title: Antagonistic Effects of Insulin and TGF-β3 during Chondrogenic Differentiation of Human BMSCs under a Minimal Amount of Factors.
abstract: Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-β (TGF-β) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-β-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-β3 or in combination, and found that TGF-β3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-β3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-β3 significantly decreased the TGF-β3-induced increase in mRNA levels of the direct downstream factor of TGF-β/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-β3 alone. On the other hand, insulin/TGF-β3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-β3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-β3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.
authors: Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors: Emilio Satoshi
authors: ES
authors: Hara
authors: Mitsuaki
authors: M
authors: Ono
authors: Yuya
authors: Y
authors: Yoshioka
authors: Junji
authors: J
authors: Ueda
authors: Yuri
authors: Y
authors: Hazehara
authors: Hai Thanh
authors: HT
authors: Pham
authors: Takuya
authors: T
authors: Matsumoto
authors: Takuo
authors: T
authors: Kuboki
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D020219:Chondrogenesis; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D060066:Protein Interaction Maps; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000378:metabolism*
chemical_list: D007328:Insulin; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053782:Transforming Growth Factor beta3; D051057:Proto-Oncogene Proteins c-akt