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.

PURPOSE: 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.

MATERIALS AND METHODS: In order to obtain secretome, hDPSCs were conditioned with serum-free alpha-MEM in an incubator containing 37°C, 5% CO2 , 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.

RESULTS: 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.

CONCLUSION: 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.