Cell surface heparan sulfate proteoglycans control the response of renal interstitial fibroblasts to fibroblast growth factor-2.
BACKGROUND
While the progression of renal disease to end stage is strongly correlated with tubulointerstitial changes, the control of the fibrotic process within the interstitium is poorly understood. Basic fibroblast growth factor (FGF-2) has been implicated as a major growth factor involved in fibroblast activation and extracellular matrix synthesis. Furthermore, in many cells, the activity of FGF-2 is controlled by a low-affinity but high-capacity interaction with heparan sulfate (HS) proteoglycans (PGs), such as members of the syndecan family. These molecules are likely to be central to the control of interstitial fibrosis, but as yet, there has been no characterization of their synthesis by interstitial cells.


METHODS
The expression of HSPG on the surface of NRK 49F fibroblasts was demonstrated by immunohistochemistry and by metabolic labeling with [(35)S]-sulfate. HSs were characterized by specific enzymatic digestion, size exclusion chromatography, and anion exchange chromatography. The mRNA for syndecan 1 through syndecan 4 in the fibroblasts was detected by semiquantitative reverse transcription-polymerase chain reaction. Fibroblast proliferation was measured by the MTT assay.


RESULTS
Immunohistochemistry and [(35)S]-sulfate-labeling demonstrated that renal fibroblasts expressed HSPGs on their surface. Furthermore, enzymatic removal of these HS (but not chondroitin sulfate) glycosaminoglycan (GAG) chains, or inhibition of GAG sulfation, abolished the proliferative response of both NRK cells and primary human cortical fibroblasts to FGF-2 but not to platelet-derived growth factor. The addition of conditioned medium, containing HS-GAG fragments, restored the proliferative response to FGF-2, confirming the specificity of the interaction. Finally, the mRNA for all four syndecans was detected in the fibroblasts, and that for syndecan 1 in particular was up-regulated by FGF-2.


CONCLUSIONS
The present study demonstrates that the expression of cell surface HSPG was essential for the proliferation of renal fibroblasts in response to FGF-2, and therefore may play a major role in the development and persistence of a proliferating phenotype during interstitial nephritis.
Clayton
A
A
Institute of Nephrology, University of Wales College of Medicine, Cardiff, Wales, United Kingdom.
Thomas
J
J
Thomas
G J
GJ
Davies
M
M
Steadman
R
R
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D019812:Heparan Sulfate Proteoglycans / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009395:Nephritis, Interstitial / Q000378:metabolism*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013462:Sulfur Radioisotopes; D053668:Syndecan-1; D053669:Syndecan-2; D053670:Syndecan-3; D053671:Syndecan-4; D053667:Syndecans
D019812:Heparan Sulfate Proteoglycans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; C508241:SDC1 protein, human; C508245:SDC2 protein, human; C508301:SDC3 protein, human; C508304:SDC4 protein, human; D013462:Sulfur Radioisotopes; D053668:Syndecan-1; D053670:Syndecan-3; D053671:Syndecan-4; D053667:Syndecans; D016222:Fibroblast Growth Factor 2; D053669:Syndecan-2