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TRPC3 Channels Regulate Expression Of Fibronectin Via NFκB Signling In Response To Cyclic Strain And Contribute To Hypertrophic Scar Formation
Kenichiro Kawai, M.D.,Ph.D1, Hisako Ishise, M.D.1, Barrett Larson, M.D.2, Soh Nishimoto, M.D.,Ph.D1, Toshihiro Fujiwara, M.D.,Ph.D1, Masao Kakibuchi, M.D.,Ph.D1.
1Hyogo College of Medicine, Nishinomiya, Japan, 2Stanford University, Stanford, CA, USA.
In the cutaneous wound healing process, fibronectin is robustly secreted from fibroblasts. Fibronectin serves a variety of important functions in the wound healing process beyond just providing as a supporting scaffold. Fibronectin induces cell differentiation, migration, coagulation and formation of ECM, which ultimately serves to promote wound contraction. The expression of fibronectin is under complex control. Among the transcriptional factors interacting with the fibronectin promoter, Nuclear factor-kappa B (NFκB) also regulates the transcriptional activity of fibronectin.
It is well known that mechanical stimuli induce hypertrophic scarring. We previously reported that Transient Receptor Potential Canonical 3 (TRPC3) is highly expressed in hypertrophic scar tissue and plays a role in mechanotransduction. In this study, we investigated the link between TRPC3 activation and the expression of fibronectin via NF-κB signaling.
TRPC3 overexpressing NIH3T3 mouse fibroblasts were created with a PMXS system. Either TRPC3 overexpressing or control-vector transfected fibroblasts were seeded on silicon chambers (3×3 cm) at density of 8×105 cells/chamber. These fibroblasts were mechanically stretched for 24 hours at a frequency of 10 cycles/min with/without either Pyr3 (TRPC3 inhibitor) or Wedeloacton (NFκB inhibitor). Then, the expression level of fibronectin was examined with real-time PCR and Western Blotting. The activity of NFκB in fibroblasts in response to mechanical stretching was also investigated using Western Blot analysis using an anti-phospho NFκB antibody. The translocation of NFκB into nuclei in response to mechanical stretching was assessed using immunocytochemistry and an EMSA assay performed on cell lysates obtained after stretching.
Immunohistochemistry was performed on mouse dermal wounds that had been transplanted with either TRPC3 overexpressing fibroblasts, control fibroblasts, or normal saline. Either TRPC3 overexpressing fibroblasts, control vector transfected fibroblasts (1×106 cells /mouse) or the same amount of normal saline were injected into the entire dorsal area (dermal and subdermal layers) of 8 week old female Balb/c nu/nu mice. At 10 days posttransplantation, bilateral dorsal cutaneous wounds were created. The rate of wound closure was measured and fibronectin expression was analyzed using immunohistochemistry at 9 days post-wounding .
TRPC3 over-expressing fibroblasts that were subjected to cyclic stretching forces showed robust expression levels of fibronectin. Mechanical stretching on TRPC3 overexpressing fibroblasts induced the activation of nuclear factor-kappa B (NFκB). The activation of NFκB that occurred upon stretching was attenuated by pharmacological blockade of TRPC3 or NFkB. Moreover, transplantation of TRPC3 overexpressing fibroblasts into mice promoted wound contraction and increased fibronectin levels in vivo.
TRPC3 channel activate NFkB signaling pathways in response to mechanical stimuli. The activation of NFkB signaling leads to enhanced production of fibronecin, which may account for hypertrophic scarring in the skin. In conclusion, the TRPC3 channel is a potential mechanical force transducer that plays an important role in the pathogenesis of hypertrophic scarring. The results presented here indicate that TRPC3 may be a potential therapeutic target for fibrotic diseases, as well as hypertrophic scarring.
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