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We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.

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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...

Pseudomonas aeruginosa is an opportunistic bacterial pathogen implicated in a variety of devastating conditions. Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions. ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of P. aeruginosa. This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells. The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated P. aeruginosa invasion. Mutation of lasA and/or lasB reduced P. aeruginosa invasion, which was not fully restored by extracellularly added LasB, P. aeruginosa conditioned medium containing LasA and LasB, or EGTA pretreatment of cells. This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity. Upon mutation of lasA and/or lasB, steady-state levels of ExoS and ExoT were increased in culture medium of P. aeruginosa grown under conditions stimulatory for these toxins. The increase in ExoS was significantly correlated with reduced invasion. In vitro experiments showed that purified LasB degraded recombinant ExoS. Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells. By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined. mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. There are an estimated 9 million new cases of infection caused by this organism each year, accounting for more than 2 million deaths annually (3). This makes M. tuberculosis one of the leading causes of death due to a single infectious agent. Humans are the only known reservoir of M. tuberculosis, and as such, part of its success as a pathogen lies in its ability to maintain a significant persistent reservoir within the population. Latently infected individuals are asymptomatic and are harder to treat, thereby complicating measures to control the spread of disease (27). In addition, the emergence of multi-and extensively drug-resistant strains has further compromised established strategies for treatment. A better understanding of the mechanisms by which M. tuberculosis establishes persistent infection is urgently needed so that better therapeutics for the latently infected population can be developed.M. tuberculosis is a facultative intracellular pathogen that primarily infects alveolar macrophages. The organism survives within these cells by blocking key steps in the phagosome maturation process (38). Infection by M. tubercul...

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 *

PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.