| The increased marrow fat and reduced spongy bone mass can besimultaneously observed in osteoporotic bones, suggesting that adipogenicdifferentiation may play a critical role in osteoporosis. Osteoblasts andadipocytes are derived from a common progenitor-bone marrowmesenchymal stem cells (MSCs). In normal physiological conditions, thebalance between the osteogenic and adipogenic differentiation of MSCsdepends on a variety of hormones and transcription factors. However,during the process of osteoporosis, an imbalance in the production ofbone-forming and fat-forming cells exists in the bone marrow, andadipocytes may be generated at the expense of osteoblastes, thereby resultin increased fat content and decreased bone volume. Therefore, increasingadipogenesis may be associated with the development and deterioration ofosteoporosis.The differentiation of MSCs to mature adipocyte involves two phases consisted of the commitment of MSCs to preadipocytes and the process ofterminal adipocyte differentiation during which preadipocytes mature intoadipocytes. It is possible, therefore, that induction of preadipocytes intoosteoblasts may be a therapeutical approach of osteoporosis with which toeither prevent further increases in adipocyte formation or divert existingadipogenic committed cells to become more osteoblastic with a resultingincrease in functional bone cells. However, it is still unclear whichcytokines can exert this function.Recently, it was shown that bone morphogenic protein (BMP) mayinduce preadipocytes to differentiate into osteoblasts. BMPs belong totransforming growth factor (TGF) superfamily，and more than20types ofBMP have been identified at present. Based on analysis of14types ofBMP, it was found that BMP9is one of the most potent BMPs in inducingosteogenic differentiation of MSCs both in vitro and in vivo. However, it isunknown whether BMP9can induce adipogenic committed preadipocytesto differentiate into osteoblast.Here, we explored whether BMP9could induce osteogenicdifferentiation of3T3-L1preadipocytes and found that BMP9effectivelyinduced early and late osteogenic makers, such as alkaline phosphatase(ALP), osteopontin (OPN) and osteocalcin (OC) and promotedmineralization in3T3-L1preadipocytes. Meanwhile, we also found thatBMP9was able to induce adipogenic differentiation of3T3-L1cells, as evidenced by increased lipid accumulation. Additionally, the in vivoexperiments showed that BMP9can induce ectopic bone forming of3T3-L1preadipocyes.Though BMP9has the most potent osteoinductive activity, it isconceivable that other signaling molecules may be also required to enhanceBMP9-induced bone formation in preadipocytes. Retinoic acids (RAs) area group of natural and synthetic derivatives of vitamin A that play a criticalrole in embryological development and maintenance of vital organs in adult.RA has been shown to be able to enhance BMP-induce osteogenesis andinhibit BMP-induce adipogenesis. Hence, we also explored whether RAsignaling had any effect on BMP9-induced differentiation of3T3-L1preadipocytes in order to further regulate the balance between osetogenesisand adipogenesis induced by BMP9. We showed that all-trans retinoic acid(ATRA), the abundant form of RA, played a positive role in BMP9-inducedosteogenic differentiation and an inhibitory role in BMP9-inducedadipogenic differentiation in vitro. Similarly, ATRA enhancedBMP9-induced bone formation with a decrease in adipocyte accumulationin vivo. Mechanistically, ATRA was shown to induce BMP9expression andenhance the BMP9-induced BMPR-Smad reporter activity and Smad1/5/8nuclear translocation. ATRA also promoted BMP9-induced osteogenictranscription factors and suppressed BMP9-induced adipogenictranscription factors. Additionally, BMP9and ATRA exerted a synergistic effect on activating Wnt/β-catenin signaling. Knockdown of β-cateninabolished the ATRA potentiation effect on BMP9-induced ALP activity andblocked the inhibitory effect of ATRA on BMP9-induced lipidaccumulation in preadipocytes. Activation of Wnt/β-catenin signaling byoverexpression of β-catenin and Wnt ligands can enhance BMP9-inducedosteogenic differentiation and inhibit BMP9-induced adipogenicdifferentiation in3T3-L1preadipocytes. In addition, we found that BMP9can regulate various Wnt ligands and receptors in preadipocytes, indicatingthat Wnt/β-catenin signaling was involved in BMP9-induced differentiationof preadipocytes. Lastly, ATRA and BMP9synergistically repressedglycogen synthase kinase3β(GSK3β) activity and promoted Aktphosphorylation, and inhibited expression of phosphatase and tensinhomologue deleted on chromosome10(PTEN) that antagonizesphosphatidylinositol-3-kinase (PI3K) function, suggesting thatWnt/β-catenin signaling was activated at least partly throughPI3K/Akt/GSK3βpathway. |
