Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins.

BACKGROUND: 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). RESULTS: Cells are able to adhere to and spread on III1-C coated on a dish. Both beta1 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 beta1 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 beta1 and alpha5 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. CONCLUSIONS: The 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.

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly.

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.

Stimulation of transcription factors NF kappa B and AP1 in endothelial cells subjected to shear stress.

Hemodynamic shear stress forces influence several important endothelial genes associated with arterial relaxation, pro/anti-coagulation, and growth control; however, the regulatory pathways remain unclear. Here we demonstrate stimulation of DNA binding activities of nuclear factor kappa B (NF kappa B), a member of the Rel Family of transcription factors, and nuclear factor activator protein-1 (AP-1) following exposure of endothelial cells to unidirectional shear stress in laminar flow. NF kappa B binding was stimulated within 30 minutes, reaching and maintaining maximal levels at 1 hour. DNA binding activity was inhibited by pre-incubation of nuclear extract with antibody directed against NF kappa B p65 subunit. AP-1 binding activity was biphasic, rising fourfold within 20 minutes and returning to basal levels before steadily increasing by 2 hours to a high level relative to basal values. These protein kinase C-coupled transcriptional factors may modulate endothelial genes that are shear stress-responsive and that possess appropriate binding sites in the promoter region.

Conformationally specific enhancement of receptor-mediated LDL binding and internalization by peptide models of a conserved anionic N-terminal domain of human apolipoprotein E.

In this paper, we test the hypothesis that peptide models of a highly conserved domain of apolipoprotein E (amino acids 41-60 in human apo E) modulate the binding and internalization of LDL to cell surface receptors in a conformationally specific manner. Three peptides were compared: peptide I containing the natural sequence of amino acids 41-60 of human apo E; peptide III containing side-chain lactam cross-links designed to enhance alpha-helical structure; and peptide II containing cross-links designed to prevent formation of alpha-helices. Peptide III was shown previously to consist of two short alpha-helical domains linked by a turn and to have more alpha-helical content than peptide I, while peptide II was shown to have less helical content than either peptide III or I(Luo et al., 1994). Peptide III induced a 30-fold increase in the specific binding of 125I-LDL to normal human skin fibroblasts and a 60-fold increase in the binding to fibroblasts lacking the LDL-R. This same peptide also restored the binding to normal fibroblasts of 125I-LDL from a patient with familial defective apolipoprotein B, the R3500-->Q mutation. Analysis of binding indicated an increase in the apparent number of binding sites, with little effect on the affinity of 125I-LDL for the cell surface. Heparinase treatment of the cells did not abrogate this effect, suggesting that the increased binding is not mediated by cell surface glycans. LDL internalization but not degradation was also increased by peptide III. Similar but smaller effects were also induced by peptide I. Peptide II was much less active than peptide I or III. Thus, the order of biological activity was the same as the order of alpha-helical content, i.e., peptide III > peptide I > peptide II. These results suggest a hitherto unknown biological function for a highly conserved domain of apolipoprotein E, and this bioactivity was shown by peptide models to be specific to the alpha-helical conformation.