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The aim of this study investigates whether the secretome collected from human dental pulp stem cells (hDPSCs) transfected with transforming growth factor-beta1 (TGF-β1) is related to CD44 expression of fibroblasts and canonical smad signaling pathway via proteomic analyzes. In order to obtain secretome, hDPSCs were conditioned with serum-free alpha-MEM in an incubator containing 37°C, 5% CO 2 , and humidity for 18–24 h. Proteins in control and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. CD44 expressions in fibroblasts were evaluated by real time-PCR, western blot, and immunofluorescent staining. The relationship of canonical smad pathway and CD44 was analyzed by western blot and LC-MS/MS. Cell cycle, proliferation and wound healing tests were performed in the secretome groups. Venn diagram was showed 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. CD44 gene and protein expressions were increased in fibroblasts treated with TGF-β1 secretome. Relationship between targeted protein data showed that activation of the canonical TGF-β1/Smad pathway was up-regulated CD44 expression in fibroblasts. The canonical smad pathway-mediated upregulation of CD44 may increase the mitotic activity, proliferation, and wound healing potential in fibroblasts. While TGF-β1-transfected hDPSC secretome may be a potential therapeutic candidate in regenerative connective tissue therapies as it induces fibroblast activation, anti-TGF-β1-based therapies would be considered in histopathological conditions such as pulmonary fibrosis or hepatic fibrosis.

connective tissue research

Comparative proteomics analysis of transforming growth factor-beta1-overexpressed human dental pulp stem cell-derived secretome on CD44-mediated fibroblast activation via canonical smad signal pathway