Siobhan Corbett, MD CAB 7322 732-235-7348 corbetsi@umdnj.edu

Following tissue injury, the formation of a provisional extracellular matrix (ECM) from both extravasated and secreted structural proteins plays a critical role in the wound repair process. The principle protein component of this "injury-associated" matrix is fibronectin (FN). Fibronectin is a multifunctional, adhesive glycoprotein that has wide tissue distribution and is essential for normal development and tissue repair. Cells secrete FN as a disulfide-bonded dimer that binds principally to integrin cell surface receptors. Integrin-FN interactions allow unfolding of the soluble protein and its assembly into a detergent-insoluble fibrillar matrix that can modulate both cell behavior and tissue architecture. Further, the FN matrix serves as the template for the deposition and assembly of other fibrillar proteins, including collagen, fibrinogen and thrombospondin-1. In this fashion, FN polymerization regulates both the formation and stabilization of the protein scaffold required for wound repair.

New tissue in a cutaneous wound consists of a neoepidermis and underlying granulation tissue. The granulation tissue is rich in FN, which by seven days post-injury has formed a dense interconnecting matrix that links adjacent fibroblasts. The appearance of the FN matrix in vivo is temporally related to the initiation of wound contraction and remodeling, suggesting that these events are causally related. In vitro, FN has been shown to stimulate the contractility of cells embedded in both 3D collagen and fibrin gels. FN-mediated contractility in this context is dependent on the active polymerization of FN, indicating that FN fibril assembly is an important regulator of force generation. These studies did not determine however, whether interconnected FN fibrils in the absence of fibrin or collagen could also support tissue contraction.

To address this question, we have used a 3D model system in which cells were cultured as multilayered aggregates without the presence of exogenous stroma. Cells grown in this fashion readily assembled a FN matrix that promoted strong intercellular cohesion and compaction of cells into spherical aggregates. These results suggest that the organization of FN matrices in the wound can initiate the process of contraction prior to the deposition of collagen. Therefore, it is logical that increased FN matrix deposition can accelerate contraction and if uncontrolled, could lead to pathologic scarring. Given the pivotal importance of FN matrix assembly to a variety of biologic processes, an understanding of the cellular mechanics of FN fibril formation and how they are regulated will have broad impact.

The α5 integrin cytoplasmic domain regulates FN matrix assembly We have hypothesized that the generation of force by the FN-binding integrin, α5β1 is regulated by the cytoplasmic domain of the α5 integrin subunit. To test this hypothesis, we constructed mutant α5 integrin subunits composed of the extracellular, transmembrane, and first 8 amino acids of the cytoplasmic domain up to an including the highly conserved amino acid sequence GFFKR. This amino acid sequence is present in almost all α integrin subunits and is thought to be important for heterodimer stability. The remaining amino acid sequence was left intact, truncated or replaced with that of the α2 integrin subunit, or the α4 integrin subunit. The chimeric cDNAs were expressed individually in CHOB2 cells that are null for endogenous α5. When FN matrix assembly was examined, we detected clear, significant differences in FN polymerization, demonstrating conclusively that the α5 integrin subunit regulates FN matrix assembly.

FN matrix assembly regulates α5β1-mediated cell cohesion: We have previously demonstrated that the α5β1-integrin mediates strong intercellular cohesion of 3D cellular aggregates. To further investigate the mechanism of α5β1-mediated cohesivity, we used the cell lines described above to analyze α5β1-integrin-mediated cell aggregation, compaction and cohesion. We then correlated this behavior with FN matrix assembly. We found that cells cultured in the absence of an external supporting substrate can assemble a FN matrix that promotes integrin-mediated cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction of cell aggregates, leading to cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.

Taken together, this work represents a paradigm shift in our understanding of the role of FN matrix assembly in wound healing. We show that FN-α5β1 interactions alone confer strong intercellular cohesion, allowing the compaction of 3D cellular aggregates. Further, we show that the α5 cytoplasmic tail is important to this process in so far as it regulates FN matrix assembly. Factors that affect FN matrix assembly therefore, are likely to be important determinants of both wound contraction and matrix composition.

Our laboratory is currently investigating the regulation of the cell surface expression of the α5β1 integrin and correlating α5β1 expression with FN matrix assembly using both biochemical assays and immunofluorescent microscopy.

Recent publications:

Robinson, E., Zazzali, K.M., Corbett, S.A. and Foty, R.A. (2003) A Novel Role for α5β1 Integrin in Mediating Cohesivity of Three-Dimensional Aggregates. J. Cell Science.
116: 377-86..

Ly, D.P., Zazzali, K.M., and Corbett, S.A (2003) De novo expression of the integrin
α5β1 regulates αvβ3-mediated adhesion and migration on fibrinogen. J. Biol. Chem. 278(24):21878-85..

Patel, T.R. and Corbett, S.A. (2003) Mevastatin suppresses LPS-induced Rac activation in the human monocyte cell line, THP-1. Surgery 134 (2): 306-311.

Patel, T.R. and Corbett, S.A. (2004) Simvastatin suppresses LPS-induced Akt phosphorylation in the human monocyte cell line, THP-1. J. Surg. Research.116(1):116-20.

Robinson, E., Foty, R.A., and Corbett, S.A. (2004). Fibronectin matrix assembly regulates α5β1-mediated cell cohesion. Mol. Biol. Cell. 15(3):973-81.

Ly, D.P.and Corbett, S.A (2004) The integrin α5β1 regulates αvβ3-mediated
Extracellular Signal-Regulated Kinase activation. J. Surg. Research. In Press.

Winters, B.S., Robinson, E.E., Mohan Raj, B.K.,Foty, R.A., and Corbett, S.A. (2004) 3D Culture Modulates a5b1 Integrin Activity by Regulating the Expression of Raf-1. Submitted.