The generation of tension in granulation tissue undergoing contraction is believed to be a cell-mediated event. In this study we used attached collagen lattices as a model system for studying the cellular mechanisms of tension generation by fibroblasts in an extracellular matrix. Fibroblasts in attached collagen lattices developed stress fibers, surface associated fibronectin fibrils, and a fibronexus-like transmembrane association interconnecting the two structural components. Release of the attached collagen lattice from its points of attachment resulted in a rapid, symmetrical contraction of the collagen lattice. Rapid contraction occurred within the first 10 minutes after release of the lattice from the substratum, with greater than 70% of the contraction occurring within the first 2 minutes. Rapid contraction resulted in a shortening of the elongate fibroblasts and compaction of the stress fibers with their subsequent disappearance from the cell. Cytochalasin D treatment prior to release disrupted the actin cytoskeleton and completely inhibited rapid contraction. The removal of serum prior to release inhibited rapid contraction, while the re-addition of serum restored rapid contraction. These results demonstrate that fibroblasts can develop tension in an attached collagen lattice and that upon release of tension the fibroblasts undergo contraction resulting in a rapid contraction of the collagen lattice. Fibroblast contraction is dependent upon an organized actin cytoskeleton and is promoted by the presence of serum.

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Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor beta-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors-A and -B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively-active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1 differentiated myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of myofibroblast differentiation in response to TGF-β1.

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-SMA expression in smooth muscle cells. A Lac Z transgene construct from -2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous alpha-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of alpha-SMA expression in myofibroblasts in granulation tissue.

Previous studies have suggested the existence of a membrane-associated serine protease expressed by mammalian preimplantation embryos. In this study, we have identified hepsin, a type II transmembrane serine protease, in early mouse blastocysts. Mouse hepsin was highly homologous to the previously identified human and rat cDNAs. Two isoforms, differing in their cytoplasmic domains, were detected. The tissue distribution of mouse hepsin was similar to that seen in humans, with prominent expression in liver and kidney. In mouse embryos, hepsin expression was observed in the two-cell stage, reached a maximal level at the early blastocyst stage, and decreased subsequent to blastocyst hatching. Expression of a soluble form of hepsin revealed its ability to autoactivate in a concentration-dependent manner. Catalytically inactive soluble hepsin was unable to autoactivate. These results suggest that hepsin may be the first serine protease expressed during mammalian development, making its ability to autoactivate critical to its function.