Tumor Necrosis Factor-α Inhibits Transforming Growth Factor-β-Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-beta 1 (TGF-beta 1) and tumor necrosis factor-alpha (TNF-alpha) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-beta 1 and TNF-alpha. Protein levels of alpha-smooth muscle actin (alpha-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. alpha-SMA and actin fibers were identified by immunofluorescence. TGF-beta 1, TNF-alpha, and alpha-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-beta 1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. Results: TGF-beta 1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of alpha-SMA and ED-A-FN. Moreover, TGF-beta 1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-alpha and TGF-beta 1 significantly reduced the expression of all the above-mentioned proteins. TNF-alpha also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. Conclusions: TNF-alpha inhibits several cell responses induced by TGF-beta 1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.