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  • Lactate LA the end product of aerobic glycolysis


    Lactate (LA), the end product of aerobic glycolysis, has been long neglected and seen as a byproduct until recently the findings for its important role in global gene transcription, cancer progression, functional polarisation of immune LY3023414 sale and the sustenance of stem cells [[7], [8], [9]]. Lactate transfers its signal into the cell via its receptor or transporter mediated signal transduction [8]. For example, its transporter MCT-1 mediated lactate transportation into macrophages promotes polarisation via Hif-1α stabilization and the resulting increased production of vascular endothelial growth factor (VEGF), which further enhances tumor growth [7,8]. LA also binds with its receptor GPR81, a Gαi protein coupled receptor, then mediates the insulin-induced reduction of lipolysis and regulates the lipid metabolism homeostasis [[10], [11], [12], [13]]. There is evidence that LA alters cellular responses in inflammatory environments, for example, LA suppresses the effect of IL-33 on primary human mast cells in an MCT-1 dependent manner [14]. In our previous study, we found that lactate was transported into cell by MCT-1 and plays a role in osteoblast differentiation via the stabilization of Hif-1α [15]. Whether LA affects osteoblast differentiation that induced by bone formation inducing factors is not clear.
    Materials and methods
    Results We have revealed that lactate (LA) in culture environment induced osteoblast differentiation [15]. Whether LA had any effect on osteoblast differentiation that induced by PTH was still not clear. In pre-osteoblast cell MC3T3-E1, PTH treatment stimulated the formation of ALP positive cells, which further increased in cells co-treated with LA (Fig. 1A). In consistence, the ALP activity (Fig. 1B) and mRNA level of Alp (Fig. 1C), Runx2 (Fig. 1D) also increased in cells with LA and PTH co-treatment. The intake of LA into the cell via the MCT-1 induced osteoblast differentiation [15]. To our surprise, treated cell with α-CCA or AZD3965, inhibitors for MCT-1, both further increased the mRNA level of Alp in cells with LA and PTH co-treatment (Supplementary Fig.1). However, the gene set enrichment analysis (GSEA) for differentiated genes from the microarray analysis revealed that the target genes for transcription factor CREB were enriched in LA and PTH co-treatment cells (Supplementary Fig.2 and Supplementary Table1). In consistence, the activity of CREB-luciferase was increase 4.89 fold (P<0.05) in LA and PTH co-treatment cells when compare with PTH treated MC3T3-E1 cells (Fig. 1E). As CREB was the principal transcriptional factor for PTH signaling in bone cells and important for osteoblast differentiation [19,20], we then try to find whether LA affects the downstream signaling for PTH. The activity of the Erk1/2, p38, Akt and β-Catenin were firstly measured in LA treated MC3T3-E1 cells and we found that the phosphorylation of Erk1/2 was activated with 5 min LA treatment but was inhibited at 15 min and recovered to normal after 1 h LA treatment (Fig. 2A and Supplementary Fig.3A). To compare, LA had no effect on β-Catenin activation but increased the activity of p38 and Akt in a time dependent manner (Fig. 2A and Supplementary Fig. 3B–D). Moreover, LA had no effect on the activation of Erk1/2 and β-Catenin but further induced the phosphorylation of Akt and p38 that induced by PTH treatment (Fig. 2B and Supplementary Fig.4). We found p38 inhibitor decreased the ALP activity and Alp mRNA levels (from 14.5 fold to 4.4 fold, P<0.01) in LA and PTH co-treatment cells (Fig. 2C–D). The MK-2206, an Akt inhibitor, also decreased the Alp mRNA levels and Alp activity in LA and PTH co-treatments cells (Fig. 2C–D). However, the Erk1/2 inhibitor further increased and β-Catenin inhibitor had no LY3023414 sale effect on Alp expression and ALP activity that induced by LA and PTH co-treatment (Supplemenatry Fig. 5). We further try to find the upstream regulators that involved in the regulation of LA on these signaling. MC3T3-E1 cells expressed GPR81 (data not shown), a Gαi protein coupled receptor for LA [13]. We firstly used pertuss toxin (PTX) to interrupt the Gαi function and found that the activation of Erk1/2 by LA treatment were inhibited but the activation of p38 and Akt was not affected (Supplementary Fig.6A). Moreover, PTX treatment further increased the Alp mRNA level in LA and PTH co-treated cells (Supplementary Fig.6B). ROS plays a role in the activation of p38 upon LA treatment [9], however, anti-oxidant NAC had no effect on p38 phosphorylation in LA treated cells (data not shown). Then PKA inhibitor (H-89) was used and p38 signaling that induced by LA treatment was inhibited, however, the activation of Akt were not affected (Fig. 3A and Supplementary Fig. 7A-B). Gi coupled receptor also activates the PKC signaling via the Gβγ-PLC signaling cascade [21]. We further treated the cell with PKC inhibitor and found the phosphorylation of Akt that induced by LA treatment was inhibited (Fig. 3A and Supplementary Fig. 7A-B). The mRNA level of Alp and the activity of ALP also decreased (Fig. 3B–C). Moreover, the induced CREB-luciferase activity in LA and PTH treatment cells was also inhibited with PKC inhibitors (Fig. 3D). Treated the cells with GDPβs, the inhibitor for Gβγ protein or U73122, the inhibitor for PLC, impaired the Akt activation (Fig. 4A–B) and decreased Alp mRNA expression (Fig. 4C) upon LA and PTH co-treatment.