Interleukin 6 (IL-6), oncostatin M (OSM) and leukaemia-inhibitory factor (LIF) share a common signal-transducing subunit in each of their receptors and thus mediate an overlapping spectrum of biological activities. Although all of these cytokines stimulate the production of α1-proteinase inhibitor (α1-PI) in hepatocyte-derived cells, only OSM is able to up-regulate levels of this inhibitor in epithelial cells originating from the lung. In this study we characterized human lung-derived epithelial-like HTB58 cells for their ability to synthesize α1-PI after treatment with IL-6, OSM and LIF. The results demonstrate that the resistance of HTB58 cells to the effects of IL-6 and LIF was not because of a lack of their individual functional receptors and suggest that OSM utilizes two different receptors, gp130/LIF receptor and gp130/OSM receptor, in lung-derived epithelial cells.
CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan, has been implicated in many adhesion‐dependent cellular processes including tumor growth and metastasis. Soluble CD44 has been identified in the serum of normal individuals. Furthermore, tumor progression is often associated with marked increases in plasma levels of soluble CD44. Release of soluble CD44 by proteolytic cleavage (shedding) of membrane‐anchored CD44 is likely to alter cellular responses to the environment due to modification of the cell surface and the potential for soluble CD44 to influence CD44‐mediated hyaluronan binding to cell surfaces. Cellular activation is typically required to induce hyaluronan binding to cell surface CD44 but the affinity of endogenous soluble CD44 for hyaluronan remains unknown. In this study, we demonstrate that oncostatin M and transforming growth factor β1 (TGF‐β1) which stimulate hyaluronan binding to HTB58 lung epithelial‐derived tumor cells, also induce the release of soluble CD44. Interestingly, soluble CD44 released by oncostatin M‐treated cells retained the ligand‐binding properties of the membrane‐anchored receptor. In contrast, soluble CD44 released from TGF‐β1‐treated HTB58 cells differed in its hyaluronan‐binding capacity from cell surface CD44 expressed on TGF‐β1‐stimulated cells. These data indicate that the mechanisms that regulate the generation of soluble CD44 may also govern the binding of the released receptor to hyaluronan and therefore determine the impact on CD44‐dependent physiologic and pathologic processes.
Abstract Chemerin, a ligand for seven-pass transmembrane, G protein-associated receptor CMKLR1 requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically-processed chemerin selectively attracts specific subsets of immunoregulatory antigen presenting cells, such as immature plasmacytoid dendritic cells (pDC) and macrophages that express CMKLR1. Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins that comprise inhibitors of cysteine proteinases (cystatins). Cysteine proteinases of the papain-like family are well characterized proteolytic enzymes, best known for their role in intracellular proteolytic pathways. Nevertheless, recent studies show that lysosomal cathepsins can also exert their proteolytic activity at extracellular sites in secreted or cell surface-bound forms. Extracellular cathepsins contribute to a variety of pathophysiological processes, including inflammation and neoplastic progression. Homology between chemerin and cystatins suggests that chemerin interacts directly with cysteine proteases. In this work, we demonstrate that chemerin does not display substantial inhibitory activity against human cysteine proteases. Instead, chemerin was found to be a new substrate for these proteases. The interaction between chemerin and the proteases resulted in the generation of potent attractant activity, eliciting a robust chemotactic response by CMKLR1 transfectants as well as human blood-derived PDC. Our data demonstrate that cathepsins may contribute to immune responses through the recruitment of pDC.
Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.
Psoriasis, which involves mast cells, is a chronic inflammatory skin disorder whose pathophysiology is still not fully understood. We investigated the role of secretory leukocyte protease inhibitor (SLPI), a potential inhibitor of mastocyte serine proteases, on mast cell-dependent processes of relevance to the skin barrier defense in psoriasis. Here, we demonstrate that the dermal mast cells of patients with psoriasis express SLPI but not those of healthy donors. Moreover, SLPI transcripts were found to be markedly upregulated in murine mast cells by mediators derived from psoriasis skin explant cultures. Using mast cells from SLPI-deficient mice and their SLPI+ wild-type controls, we show that SLPI inhibits the activity of serine protease chymase in mastocytes. SLPI was also found to enhance the degranulation of mast cells activated via anti-IgE Abs but not Mrgprb2 ligands. Finally, we demonstrate that the expression and function of Mrgprb2 in mast cells are suppressed by a normal and, to a larger extent, psoriatic skin environment. Together, these findings reveal mechanisms underlying FcεRI- and Mrgprb2-dependent mast cell function that have not been described previously.
Recent studies suggest that the anti‑diabetic drug metformin may reduce the risk of cancer and have anti‑proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune‑deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0‑G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin‑dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.
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