of FGF-2. Treatment with the proteosome inhibitor lactacystin (50 μg/mL) did not influence
FGF-2 accumulation in either native or HSPG-deficient
VSMC (data not shown).
</p></sec><sec id="d7e783"><title>Discussion</title><p>HSPG modulation of FGF-2 stability in the extracellular
environment has been extensively characterized (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00038" ref-type="bibr"></xref>, <xref rid="bi992243db00039" ref-type="bibr"></xref></named-content></italic>). In
the extracellular matrix and basement membranes of tissues,
HSPG regulate both the bioavailability and the half-life of
growth factors by direct physical interaction (<italic toggle="yes"><xref rid="bi992243db00039" ref-type="bibr"></xref></italic>). Heparan
sulfate and heparin bind and inhibit FGF-2 proteolysis. It
has been difficult to establish an intracellular role for HSPG
in modulating FGF-2 activity. This has largely been due to
the fact that HSPG increase the affinity of FGF-2 for its cell
surface receptors and thereby influence the binding and
internalization of FGF-2 over the entire course of uptake.
Dissecting the role of HSPG in specific steps governing
growth factor function distinct from that governed by cell
surface receptors has been limited. HSPG have been shown
to be constitutively internalized and degraded, by both
lysosomal-mediated and nonlysosomal pathways, in rat
ovarian granulosa cells <italic toggle="yes">(24, 40)</italic>. These previous studies
suggest that the constitutive internalization of HSPG may
influence growth factor internalization.
</p><p>FGF-2 undergoes internalization and degradation once it
has gained access to the intracellular environment <italic toggle="yes">(1, 11,
14, 15</italic>). The role of HSPG in the regulation of intracellular
processing of FGF-2 has not been extensively studied. In
the present study, the role of HSPG in modulating the
intracellular half-life of FGF-2 was investigated. The consequences of the presence and absence of HSPG in FGF-2
processing within VSMC was analyzed. Native and HSPG-deficient VSMC were exposed to FGF-2 for various times,
to allow for uptake and processing of growth factor. Native
VSMC incorporated higher levels of FGF-2 (Figure <xref rid="bi992243df00002"></xref>). The
increase in FGF-2 incorporation was largely due to the high
accumulation of LMW forms of processed FGF-2, which
accumulated to high levels within native and not HSPG-deficient VSMC (Figures <xref rid="bi992243df00001"></xref> and <xref rid="bi992243df00002"></xref>). LMW fragments of
FGF-2 have been shown to be generated in other cell types
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00014" ref-type="bibr"></xref>, <xref rid="bi992243db00015" ref-type="bibr"></xref></named-content></italic>). Both native and HSPG-deficient VSMC were
capable of processing FGF-2. However, LMW fragments
were stabilized in native VSMC but not in HSPG-deficient
cells. It is possible that HSPG either directly interact with
LMW fragments of FGF-2 or direct these fragments to
intracellular compartments where they are protected from
further processing.
</p><p>The increased accumulation of LMW fragments of FGF-2
within native VSMC is likely the result of increased
production and decreased degradation of these fragments
(Figure <xref rid="bi992243df00008"></xref>). The pulse chase experiments demonstrated that
degradation of FGF-2 from 18 to 16 kDa occurred relatively
rapidly (with respect to other processes) in both native and
HSPG-deficient VSMC and proceeded until LMW intermediates of FGF-2 were formed (Figures <xref rid="bi992243df00001"></xref> and <xref rid="bi992243df00005"></xref>). This was
consistent with previous results with venular endothelial cells
(<italic toggle="yes"><xref rid="bi992243db00014" ref-type="bibr"></xref></italic>). However, the further removal of LMW fragments was
significantly slowed in native compared to HSPG-deficient
cells. Thus, it is possible that an intracellular complex of
FGF-2 and heparan sulfate inhibits further processing of
FGF-2 within native cells. It may also be that excretion
reactions are operating under different regimes in native
versus HSPG-deficient VSMC. In native VSMC, where
LMW fragment levels are high, the excretion reaction may
be limited by other components. In HSPG-deficient VSMC,
where LMW fragment levels are low, the excretion reaction
may be directly dependent on LMW fragment concentrations.
<fig id="bi992243df00008" position="float" orientation="portrait"><label>8</label><caption><p>Kinetic model of FGF-2 processing in native versus HSPG-deficient VSMC. The various kinetic constants determined from the analyses described in Figures <xref rid="bi992243df00001"></xref>−6 and Tables <xref rid="bi992243dt00001"></xref> and <xref rid="bi992243dt00002"></xref> were incorporated into a simplified model of FGF-2 processing. Native cells containing HSPG (A) are characterized by increased generation and decreased decay of LMW fragments of FGF-2, suggesting that these species are separate from the bulk of FGF-2 that is degraded and excreted as TCA-soluble material. HSPG-deficient cells (B) are characterized by slow formation and rapid decay of LMW fragments such that these species do not appear to be separate from the bulk processing of FGF-2 to small degradation products. Thus the model is consistent with a process where HSPG cause the selective buildup of intracellular pools of LMW fragments of FGF-2.<italic toggle="yes">k</italic><sub>obs</sub> represents the observed first-order rate constant
derived from the pulse chase analysis of LMW fragment clearance
(Figure <xref rid="bi992243df00003"></xref>).</p></caption><graphic xlink:href="bi992243df00008.tif" position="float" orientation="portrait"></graphic></fig></p><p>The rate constants derived by modeling the uptake and
processing of FGF-2 as a set of first-order differential
equations suggest that the greatest difference between native
and HSPG-deficient VSMC was in the formation of LMW
fragments, as reflected by the values of <italic toggle="yes">k</italic><sub>LMW</sub> (Table <xref rid="bi992243dt00002"></xref>). The
model results further suggest that the rapid generation of
TCA-soluble excreted fragments of FGF-2 in both native
and HSPG-deficient cells, as characterized by the relatively
large values of <italic toggle="yes">k</italic><sub>deg</sub>, is distinct from the degradation of the
LMW fragments. That is, under both conditions the rate
constants for degradation product generation were similar
and much larger than the apparent rate constants describing
the loss of LMW fragments (Table <xref rid="bi992243dt00002"></xref>, Figure <xref rid="bi992243df00008"></xref>). Thus, the
LMW fragments appear to represent a selectively stable
subpool of internal FGF-2 that is preferred in the presence
of HSPG. Since the intracellular levels of the 18 and 16 kDa
FGF-2 were similar in both native and HSPG-deficient
VSMC, it is unlikely that the increased kinetics of LMW
FGF-2 fragment formation in native cells resulted from
increased reactant (18 and 16 kDa FGF-2) concentration
driving LMW formation. Instead it appears that FGF-2
uptake by lysosomes was occurring at levels nearing saturation for both native and HSPG-deficient VSMC and was not
limiting. These data strongly suggest that HSPG specifically
modulate intracellular events governing FGF-2 processing.
HSPG might act to direct FGF-2 or LMW fragments to other
intracellular compartments where rapid degradation is slowed.
</p><p>To assess whether the increased retention of FGF-2 in
native compared to HSPG-deficient cells required lysosomal
cleavage, the effect of the lysosomal inhibitor chloroquine
(50 μM) was evaluated. The addition of chloroquine to the
medium of VSMC reduced the levels of FGF-2 incorporation
in native cells to those found in HSPG-deficient VSMC
(Figure <xref rid="bi992243df00007"></xref>). Whereas chloroquine reduced FGF-2 incorporation in native VSMC, it had virtually no effect on HSPG-deficient cells. These data suggest that a proteolytic event
was required for the high retention of FGF-2 in native
VSMC. The appearance of FGF-2 in the nuclear fraction
was unaffected by chloroquine treatment in both native and
HSPG-deficient VSMC. This was consistent with existing
literature as well as data from our laboratory confirming that
high molecular weight 18 kDa FGF-2 is shuttled to this
compartment (<italic toggle="yes"><xref rid="bi992243db00011" ref-type="bibr"></xref></italic>).
</p><p>The retention of FGF-2 for such prolonged periods of time
may serve a biological function. It still remains to be
determined whether FGF-2 interacts with other intracellular
factors to regulate cellular activity. The data presented here
strongly suggest that FGF-2 interacts with heparan sulfate
once internalized. It may be that the degradation of components involved in a complex of FGF-2/FGFR/HSPG is a
requisite for retention of LMW fragments of FGF-2. For
example, the processing of either receptor or HSPG may also
be required. Furthermore, the processing of either of these
moieties may provide distinct biological functions independent of FGF-2.
</p><p>The biological function of cell surface receptors and the
ligands that interact with them may not be limited to the
signaling generated at the cell surface. Internalized cell
surface receptors and ligands might have important biological
functions. As cells have evolved into very efficient processors
of signals, the question arises as to why some growth factors
are retained for such prolonged periods after their cell surface
role has been fulfilled. Considering that both epidermal
growth factor (EGF) and FGF-2 have been shown to activate
the MAPK signaling cascade (<italic toggle="yes"><xref rid="bi992243db00041" ref-type="bibr"></xref></italic>), it may be that differences
in biological action between these mitogens arise from the
intracellular processing they undergo. In particular, the
persistence of growth factor-mediated signals might ultimately dictate biological response. Indeed, the effects of EGF
and nerve growth factor (NGF) on the proliferation and
differentiation of PC12 cells represents a classic example of
the role for signal persistence (<italic toggle="yes"><xref rid="bi992243db00042" ref-type="bibr"></xref></italic>). Whereas both EGF and
NGF induce a rapid activation of the MAPK pathway, NGF
treatment induces a prolonged activation and differentiation
while EGF induces proliferation. Hence, the role of HSPG
may be to act as a cellular switch between immediate and
prolonged signal activation by heparin-binding growth factors
such as FGF-2. In the absence of HSPG, FGF-2 can interact
with and activate its receptor (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi992243db00008" ref-type="bibr"></xref>, <xref rid="bi992243db00043" ref-type="bibr"></xref></named-content></italic>), yet in the presence of
HSPG, FGF-2 might be able to mediate prolonged biological
responses through intracellular processes. While the role of
internal FGF-2 or fragments of FGF-2 has yet be to
demonstrated, it is intriguing to speculate that internalized
FGF-2 has novel activities. Furthermore, the large amount
of chemical diversity within heparan sulfate as well as the
many HSPG core proteins could provide an extremely
sensitive system for cells to use to generate unique responses
to ubiquitous growth factors.
</p></sec></body><back><ack><title>Acknowledgments</title><p>The work was presented as part of a Ph.D. dissertation
(G.V.S.) to the Department of Biochemistry. Thus, we thank
the Ph.D. dissertation advisory committee and other members
of the department for helpful suggestions and valuable
discussions.
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Chem.</source><year>1994</year><volume>269</volume><fpage>3976</fpage><lpage>3984</lpage></element-citation></ref><ref id="bi992243dn00001"><mixed-citation><comment>Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FGF-2, fibroblast growth factor 2; FGFR, fibroblast growth factor receptor; HSPG, heparan sulfate proteoglycans; LMW, low molecular weight; MAPK, mitogen-activated protein kinases; NGF, nerve growth factor; TCA, trichloroacetic acid; VSMC, vascular smooth muscle cells.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Mechanisms of Fibroblast Growth Factor 2 Intracellular Processing: A Kinetic Analysis of the Role of Heparan Sulfate Proteoglycans†</title></titleInfo><name type="personal"><namePart type="family">SPERINDE</namePart><namePart type="given">Gizette V.</namePart><affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">NUGENT</namePart><namePart type="given">Matthew A.</namePart><affiliation>Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118</affiliation><affiliation> Correspondence should be addressed to this author at the Department of Biochemistry, Room K225, Boston University School ofMedicine, 716 Albany St., Boston, MA 02118. Fax 617 638-5339;E-mail nugent@biochem.bumc.bu.edu.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2000-03-10</dateCreated><dateIssued encoding="w3cdtf">2000-04-04</dateIssued><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><note type="footnote" ID="bi992243dAF2"> This work was supported in part by Grants R01HL56200 and P01HL46902 from the National Institutes of Health and by Departmental grants from the Massachusetts Lions Eye Research Fund and Research to Prevent Blindness, Inc.</note><abstract>The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 °C for prolonged periods (0−48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4−10 kDa) were detected that accumulated to much higher (∼10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse−chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native (∼18 h) compared to HSPG-deficient cells (∼4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.</abstract><note type="footnote" ID="bi992243dAF2"> This work was supported in part by Grants R01HL56200 and P01HL46902 from the National Institutes of Health and by Departmental grants from the Massachusetts Lions Eye Research Fund and Research to Prevent Blindness, Inc.</note><relatedItem type="host"><titleInfo><title>Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>Biochemistry</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0006-2960</identifier><identifier type="eISSN">1520-4995</identifier><identifier type="acspubs">bi</identifier><identifier type="coden">BICHAW</identifier><identifier type="uri">pubs.acs.org/biochemistry</identifier><part><date>2000</date><detail type="volume"><caption>vol.</caption><number>39</number></detail><detail type="issue"><caption>no.</caption><number>13</number></detail><extent unit="pages"><start>3788</start><end>3796</end></extent></part></relatedItem><identifier type="istex">CF0F213D60C325EE792FA567983A500DA0EECBC5</identifier><identifier type="ark">ark:/67375/TPS-9DV1S2MW-W</identifier><identifier type="DOI">10.1021/bi992243d</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2000 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">ACS</recordContentSource><recordOrigin>Copyright © 2000 American Chemical Society</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/record.json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/annexes.gif</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/CF0F213D60C325EE792FA567983A500DA0EECBC5/annexes/tiff</uri></json:item><json:item><extension>eps</extension><original>true</original><mimetype>image/x-eps</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-9DV1S2MW-W/annexes.eps</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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