Abstract
BackgroundThe first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C).ResultsCells are able to adhere to and spread on III1-C coated on a dish. Both β1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block β1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin β1 and α5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C.ConclusionsThe results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.
Highlights
The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis
The effect of III1-C on cell morphology was seen even when the plates had not been coated with fibronectin (Fig. 1A, panel C, and Fig. 1B), indicating that intact fibronectin was not required for cell attachment and spreading when III1-C was in the culture medium
The results presented above indicate that a heparan sulfate proteoglycan (HSPG) or dermatan sulfate proteoglycan on the surface of rat aortic smooth muscle cells (RASMCs) may serve as a receptor for III1-C
Summary
The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C). Fibronectin can control many aspects of cell behavior, including cell growth, migration and differentiation [1]. Fibronectin exists in the blood as a dimer, but in tissues it is in the form of an insoluble fibrillar matrix. High concentrations of dimeric fibronectin enhance cell migration whereas high concentrations of fibrillar fibronectin reduce cell migration [2]. Inhibition of fibronectin matrix assembly has been
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