αvβ5 Integrin Receptor-mediated Endocytosis of Vitronectin Is Protein Kinase C-dependent

Abstract

Previous studies have demonstrated that the alpha v beta 5 integrin receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the beta 5 integrin or exogenous heparin, suggesting that both integrin receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha v beta 5 integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-alpha v beta 5 antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the alpha v beta 5 integrin following activation of protein kinase C.

Vitronectin's Basic Domain is a Syndecan Ligand which Functionsin transto Regulate Vitronectin Turnover

“…These data as well as our previous data (58,60,88) indicate that vitronectin's basic domain may be , after which wells were blocked with 1% BSA for 1 h at 37 • C. HT-1080 cells that had been pretreated with and without sodium chlorate (CLO 3 ) for two days (see Materials and Methods) were lifted with Cellstripper TM , washed three times with DMEM/F12 containing 0.2% BSA, then seeded onto protein coated wells at 6 × 10 5 cell/ml. Cells were allowed to adhere for 1 h at 37 • C, then washed with PBS and fixed in 3% paraformaldehyde.…”

“…Previous studies from our laboratory have shown that vitronectin degradation is dependent upon an RGD mediated interaction with integrin αvβ5 (49,59) and is sensitive to the addition of exogenous heparin, as well as treatment with sodium chlorate or heparitinase (60,88), suggesting that vitronectin's turnover is also dependent upon its interaction with a heparan sulfate proteoglycan. To determine whether vitronectin's basic domain is required for its turnover from the extracellular environment, HT-1080 cell layers were incubated with increasing concentrations of 125 I labeled rVn and assayed to determine the level of binding and degradation.…”

Pharmacoproteomic Analysis of a Novel Cell-permeable Peptide Inhibitor of Tumor-induced Angiogenesis

“…There are several reports that fibrinogen bound to the surfaces of A549 cells is internalized by endocytosis via RGD-dependent binding to integrin ␣ v ␤ 3 (65). Additionally, the internalization and degradation of matrix-bound vitronectin are mediated by integrin ␣ v ␤ 5 and involve protein kinase C (66). Integrin ␣ v ␤ 5 is internalized in its active, vitronectinbound form (67, 68) through clathrin-coated pits (69).…”

Internalization of FimH+ Escherichia coli by the human mast cell line (HMC-1 5C6) involves protein kinase C

“…Given the significant roles of PKC in the modulation of phagocytosis in other cell types [17][18][19] and the unique effects of PMA, a PKC activator, on the cytoskeletal rearrangement in mast cells [16], it is likely that PKC may modulate bacterial internalization by mast cells. To investigate the roles of PKC in FimH ϩ E. coli internalization by HMC-1 5C6 cells, we used PMA, a well-characterized activator for cPKC and nPKC members, to stimulate mast cells for 1 h. The activator was then completely removed by washing before adding bacteria.…”

“…Its activation induces a pattern of cytoskeletal rearrangement in mast cells that is quite different from that seen in other cell types [16]. Moreover, it is known that PKC facilitates several internalization processes including Fc␥ receptor-mediated phagocytosis in monocytes [17], integrin receptor-mediated endocytosis in fibroblasts [18], and oxidant-dependent ingestion of IgGopsonized targets in neutrophils [19]. However, it is also noteworthy that PKC has been reported to down-regulate a number of internalization processes such as phagocytosis of zymosan by macrophages [20].…”