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Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation. INTRODUCTIONMyofibroblast differentiation from fibroblasts is a critical component of the healing process. Regenerative healing (without scarring) results from the successful execution of what have been characterized as three distinct phases of wound healing. In the first phase, fibroblasts that migrate into the wound secrete proteases, extracellular matrix (ECM) molecules, and growth factors. In the second phase, fibroblasts differentiate into nonmotile, wound-contracting myofibroblasts that also secrete ECM proteins and remodel the ECM (Jester et al., 1995;Mohan et al., 2003;Netto et al., 2005). In the third phase, after wound closure, myofibroblasts usually disappear by apoptosis (Desmouliere et al., 1995). Pathological states such as hypertrophic scars, liver cirrhosis, idiopathic lung fibrosis, and glomerulosclerosis are characterized by the persistence of myofibroblasts, which contribute to disease progression by overproduction of ECM and by excessive contraction (Desmouliere et al., 2003;Gabbiani, 2003).To better understand the molecular basis for the fibroblast to myofibroblast transition, we have focused on the role of the urokinase plasmingen activator (uPA) pathway during wound healing. uPA is an extracellular serine protease that binds to its receptor, uPAR, and generates plasmin from plasminogen at the cell-matrix interface. Plasmin is a broadspectrum protease that not only cleaves fibrin and other ECM proteins but also promotes cell migration by activating matrix-sequestered metallopr...

Maspin is a 42-kDa protein synthesized by normal epithelial cells of a variety of mammalian organs such as mammary gland, prostate, skin, and cornea (1, 2). Synthesis of maspin has also been identified in the non-epithelioid cells of the corneal stroma, both in situ and in cell culture (2). The expression of maspin, however, is lost after the second passage of cultured stromal cells. These later passage cells mimic the wound-activated stromal fibroblasts, which are much more mobile than stromal cells in the normal corneas. Expression of maspin is also lost or down-regulated in many invasive carcinoma cells (3, 4). Down-regulation of maspin in carcinoma tissues correlates with progression and metastasis of tumors (5-7). Both the later passage corneal stromal cells and the invasive carcinoma cells respond to exogenously added maspin (2, 3).Several biological activities of maspin have been characterized, which suggest a role for maspin as a tumor suppressor and an inhibitor of angiogenesis. Addition of recombinant maspin or transfection of the maspin gene into carcinoma cells inhibits cell migration and invasion in vitro and reduces tumor growth and metastasis in vivo (3,8). Maspin also inhibits the in vitro migration and proliferation of endothelial cells and blocks basic fibroblast growth factor-induced neovascularization in the rat corneal pocket model (9). To date, the underlying mechanisms of maspin action on inhibition of tumor invasion and angiogenesis are not well established. Nonetheless, maspin can regulate adhesion of cultured corneal stromal cells and carcinoma cells to extracellular matrix (ECM) 1 molecules (2, 10). Pretreatment with recombinant maspin increases adhesion of cultured corneal stromal cells to several ECM molecules, including type I and type IV collagen, laminin, and fibronectin (2), whereas it induces adhesion of carcinoma cells only to fibronectin and not to gelatin, laminin, type I, and type IV collagens or fibrinogen (10). Stimulation of cell-ECM adhesion by maspin likely involves a mechanism by which maspin upregulates expression of integrins, because the level of ␣ 5 integrin (the ␣ component of the fibronectin receptor) on the cell surface is induced in carcinoma cells pretreated with recombinant maspin (10).Maspin shares sequence homology with the serpins (serine protease inhibitors) of the ovalbumin-type subfamily, which includes ovalbumin, plasminogen activator inhibitor-2, squamous cell carcinoma antigen (SCCA), and bomapin (PI10) (11). Most serpins are inhibitors of specific proteases that react with an exposed reactive site loop (RSL) at the top of the molecule *

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase-dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 microm filter to remove the myotubes, frozen at -80 degrees C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a-actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle-shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190 -202 and 260 -275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and ␤1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-␤1 integrin mega-complex that regulates mammary epithelial cell adhesion.Maspin is a non-inhibitory serine protease inhibitor (serpin) 4 that was originally identified as a type II tumor suppressor protein in mammary epithelial cells (1). One major tumor suppressor function of maspin is suppression of tumor cell motility, as it inhibits tumor cell migration/invasion in vitro and suppresses metastasis in mouse models (1-7). Several studies show that pericellular maspin inhibits cell motility by enhancing cell adhesion (2,3,8,9). In addition to its tumor suppressing functions, our laboratory showed that maspin is also essential for normal fetal development as maspin knock-out mice are embryonic lethal during the peri-implantation stage partially due to disrupted visceral endodermal cell adhesion (10). The underlying molecular mechanism by which maspin regulates cell adhesion is currently unknown and under intense investigation. To date, there are two proposed pathways utilized by maspin to increase cell-extracellular matrix (ECM) adhesion; that is, the plasminogen activation system and ␤1 integrin signaling (9, 11-13).The plasminogen activation system is believed to be a central player in several different processes important for tumor progression and metastasis (14 -16). In this system urokinase-type plasminogen activator (uPA), a serine protease, binds to its glycosylphosphatidylinositol-anchored receptor (uPAR) and readily activates plasminogen to initiate a protease cascade resulting in localized ECM degradation for the purpose of cell migration (17, 18). It has been suggested that maspin integrates into the plasminogen activation system. Maspin inhibits prostate carcinoma cell migration and invasion by strengthening mature focal adhesion contacts, reducing uPA activity by internalizing the maspin-uPA-uPAR complex and by binding to pro-uPA, thus blocking its activation (12). Although maspin is classified as a serpin and decreases pericellular uPA activity, maspin does not directly inhibit uPA proteolytic activity (19 -21). Together, these studies demonstrated that maspin can reduce prostate carcinoma cell migration and invasion by internalization of cell surface maspin-uPA-uPAR complexes.The second pro...