Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the av.83 integrin. In this work, we studied human smooth muscle cells varying in avB83 levels to identify additional osteopontin receptors. We report that, in addition to 0avP3, both a435 and aci31 are osteopontin receptors. Moreover, the presence or absence of a433 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of a4v33-deficient cell populations to osteopontin was only half that of cells containing av43, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface C433. Although av.I3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of av./5 and av4l as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing f3, 83, and j3, migration in response to osteopontin appears to depend on a4v.3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions. (J. Clin. Invest. 1995. 95:713-724.)

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

Neutrophils and monocytes, but not lymphocytes, adhered strongly to plastic surfaces coated with GMP140, a protein of endothelial cells and platelets. This adhesion of neutrophils was mediated by GMP140 and not by the CD18 integrin complex. By contrast, GMP140 in solution inhibited the CD18-dependent adhesion of tumor necrosis factor-alpha-activated neutrophils to plastic surfaces and resting endothelium, but not of resting neutrophils to tumor necrosis factor-alpha-activated endothelium. Thus, the binding of a soluble form of an adhesion protein selectively inhibited another set of adhesive events. Soluble GMP140 may be important in maintaining the nonadhesiveness of neutrophils in the circulation and may serve to limit inflammatory reactions.

The respiratory burst of blood neutrophils has a critical role in the destruction of microorganisms and tissue damage in inflammation. Neutrophils adhere in a dosedependent fashion to granule membrane protein 140 (GMP140), a member of the LEC-CAM (lectin/epidermal growth factor/complement-binding domain cell adhesion molecule) family of adhesion proteins when it is immobilized onto plastic surfaces. Adherence to GMP140 was associated with less superoxide anion generation than adherence to other surfaces, an effect that is especially remarkable after activation of neutrophils with tumor necrosis factor a, an agent that on other surfaces promotes adhesion and spreading. However, on GMP140 the cells fail to spread and instead remain rounded and refractile. Neutrophils adhering to GMP140 were also deficient in superoxide anion generation to formylmethionylleucylphenylalanine. Furthermore, fluid-phase GMP140 also inhibited the superoxide generation by neutrophils stimulated by tumor necrosis factor a. The effect of GMP140 was reversible by washing and was inhibited by anti-GMP140 Fab antibody. GMP140 appears to be a natural antiinflammatory molecule that may prevent the inappropriate activation of neutrophils in the circulation.

Figure 2 summarizes our current interpretation of data concerning signals from the activated PDGF receptor involved in directed migration and proliferation of human arterial SMC. Binding of PDGF (PDGF-BB or PDGF-AA) causes PDGF-receptor dimerization, tyrosine autophosphorylation, and subsequent binding of several molecules containing SH2 domains to the activated receptor. Binding and activation of PLC gamma by the PDGF receptor leads to PIP2 hydrolysis, resulting in generation of diacylglycerol (DAG) and IP3. Subsequently, intracellular levels of calcium are elevated as a result of IP3-mediated calcium release from intracellular compartments. The decreased levels of PIP2 and increased levels of calcium both favor actin-filament disassembly by inducing capping of actin-filament barbed ends and actin-monomer sequestration. A localized, and transient, actin-filament disassembly enables the cell to extend filopodia towards PDGF, thereby enabling chemotaxis to take place. At a later time and/or in a different compartment, actin-filament assembly is promoted by PDGF by a mechanism that is not completely understood, but that may involve small GTP-binding proteins, such as Rho, and formation of DAG. Migration on collagen requires functional alpha 2 beta 1 integrins, which may either constitute a permissive state required for a cell to migrate, or which may be actively involved in intracellular signals leading to migration. PDGF-induced DNA synthesis and proliferation involves activation of Ras, MAP kinase kinase, and MAP kinase. Cross-talk between PKA signaling and tyrosine-kinase receptor signaling results in PKA inhibition of the MAP kinase cascade, probably at the level of Raf. Activation of PI 3-kinase, or a PI 3-kinase-like enzyme, is also likely to contribute to the mitogenic effects of PDGF in these cells (Bornfeldt, unpublished observation). What determines if a SMC will migrate and/or proliferate in response to PDGF? Results are starting to emerge that show regulation of expression of molecules involved in intracellular signaling with different phenotypic states of SMC. For example, expression of PLC gamma is very low in intact vascular wall (where SMC show a "contractile phenotype"), and induced when SMC are converted to a "synthetic phenotype" in culture. Proliferation and expression of MAP kinase, but not calcium signaling, appear to be regulated by the extracellular matrix, and the profile of integrin expression is different in SMC in culture compared to SMC in the vascular wall. Thus, the relation between expression of signaling molecules involved in migration and signaling molecules involved in proliferation, as well as cross-talk between different signal-transduction pathways, may determine the net effect of PDGF.