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.

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Crossfostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect the broad-spectrum MMP inhibitor BB-94 has when applied topically to full-thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by latent transforming-growth factor-β1 (TGF-β1). BB-94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB-94 dramatically increased the level of latent and active MMP-9. The increased levels of active MMP-9 may eventually overcome the ability of BB-94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB-94 was also found to inhibit the ability of latent TGF-β1 to promote the formation and function of myofibroblasts. These results suggest that BB-94 could delay wound closure through a two-fold mechanism; by blocking keratinocyte migration and thereby blocking necessary keratinocytefibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF-β1.