Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation.
The growth promoting activity of the subendothelial extracellular matrix (ECM) is attributed to sequestration of basic fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The present study focuses on the growth promoting activity and bFGF binding capacity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintained in the presence of chlorate, an inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM containing 10-15% of the sulfate normally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of 125I-bFGF to sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to release by heparin. Incubation of 125I-bFGF on top of native ECM resulted in dimerization of the ECM-bound bFGF, but there was a markedly reduced binding and dimerization of bFGF on sulfate-depleted ECM. ECM produced in the presence of chlorate contained a nearly 10-fold less endogenous bFGF as compared to native ECM and exerted little or no mitogenic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulated the proliferation of chlorate-treated EC seeded on native ECM, suggesting its interaction with ECM-bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin had no effect on chlorate-treated cells seeded in contact with sulfate-depleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated with the cell surface and ECM act in concert to regulate the bioavailability and growth promoting activity of bFGF. While HS in the subendothelial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to high affinity signal transducing receptors.
Miao
H Q
HQ
Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel.
Ishai-Michaeli
R
R
Atzmon
R
R
Peretz
T
T
Vlodavsky
I
I
D016475:3T3 Cells; D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects*; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D016222:Fibroblast Growth Factor 2; D006493:Heparin