A Complex Mechanism Of Extracellular Matrix Induction By Er Chaperone Calreticulin And Tgf-β For Tissue Regeneration
Leslie I. Gold, Unnati M. Pandya, Julien Daubriac, Ana Tellechea, Miguel M. Manzanares, Chinaza Egbuta.
New York University School of Medicine, New York, NY, USA.
BACKGROUND: A lack of extracellular matrix (ECM) synthesis for neodermal formation is a hallmark of impaired wound healing such as in diabetic foot ulcers (DFUs). We have shown that topical application of calreticulin (CRT) markedly enhances the rate and quality of wound healing in porcine and diabetic mouse models by promoting cellular migration and proliferation of keratinocytes for re-epithelialization, recruitment and anti-microbial action of macrophages, and migration, proliferation, and ECM induction by fibroblasts for reconstitution of the defect. Using CRT null mouse embryo fibroblasts (MEFs), it was previously shown that TGF-β-induction of collagen and fibronectin requires intracellular CRT (iCRT) for expression and processing of these ECM proteins. Since exogenous CRT (eCRT) and TGF-β both induce these same ECM proteins, we hypothesized that CRT might signal protein synthesis of ECM proteins through release of TGF-β. RESULTS: We show that CRT treatment of human neonatal fibroblasts induced TGF-β1/TGF-β3 proteins within 3 hours whereas collagen, fibronectin, elastin, smooth muscle cell actin (αSMA), and α5 and β1 integrins expression was not observed until 24 hours. Furthermore, an inhibitor of TGFβ receptor I signaling, SD208, completely blocked eCRT-mediated induction of these ECM proteins and αSMA but not the α5/β1 integrins in both neonatal and adult dermal fibroblasts thus, suggesting that only the ECM proteins and αSMA require TGF-β signaling. Additionally, CRT-mediated TGF-β1 release into media (ELISA) and ECM protein production, but not α5/β1 integrin were blocked by RAP, an inhibitor of LRP1 signaling. Importantly, eCRT did not induce ECM proteins in CRT null MEFs. CONCLUSIONS: The data supports the following schema: eCRT binds LRP1 receptor, LRP1 signaling induces TGFβ1/3, TGF-β1/3 are released from cells and, in an autocrine manner, bind TGF-β receptors to induce iCRT-dependent induction of ECM proteins. Although ECM protein induction by eCRT involves TGF-β, the responses are attenuated and mimic tissue regeneration without scaring implicating eCRTs strong therapeutic potential for DFUs.
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