||Mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into several distinct lineages. We have reported an immortalized line of human MSCs (hMSCs), KP-hMSCs, which undergo adipogenesis in adipocyte induction medium (AIM) with IBMX, a phosphodiesterases inhibitor, which was reported to stimulate cAMP-dependent protein kinase activity (cAMP-PKA). In current study, we further hypothesized the involvement of cAMP-PKA pathway in adipogenesis of hMSC. We investigated the involvement of several modulators of cAMP-PKA pathway, including stimulators (forskolin, Sp-cAMP), inhibitors PKI, and combination of both in the adipogenesis of hMSC.
We demonstrated that KP-hMSCs when cultured in AIM with the stimulators of PKA enhance the differentiation into adipocytes, and also increased the expression of markers for adipocyte (PPARγ2 and LPL) and decreased the expression of makers for osteoblast (Runx2, Alk-p and Op). On the other hand, KP-hMSCs when cultured in AIM with the inhibitors of PKA decreased the expression of markers for adipocytes and increased the expression of makers for osteoblast. We further found that the secretion of leptin and the mRNA expression of leptin by KP-hMSCs decreased as the addition of PKA stimulators and increased as the addition of PKA inhibitors. Because we speculated that leptin played a role in cAMP-PKA mediated regulation of adipogenesis and osteogenesis in KP-hMSCs, we added leptin into AIM with forskolin and found leptin reversed the effects of forskolin on adipogenesis and osteogenesis in a dose-dependent manner.
KP-hMSCs transfected with DN (dominant negative) CREB was found to have an increase in CREB protein level, however, not only underwent adipogenesis of hMSC but also amplified mRNA expressions of Runx2 or leptin in AIM with forskolin at three day. On the other hand, mRNA expression of PPARγ2 was downregulated by DN CREB. Beside, DN CREB also increased RNAKL/OPG ratio in mRNA level. From the current results, KP-hMSCs may be used to elucidate molecular signaling on the regulation of adult adipogenesis and osteogenesis.