Following injury, the formation of a provisional extracellular matrix (ECM) from extravasated plasma proteins is a critical step in the wound repair process. The interactions between cell surface receptors and this """"""""injury-associated"""""""" matrix provide important cues that can modulate the cellular response at the injury site leading to alterations in cell growth and gene expression. The long-term goal of this project is to define the molecular mechanisms of ECM-mediated signaling required for tissue repair. We hypothesize that fibronectin (FN), an essential adhesive component of the provisional ECM, is a key determinant of cell growth and survival during wound healing. We propose that FN modulates these important cellular behaviors through its interaction with the integrin receptor alpha5beta1. To determine the molecular mechanisms, both structural and biochemical, that are required for this process, we have developed an in vitro model that allows the incorporation of FN molecules into a three- dimensional (3D) fibrin matrix that mimics the provisional ECM found in vivo. We will use this system to test the hypotheses that: 1) the incorporation of FN into fibrin matrices enhances alpha5beta1-mediated matrix contraction by modulating cell motility and contractile force generation 2) alpha5beta1-FN interactions promote cell shape change and are essential for cell growth and survival in FN-fibrin matrices 3) the cytoplasmic domain of the alpha5 integrin subunit regulates FN-fibrin matrix contraction. The experiments outlined above are the first to specifically examine FN- mediated cell signaling in 3D culture. As such, they will provide important information regarding the determinants of cell behavior in an environment that closely approximates the early provisional wound matrix. This will improve our understanding of the fundamental processes of the injury response and contribute to the development of effective therapeutic strategies for acute and chronic injury.
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