Modulation of cell-fibronectin matrix interactions during tissue repair.

Environmental signals from the extracellular matrix (ECM) are transmitted by cell surface receptors that connect to the actin cytoskeleton and to multiple intracellular signaling pathways. To dissect how the ECM regulates cell functions, we are using a three-dimensional (3D) fibrin-fibronectin matrix, resembling the wound provisional matrix. Fibroblasts adhere to fibronectin in this matrix via concomitant engagement of alpha 5 beta 1 integrin receptors and syndecan-4, a transmembrane proteoglycan. An adhesive phenotype is developed with actin stress fibers and activation of focal adhesion kinase (FAK) and Rho GTPase. Lack of syndecan-4 engagement, as occurs in the presence of the ECM protein tenascin-C, promotes a motile phenotype; FAK and Rho signaling are downregulated and filopodia are extended. Fibronectin matrices have distinct effects on two other receptors: alpha 4 beta 1 and beta v beta 3 integrins. Although alpha 4 beta 1 does not naturally support strong cell interactions with a fibrin-fibronectin matrix, binding is dramatically enhanced by proteolytic cleavage of fibronectin. Conversely, activity of alpha v beta 3 is stimulated by multimeric fibronectin fibrils showing that the organization of fibronectin differentially affects integrin functions. Thus, deposition of additional ECM components, expression of co-receptors for ECM, cleavage of adhesive proteins, and the architecture of the ECM microenvironment are different mechanisms for modulating cell responses to fibronectin matrix.

Ligand density and integrin repertoire regulate cellular response to LPA.

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance alpha5beta1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for alpha4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of alpha4beta1-expressing CHOalpha4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOalpha4 and human A375 melanoma cells in an alpha4beta1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for alpha4beta1. LPA signaling combined with alpha4beta1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.

Fibronectin fragmentation promotes alpha4beta1 integrin-mediated contraction of a fibrin-fibronectin provisional matrix.

In injured tissues, the fibrin-fibronectin (FN) provisional matrix provides a framework for cell adhesion, migration, and repair. Effective repair and remodeling require a proper balance between extracellular matrix (ECM) deposition, contraction, and turnover. We utilized a three-dimensional (3D) fibrin-FN provisional matrix model to determine the contributions of the FN-binding integrin receptors alpha5beta1 and alpha4beta1 to matrix contraction. CHOalpha5 cells expressing alpha5beta1, a receptor for FN's RGD cell-binding domain, were highly contractile, and cells were well spread on a 3D fibrin-FN matrix. In contrast, CHOalpha4 cells expressing the alpha4beta1 receptor for FN's alternatively spliced V region attached less efficiently to FN and were deficient in fibrin-FN matrix contraction. Surprisingly, cell adhesion and matrix contraction by CHOalpha4 cells were dramatically enhanced, to levels equivalent to CHOalpha5 cells, when proteolyzed FN was used in place of intact FN in the fibrin-FN matrix. Similar enhancement was observed when ligand binding by alpha4beta1 integrins was activated by treatment with Mn(++), but not by stimulation of actin organization with LPA. Therefore, alpha4beta1-dependent cell responses to the provisional matrix are modulated by cleavage of matrix components.

Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4.

Syndecan-4 is a ubiquitously expressed heparan sulfate proteoglycan that modulates cell interactions with the extracellular matrix. It is transiently up-regulated during tissue repair by cells that mediate wound healing. Here, we report that syndecan-4 is essential for optimal fibroblast response to the three-dimensional fibrin-fibronectin provisional matrix that is deposited upon tissue injury. Interference with syndecan-4 function inhibits matrix contraction by preventing cell spreading, actin stress fiber formation, and activation of focal adhesion kinase and RhoA mediated-intracellular signaling pathways. Tenascin-C is an extracellular matrix protein that regulates cell response to fibronectin within the provisional matrix. Syndecan-4 is also required for tenascin-C action. Inhibition of syndecan-4 function suppresses tenascin-C activity and overexpression of syndecan-4 circumvents the effects of tenascin-C. In this way, tenascin-C and syndecan-4 work together to control fibroblast morphology and signaling and regulate events such as matrix contraction that are essential for efficient tissue repair.

Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-kappa B site on its promoter.

We report here that human monocytic/macrophage THP-1 cells express the neurokinin 1 receptor (NK-1R), and that exposure of these cells to the proinflammatory cytokine IL-1 beta increased the expression of the NK-1R gene at the mRNA and protein levels. Because IL-1 beta function involves nuclear factor kappa B (NF-kappa B) activation, these data suggest that this increase in the expression of the NK-1R gene is mediated by the NF-kappa B transcription factor. An earlier report noted that the promoter region of the human NK-1R gene contains a putative binding site for NF-kappa B [Takahashi, K., Tanaka, A., Hara, M. & Nakanishi, S. (1992) Eur. J. Biochem. 204, 1025-1033]. Here we demonstrate that this is indeed a functional NF-kappa B-binding site, and that NF-kappa B is responsible for regulating the expression of the NK-1R gene by binding to the promoter region of the NK-1R gene. To further substantiate that the observed NF-kappa B-dependent IL-1 beta induction of the human NK-1R gene is regulated via a transcriptional event through this NF-kappa B site on the NK-1R gene promoter, we transfected THP-1 cells with a luciferase promoter-reporter construct containing the 5' promoter region of the human NK-1R gene. Exposure of these cells to IL-1 beta or overexpression of NF-kappa B cDNAs resulted in a significant increase in the amount of luciferase activity that was diminished greatly in cells transfected with I kappa B alpha, the NF-kappa B inhibitor. These results directly implicate NF-kappa B in the regulation of the NK-1R gene and provide a molecular mechanism for the increase in expression of the NK-1R gene in responsive cells.