Evidence of a role for a non-matrix-type metalloproteinase activity in the shedding of syndecan-1 from human myeloma cells.

Syndecan-1 is a cell surface proteoglycan that is expressed on human myeloma cells and is thought to act as a co-receptor for certain extracellular matrix proteins and growth factors. The ectodomain of syndecan-1 is thought to be shed from the surface of myeloma cells, although the exact mechanism of release remains unclear. In this study, we used a panel of inhibitors to identify the class of proteinase responsible for shedding the soluble syndecan-1 ectodomain from human myeloma cells. Using enzyme-linked immunosorbent assay, flow cytometry and immunocytochemistry, we demonstrated that myeloma cell lines expressed syndecan-1 on their surface and that this was shed constitutively, but to a varying extent. In addition, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated a marked loss of cell surface syndecan-1 from each of the cell lines and this was associated with a corresponding increase in soluble syndecan-1. Inhibitors of serine and cysteine proteinases, and matrix-type metalloproteinases, did not inhibit constitutive or PMA-stimulated syndecan-1 shedding from JJN3 and RPMI 8226 cells. However, BB-94, a hydroxamate-based, broad-spectrum, metalloproteinase inhibitor, substantially suppressed constitutive and PMA-stimulated syndecan-1 loss from myeloma cells. These data indicate that a non-matrix-type metalloproteinase is responsible for syndecan-1 shedding from the surface of myeloma cells.

Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen.

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.

Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor.

Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.

Ionomycin-stimulated arachidonic acid release in human platelets: a role for protein kinase C and tyrosine phosphorylation.

Collagen (10-90 micrograms/ml) and ionomycin (1 microM; a calcium ionophore) each evoked rises in intracellular free calcium, protein kinase C activity and arachidonic acid release in human platelets, and as previously demonstrated for collagen, ionomycin (1 microM) stimulated protein tyrosine phosphorylation. However, at lower concentrations (60 and 250 nM) ionomycin selectively mobilised calcium. Ro31-8220 (a selective inhibitor of protein kinase C) inhibited (by 50%) ionomycin-stimulated arachidonic acid release. Genistein (an inhibitor of protein tyrosine kinases) also reduced by 50% ionomycin-stimulated arachidonic acid release. In combination, genistein and Ro31-8220 abolished ionomycin-stimulated arachidonic acid release. These findings show 1) that a rise in calcium is not sufficient, and 2) the activation of both protein kinase C and protein tyrosine phosphorylation is necessary, for full ionomycin-stimulated arachidonic acid release in human platelets.

Signals elicited from human platelets by synthetic, triple helical, collagen-like peptides.

Synthetic collagen-like peptides, of general structure [Gly-Pro-HyP]n, adopt the triple-helical structure which is essential for the platelet-reactivity of native collagens. These peptides are potent activators of platelets, stimulating platelet aggregation at much lower dose than collagen fibres, but, unlike collagen fibres, they are not recognised by the integrin alpha 2 beta 1. We have examined the ability of the synthetic peptides to activate the various signalling pathways which regulate human platelet function. The peptides are potent activators of Ca2+ mobilisation and of protein kinase C, and they stimulate tyrosine phosphorylation of some substrates preferentially. However, in contrast with native type I collagen fibres, they are unable to inhibit platelet adenylate cyclase. This suggests a mode of action for the synthetic peptides which substantially overlaps, but which is not entirely identical with, that of native collagen.

Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.

The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

The tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, inhibit the release of (3H)arachidonate from human platelets stimulated by thrombin or collagen.

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen.