Abstract
Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.
Highlights
Heparin affin regulatory peptide (HARP)1 belongs to a growing group of heparin binding extracellular regulatory molecules, and it has mitogenic and neurite outgrowth activities
The high affinity of HARP for heparin suggests that HARP may bind to heparan sulfate proteoglycans (HSPGs) present in extracellular compartments defined as cell surface and extracellular matrix (ECM)
Many growth factors bind to heparin, and the interaction of these growth factors with HSPGs at the cell surface and in the extracellular matrix is a central event in regulating the transport and effector functions of the growth factors
Summary
Materials—Cell culture reagents were from Life Technologies, Inc. Heparin lyase I (Flavobacterium heparinium; EC 4.2.2.7), heparin lyase III (F. heparinium; EC 4.2.2.8), chondroitin ABC lyase (Proteus vulgaris; EC 4.2.2.4), leupeptin, pepstatin, phenylmethylsulfonyl fluoride, heparan sulfate (HS) from bovine intestinal mucosa, keratan sulfate (KS) from bovine cornea, chondroitin sulfate A (CS-A) from bovine trachea, dermatan sulfate (DS) from porcine skin, and chondroitin sulfate C (CS-C) from shark cartilage were purchased from Sigma. Western Blot Analysis of HARP—The presence of HARP in extracellular compartments defined as cell surface and extracellular matrix was investigated by washing the cells grown to confluency in a 150-cm tissue culture dish with 2 ϫ 5 ml of 20 mM Hepes, pH 7.4, containing 2 M NaCl supplemented with 1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 1 g/ml pepstatin A, and 5 mM EDTA. Activation of HARP by Heparin in Heparitinase-treated BEL Cells— BEL cells were seeded in 48-well plates in DMEM medium supplemented with 10% (v/v) fetal calf serum as described above and incubated for 72 h. The medium was discarded, and the cells were incubated in serum-free DMEM in the presence of 1.25 ng/ml HARP (ED40: efficiency dose for 40% of maximal stimulation induced by HARP) with or without increasing concentrations of heparin (10 to 104 ng/ml) in triplicate for 18 h, and the incorporation of [methyl-3H]thymidine incorporation was determined as described.
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