Biochemical and Biophysical Research Communications
Regular ArticleTumor Necrosis Factor-α Increased the Integrin α2β1 Expression and Cell Attachment to Type I Collagen in Human Dermal Fibroblasts
Abstract
Cell adhesion molecules of human dermal fibroblasts play an important role in the processes of wound healing. The effects of tumor necrosis factor- α (TNF) on the expression of integrin β 1 subfamily in human dermal fibroblasts were examined. TNF preferentially induced the expression of α 2 β 1 integrins, receptors for collagen and laminin, in a time and dose dependent manner. Cell attachment to type I collagen increased by the treatment with TNF. However, cell attachment to fibronectin and laminin was not increased. This TNF-induced cell attachment could be reduced significantly by anti-integrin α 2 β 1 antibody. Antibodies against receptors other than α 2 β 1 integrin did not significantly reduce cell attachment. These data suggest that the increased attachment of human dermal fibroblasts to type I collagen appears to be mediated predominantly through the augmentation of integrin α 2 β 1 expression by TNF.
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Activation of protein kinase C by phorbol esters modulates α<inf>2</inf>β<inf>1</inf> integrin on MCF-7 breast cancer cells
1999, Experimental Cell ResearchCellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell–substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l,3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by α2β1integrin, since both anti-α2and anti-β1blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of α2β1integrin over a 4-h time interval. However, α2β1levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of α2β1-dependent cellular adhesion preceded the induction of α2β1cell surface expression. Northern blot analysis revealed that mRNA levels of both α2and β1subunits were increased after exposure to TPA for 4 h, indicating that the induction of α2β1mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of α2β1was independent of increased expression of α2β1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of α2β1integrin expression and of α2β1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of α2β1adhesion, we treated the cells with the Rho-GTPase inhibitorClostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the α2β1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins α2and β1and altering the avidity of the α2β1integrin by a Rho-dependant mechanism.
Deregulation of collagen phagocytosis in aging human fibroblasts: Effects of integrin expression and cell cycle
1997, Experimental Cell ResearchIntracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with increased lysomomal enzyme content in fibroblasts, we examined the regulation of collagen phagocytosis by integrin expression and the cell cycle in anin vitrofibroblast aging model. Two different fibroblast lines (CL1; CL2) at the fourth subculture were passaged up to replicative senescence to model aging processesin vitro.Cells were incubated with collagen-coated or BSA-coated green fluorescent beads for 3 h to assess α2β1-integrin-mediated or nonspecific phagocytosis, respectively. Single-cell suspensions were stained with DAPI and sulforhodamine 101 to separate cycling G1and noncycling G0cells. Staining for α2-integrin, bead internalization, and bivariate analyses of DNA/protein content were measured by three-color flow cytometry. Serum deprivation was used to induce increases in the proportion of G0cells. For G1cells, the proportion of collagen phagocytic cells was >50% for all passages and collagen beads were internalized >5-fold more frequently than BSA beads. In contrast, G0cells with diploid DNA content but low protein content exhibited greatly reduced phagocytic capacity (<10% of cells internalized collagen or BSA beads), the number of beads per cells was 4-fold less, and α2integrin expression was very low compared to G1cells. The proportion of collagen phagocytic cells and the proportion of α2-integrin-positive cells increased with transit through the cell cycle. At higher passage numbers mean cell volume and cytoplasmic granularity were reduced ∼30% but at replicative senescence cells with large surface area and subdiploid DNA predominated. The proportion of collagen and BSA phagocytic G1cells increased 1.5- and 5-fold, respectively, and the number of beads per cell increased <3-fold. However, surface α2-integrin staining remained unchanged. These data indicate that the collagen and nonspecific internalization pathways were greatly upregulated, independent of cell cycle phase, and that cellular agingin vitrostrongly influences the specificity and rate of phagocytic processes in fibroblasts. We suggest that age-related loss of collagen in connective tissues undergoing turnover may be a manifestation of a deregulated increase of collagen phagocytosis in which the net loss of degraded collagen exceeds new synthesis.
General principles of wound healing
1997, Surgical Clinics of North AmericaInjury triggers an organized and complex cascade of cellular and biochemical events that result in a healed wound. For didactic purposes, the wound healing response can be divided into three distinct but overlapping phases: (1) hemostasis and inflammation, (2) proliferation, and (3) maturation or remodeling.116 Failure or prolongation in one phase may result in delay of healing or nonclosure of the wound. Wound healing failures remain a significant clinical problem with large impact on health care costs. A better grasp of the fundamental physiology of healing results in a clearer understanding of the pathophysiologic processes that impair healing.
Expression of cytokines and their receptors by psoriatic fibroblasts. II. Decreased TNF receptor expression
1996, CytokinePsoriatic fibroblasts produce enhanced amounts of IL-6 in vitro. This state of activation may reflect an altered expression of cytokine receptors, involved in auto/paracrine induction of IL-6. Cultures of dermal fibroblasts derived from lesional psoriatic (PP) and normal control (NN) skin were therefore analysed for their ability to bind biotinylated recombinant human cytokines using flow cytometry. PP and NN fibroblasts bound negligible amounts of IL-1α and IL-1β, but clearly bound IL-4, IL-6 and TNF-α. Serum upregulated the number of NN fibroblasts which bound TNF-α, and to a lesser extent IL-6, but not the number of binding sites per cell. In contrast, this upregulation was significantly less in PP fibroblasts. This was not a result of differences in growth characteristics, receptor occupancy or an inability of stimulated PP fibroblasts to bind TNF-α. Immunocytochemistry of cells grown on slides showed that the TNF receptor type 1 (TNFR1, p55) was the predominant receptor in NN fibroblasts and was localized to the nucleus and cytoplasma. The expression of TNFR1 was clearly decreased in PP fibroblasts, which confirmed the binding studies. A slow and serum-induced shedding of TNFR1 was observed, but not of the TNFR2 (p75), in both types of fibroblasts. Confluent multi-passaged PP fibroblasts display both a decreased TNFR expression as well as an enhanced IL-6 production under serum conditions. These inherent abnormalities of PP fibroblasts imply the involvement of dermal fibroblasts in the maintenance of chronic inflammation in psoriasis.
Up-regulation of ICAM-1 expression on human dermal fibroblasts by IFN β in the presence of TNF-α
1995, FEBS LettersUnstimulated human fibroblasts show low or undetectable ICAM-1 expression. Interferon-beta (IFN-β) at concentrations of 10,100, and 1000 IU/ml in the presence of tumor necrosis factor-alpha (TNF-α) significantly increased the ICAM-I expression of fibroblasts in a dose-dependent manner. Treatment with IFN-β alone, however, did not up-regulate the ICAM-1 expression. Furthermore the attachment of peripheral blood mononuclear cells (PBMCs) to cytokine-treated fibroblasts was increased. This augmented attachment was partly inhibited by anti-ICAM-1 antibody. These results suggest that IFN-β and TNF-α may cooperatively modulate the attachment of PBMCs in the dermis.
Paracrine Responses of Cardiosphere-Derived Cells to Cytokines and TLR Ligands: A Comparative Analysis
2023, International Journal of Molecular Sciences