IL-1 enhances cell adhesion to degraded fibronectin
ABSTRACT IL-1β is a prominent proinflammatory cytokine that mediates degradation of extracellular matrix proteins through increased expression of matrix metalloproteinases, which involves a signaling pathway in adherent cells that is restricted by focal adhesions. Currently, the mechanism by which IL-1β affects cell adhesion to matrix proteins is not defined, and it is not known whether degraded matrix proteins affect IL-1β signaling. We examined adhesion-related IL-1β signaling in fibroblasts attaching to native or MMP3-degraded fibronectin. IL-1β increased cell attachment, resistance to shear force and the numbers of focal adhesions containing activated β(1) integrins. IL-1β-enhanced attachment required FAK, kindlins 1/2, and talin. MMP3-degraded fibronectin-inhibited IL-1β-enhanced cell adhesion and promoted spontaneous ERK activation that was independent of IL-1β treatment. We conclude that IL-1β enhances the adhesion of anchorage-dependent cells to MMP3-degraded fibronectin, which, in turn, is associated with deregulated cellular responses to IL-1β. These data point to a novel role of IL-1β as a proadhesive signaling molecule in inflammation that employs kindlins and talin to regulate adhesion.-Rajshankar, D., Downey, G. P., McCulloch, C. A. IL-1β enhances cell adhesion to degraded fibronectin.
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- "DDR1 over-expression enhances activated b1 integrins in focal adhesions Although DDR1 does not localize to focal adhesions (Vogel et al., 2000), we determined whether over-expression of DDR1 might influence the activation of b1 integrins and their recruitment to adhesions. Cells were plated overnight on fibrillar collagen, stained with 9EG7 (a neo-epitope antibody that binds to activated b1 integrins) (Lenter et al., 1993) and analyzed by TIRF microscopy (Rajshankar et al., 2012). Compared with wild type, DDR1 over-expressing cells contained more adhesions with activated b1 integrins (Fig. 3A; P,0.01). "
ABSTRACT: Collagen degradation by phagocytosis is essential for physiological collagen turnover and connective tissue homeostasis. The rate limiting step of phagocytosis is the binding of specific adhesion receptors, which include the integrins and discoidin domain receptors (DDR), to fibrillar collagen. While previous data suggest that these two receptors interact, the functional nature of these interactions is not defined. In mouse and human fibroblasts we examined the effects of DDR1 knockdown and over-expression on β1 integrin subunit function. DDR1 expression levels were positively associated with enhanced contraction of floating and attached collagen gels, increased collagen binding and increased collagen remodeling. In DDR1 over-expressing cells compared with control cells, there were increased numbers, area and length of focal adhesions immunostained for talin, paxillin, vinculin and activated β1 integrin. After treatment with the integrin-cleaving protease jararhagin, in comparison to controls, DDR1 over-expressing cells exhibited increased β1 integrin cleavage at the cell membrane, indicating that DDR1 over-expression affected the access and susceptibility of cell-surface β1 integrin to the protease. DDR1 over-expression was associated with increased glycosylation of the β1 integrin subunit, which when blocked by deoxymannojirimycin, reduced collagen binding. Collectively these data indicate that DDR1 regulates β1 integrin interactions with fibrillar collagen, which positively impacts the binding step of collagen phagocytosis and collagen remodeling.Biology Open 11/2013; 2(11):1148-59. DOI:10.1242/bio.20135090 · 2.42 Impact Factor
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ABSTRACT: IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα(+/+) and PTPα(-/-) mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5-3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα(-/-) mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα(KO) and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.PLoS ONE 08/2013; 8(8):e70659. DOI:10.1371/journal.pone.0070659 · 3.23 Impact Factor
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ABSTRACT: The α5β1 integrin heterodimer is involved in many cellular processes and is an anti-cancer therapeutic target. Therefore, access to quantities of protein suitable for studies aimed at understanding its biological functions is important. To this end, a large-scale protein expression system, utilizing the recombinant baculovirus/SF9 insect cell expression system, was created to produce the extracellular domain of the α5β1 integrin. An incorporated 8X-histidine tag enabled one-step nickel-column purification. Following sequence confirmation by LC-MS/MS, the conformation of the heterodimer was characterized by native dot blot and negative stain electron microscopy. Cellular transduction inhibition studies confirmed biological activity. The system allows expression and purification of α5β1 integrin in quantities suitable for an array of different experiments including structural biology.Protein Expression and Purification 08/2013; 92(1). DOI:10.1016/j.pep.2013.08.013 · 1.51 Impact Factor