Serine proteinases such as thrombin, mast cell tryptase, trypsin, or cathepsin G, for example, are highly active mediators with diverse biological activities. So far, proteinases have been considered to act primarily as degradative enzymes in the extracellular space. However, their biological actions in tissues and cells suggest important roles as a part of the body's hormonal communication system during inflammation and immune response. These effects can be attributed to the activation of a new subfamily of G protein-coupled receptors, termed proteinase-activated receptors (PARs). Four members of the PAR family have been cloned so far. Thus, certain proteinases act as signaling molecules that specifically regulate cells by activating PARs. After stimulation, PARs couple to various G proteins and activate signal transduction pathways resulting in the rapid transcription of genes that are involved in inflammation. For example, PARs are widely expressed by cells involved in immune responses and inflammation, regulate endothelial-leukocyte interactions, and modulate the secretion of inflammatory mediators or neuropeptides. Together, the PAR family necessitates a paradigm shift in thinking about hormone action, to include proteinases as key modulators of biological function. Novel compounds that can modulate PAR function may be potent candidates for the treatment of inflammatory or immune diseases.
Nerve growth factor (b-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human b-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.
Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro- and anti-inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti-inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up-regulated scavenger receptor CD163 and Gr-1, down-regulated CX(3)CR1, and shared with human GC-treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up-regulated anti-inflammatory IL-10, but not TGF-beta, and in contrast to their human counterparts, they down-regulated IL-6. Although GC-induced monocytes down-regulated CX(3)CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX(3)CR1(lo)CCR2(+)Ly6C(hi)), they differed from this physiologically occurring subset, as they remained Ly6C(med) and unactivated (CD62 ligand(++)). In addition to their immunosuppressive effects, they were CD11b(+)Gr-1(+) and expressed the IL-4Ralpha chain (CD124), a recently described, signature molecule of tumor-induced myeloid-derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma-bearing mice and indeed found parallel up-regulation of CD11b(+)Gr-1(+) and CD124 on GC-induced monocytes and MDSC. These data allow us to speculate that the GC-induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti-inflammatory effects and that similarities between GC-induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.
In the vascular system, circulating tumor cells interact with endothelial cells. Tumor-endothelial cross-talk transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state called endothelial cell activation (ECA). In the present study, we analyze the potential of metastatic tumor-derived soluble factors to transform the vascular endothelium into a prothrombotic and proinflammatory activated state. Supernatant from cultured melanoma and colon cancer cells (A375, WM9, A7, and HT-29) induced an acute activation of macrovascular and microvascular endothelial cells (human umbilical vein endothelial cells and human dermal microvascular endothelial cells) as shown by intracellular calcium flux and secretion of von Willebrand factor and interleukin-8, all markers of acute ECA. This process was inhibited using specific proteinase-activated receptor 1 (PAR1) inhibitors (RWJ-58259 and SCH-79797), indicating a mediating role for endothelial thrombin receptors. Immunofluorescence, Western blot analysis, and collagenase activity assay of tumor cells and culture supernatant revealed the presence of matrix metalloproteinase-1 (MMP-1), a recently described activator of PAR1. Inhibition of MMP-1 in supernatant from cultured tumor cells significantly attenuated ECA. Additional studies using isolated human MMP-1 (5 nmol/L) proved the presence of a functional MMP-1/PAR1 axis in tumor-endothelial communication. These findings show a new pathway of tumor-endothelial cross-talk via an intravascular MMP1/PAR1 axis in microvascular and macrovascular endothelium. Inhibition of this cross-talk may be a powerful means to prevent tumor-induced ECA and thus thrombotic and inflammatory cell adhesion. (Cancer Res 2006; 66(15): 7766-74)
Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the Cterminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA2DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.
We have assessed the suitability of retroviral vectors for gene therapy of recessive dystrophic epidermolysis bullosa (RDEB) in dogs expressing a mutated collagen type VII. Isolation and analysis of the 9 kb dog collagen type VII cDNA identified the causative genetic mutation G1906S and disclosed the interspecies conservation of collagen type VII. Highly efficient transfer of the wild-type collagen type VII cDNA to both dog RDEB and human primary RDEB collagen type VII-null keratinocytes using recombinant vectors derived from LZRS-Ires-zeo and MSCV retroviruses achieved sustained and permanent expression of the transgene product. The expression and post-translational modification profile of the recombinant collagen type VII was comparable to that of the wild-type counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Hypermotility was fully reverted in human RDEB keratinocytes, and strongly reduced in the dog RDEB cells. This observation suggests that not only infection efficiency but also high expression levels are required to ensure therapeutic efficacy in the presence of mutated gene products. Our results set the basis for preclinical gene therapy assays in the first immune-competent large animal model for an inherited skin disease and broaden the spectrum of preclinical and clinical applications of retroviral vectors in the transfer of large recombinant genes in epithelial cells.
Proteinase-activated receptors (PARs) are G-protein-coupled receptors with seven transmembrane domains that are activated by specific proteolytic cleavage of the extracellular N-terminus. To date, four PARs are known (PAR(1-4)). They are stimulated by a variety of serine proteinases. PAR(1), PAR(3) and PAR(4) are cleaved by thrombin. Both PAR(1) and PAR(4) can be activated by trypsin as well; and PAR1 can also be activated by matrix metalloproteinase-1. PAR(2) can be activated by a variety of endogenous serine proteinases with trypsin-like specificity. However, the receptor can additionally be stimulated by various proteinases produced by pathogenic organisms. It can also be inactivated by certain proteinases. PAR(2) is expressed by many cell types present in the skin, including epidermal keratinocytes, fibroblasts, endothelial cells as well as by afferent neuron terminals. Moreover, functional PAR(2) is expressed by cells crucially involved in innate and adaptive immunity such as eosinophils, neutrophils, monocytes, macrophages, dendritic cells, mast cells and T cells. Activation of the receptor leads to the production of various cytokines and chemokines which modulate skin homeostasis, immune and inflammatory responses as well as tumor surveillance.
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