DAPI hydrochloride br Discussion Recent progresses in
Discussion Recent progresses in osteoblast biology reveal that aerobic glycolysis was the main pathway to supply energy and materials that need by osteoblast differentiation . In current study, we showed that LA enhanced the effect of PTH on osteblast differentiation and revealed an unrecognized mechanism that mainly mediated by downstream Gβγ-PLC-PKC-Akt signaling of its receptor GPR81. The stimulation of osteoblast differentiation by PTH is the main cause for its bone anabolism effect and this effect can be modulated at multiple levels . The co-exist factors in culture environment as IL-6, DKK1 and Sclerostin modulated PTH activity on osteoblast [23,24]. Here with the Alp staining assay, Alp activity analysis and the detection for the expression of specific osteoblast differentiation marker genes, we found LA in culture environment enhanced the osteblast differentiation inducing effect of PTH. Taken the finding by Long et al. that PTH stimulated aerobic glycolysis in osteoblast , our data supported a positive feedback loop was exist during osteoblast differentiation process that mediated by PTH (Fig. 4D). Moreover, the lactate could also be produced from the hypoxic osteoblast or WNT3a treated DAPI hydrochloride [5,25] and in that situations the effect of PTH on osteoblast differentiation could also be enhanced (Fig. 4D). In addition to its anabolism effect, PTH also induced the expression of RNAKL in osteoblast and RANKL is a known factor for osteoclatogenesis . Further studies are essential to learn whether LA also modulates the expression of RANKL by PTH. The anabolic or catabolic effect of LA on PTH treatment in bone homeostasis should also be evaluated with in vivo mice models. Our previous study showed that in osteoblast LA was transported into the cell via MCT-1 and drove the cell into the oxidative phosphorylation (OXPHOS) which further induced osteoblast differentiation . However, treated the cells with MCT-1 inhibitors did not interrupt the osteoblalst differentiation that mediated by LA and PTH co-treatment but further increase the mRNA level of Alp in current study. Taken the findings that MCT1 expression was down regulated in PTH treated MC3T3-E1 cells (Yu. W, Unpublished data), it was plausible that the intake of LA via MCT-1 was inhibited in PTH treated cells with an undefined mechanism. It had been demonstrated that the metabolic model depend on the state of osteoblast differentiation with mature calvarial osteoblastic cells relied more on aerobic glycolysis than OXPHOS during differentiation process [2,3]. Moreover, PTH also stimulates oxidative phosphorylation in MC3T3-E1 cells . The metabolic shift between OXPHOS and aerobic glycolysis upon PTH treatment may be important for osteoblast differentiation. Unravel the mechanism underlie would be helpful to answer our unexpected findings that MCT-1 inhibition further enhanced the osteoblast differentiation in PTH and LA co-treated cells. PTH induced osteoblast differentiation via its binding to the PTHR receptor then mainly activates Gαs-cAMP-PKA dependent signaling [19,22]. With the western blotting analysis, we found that LA alone in culture environment activated the Erk1/2, p38 and Akt signaling in a time dependent manner. However, LA had no effect on the activation of Erk1/2 but further induce the activation of Akt and p38 that induced by PTH, which indicated that the activation mechanisms for these signaling are different. With specific inhibitors, we showed that the activation of Erk1/2 is GPR81-Gαi signaling dependent as its activation can be interrupted by Gαi inhibitor PTX. It had reported that LA was transported into the cell and transversed to pyruvate then activate p38 signaling via the production of ROS in OXPHOS , but p38 activation upon LA treatment in current study was PKA dependent and not ROS dependent as NAC, an anti-oxidant reagent, did not inhibited p38 activation in LA treated cells. It seems that the activation mechanisms for p38 are cell dependent and further investigation is warranted to find how LA activated p38 in osteoblast via PKA signaling. For Akt signaling, we revealed that the activation of Akt by LA treatment is Gβγ-PLC-PKC signaling dependent. Taken the findings that GPR81 signaling controlled Akt activation via PI3K in breast cancer cells , whether PI3K was involved in the regulation of LA on Akt activation in osteoblast need further study. With the findings in current study, it was important to note that p38 and Akt signalings contributed largely to the osteoblast differentiation inducing effect of LA on PTH treatment. Moreover, the Akt and p38 signaling seems crosstalk at CREB, a well defined downstream target of p38, as we found inhibitors that interrupt PKC-Akt signaling also decreased CREB transcriptional activity (Fig. 4D), and this lead to the CREB target genes enriched in LA and PTH co-treatment cells.