Effect of vitronectin on the deposition, conformation, and physiologic properties of extracellular matrix fibronectin

Thesis (Ph. D.)--University of Rochester. Dept. of Biomedical Engineering, 2011.Extracellular matrix (ECM) fibronectin directs many cellular activities that are critical for tissue development and regeneration, including cell growth, migration, and contractility. The factors that regulate the deposition of fibronectin into the ECM and govern how cells respond to ECM fibronectin during tissue repair processes are unknown. The deposition of other ECM proteins, including vitronectin and fibrin, into a provisional ECM in response to tissue injury may be one mechanism by which cellular responses to ECM fibronectin are controlled. Vitronectin has been shown to modulate the deposition of ECM fibronectin, suggesting that the presence of vitronectin during tissue regenerative processes regulates cellular responses to ECM fibronectin. The goal of the studies performed in this thesis was to examine the effects of vitronectin on the functional properties of ECM fibronectin and the deposition of fibronectin into the ECM. The data presented in this thesis show that vitronectin-adherent fibronectin-null cells produce a fibrillar detergent-insoluble fibronectin matrix when cultured in a defined serum-free media. The fibronectin matrix produced by vitronectin-adherent cells was 10-fold less effective at stimulating cell growth than the fibronectin matrix produced by collagen-adherent cells (EC50 = 3.7 ± 1.2 nM and 41.1 ± 1.17 nM for collagen- and vitronectin-adherent cells, respectively). In contrast, vitronectin- and collagen-adherent cells exhibited a similar 1.8-fold-increase in cell number when treated with a fibronectin fusion protein, GST/III-1H,8-10, demonstrating that reduced cell proliferation is not a general property of vitronectin-adherent cells. Fibronectin-null cells adhered to a vitronectin fragment containing the cell binding domain of vitronectin also exhibited a decreased growth response to fibronectin, suggesting that interactions of vitronectin with integrins on the cell surface play a role in mediating cellular responses to ECM fibronectin. However, the reduced growth response to fibronectin observed for vitronectin-adherent cells was not a general property of [alpha]v[beta]3 integrin-binding substrates, as cell adhesion to either gelatin or fibrinogen resulted in a fibronectin-mediated growth response similar to collagen-adherent cells. Nor was the less efficient fibronectin growth response of vitronectin-adherent cells due to a decreased deposition of detergent-insoluble fibronectin as addition of a fibronectin fragment that enhances the deposition of detergent-insoluble fibronectin did not enhance the fibronectin growth response of cells adherent to vitronectin. Taken together, these data suggest that fibronectin matrices formed by cells can differ in their ability to modulate cell growth and that the presence of vitronectin regulates the growth-promoting properties of ECM fibronectin. Strain-induced conformational changes in the III-1 module of ECM fibronectin may facilitate the exposure of the cryptic heparin binding domain in III-1 and may be one mechanism by which cellular responses to ECM fibronectin are regulated. The heparin binding sequence in the III-1 module of fibronectin was necessary for GST/III-1H,8-10-mediated growth of vitronectin- or collagen-adherent cells, suggesting the exposure of this sequence in matrix fibronectin is important for fibronectin-mediated growth of vitronectin- and collagen-adherent cells. A conformation-dependent anti-fibronectin mAb, L8, recognized fibronectin fibrils formed by collagen-adherent cells but not vitronectin-adherent cells during the first 24 h of fibronectin matrix assembly. These data indicate that there are conformational differences in the I-9/III-1 region of fibronectin matrices formed by vitronectin- or collagen-adherent cells and suggest that conformational differences in this region may be associated with differences in the growth-promoting properties of ECM fibronectin. Finally, the data presented in this thesis show that adhesion to vitronectin delays both the initial deposition of fibronectin into the ECM and the overall amount of fibronectin deposited into the ECM during the first 24 h of fibronectin matrix assembly when compared to collagen-adherent cells. These data suggest that vitronectin- and collagen-adherent cells may utilize different pathways of fibronectin matrix assembly. Vitronectin- and collagen-mediated fibronectin matrix assembly was inhibited by the amino-terminus of fibronectin and reagents directed against the III-1 module of fibronectin. However, addition of carboxy-terminal fragment of III-1, III-1C, selectively inhibited the fibronectin matrix assembly by vitronectin-adherent cells, suggesting that the III-1 region of fibronectin plays a different role in the assembly of a fibronectin matrix when cells are adherent to collagen or vitronectin. These data provide some of the first evidence to suggest that cells can utilize alternative pathways to assemble a fibronectin matrix. Together, these data suggest that different types of fibronectin matrices exist and are defined by their physiologic properties, their conformational properties, and the regions of fibronectin that inhibit fibronectin matrix assembly. Furthermore, these data suggest that the type of fibronectin matrix formed may be regulated by composition of the underlying ECM