In this study, we demonstrated that BMP9 up-regulates Lox in preadipocyte, the osteoblastic capability of BMP9 was strengthened by inhibiting or silencing Lox, but reduced by Lox over-expressing. The possible molecular mechanism underlying this process may be resulted from increasing the activity of Wnt/β-catenin signal through up-regulating HIF-1α when Lox was inhibited or knockdown.
It’s well known that multiprogenitor cells can be committed toward different lineages, including adipogenic, osteogenic, myogenic, and chondrogenic lineages. In the development of skeletal system, adipogenesis and osteogenesis may occurred concomitantly although they are excluded usually. The homostasis of bone is affected by the balance between adipogenic and osteoblastic differentiation. More osteogenesis may dependent on the cost of adipogenesis, and vice verse(21, 22). Bone mesenchymal stem cells (bMSCs) can be directed to either adipocyte or osteocye. One of main pathogenies about osteoporosis is more adipocyte originated from bMSCs (23). PPARγ is a well-known adipogenic transcriptional regulator. Agonist of PPARγ a serial of small molecules, which has been used as efficacious drugs for the treatment of diabetes mellitus, such as rosiglitazone and pioglitazone(24). However, one of the most notorious adverse effect of these drug is osteoporosis(25, 26). For this reason, PPARγ agonist usually prescribed for the treatment of diabetes mellitus combing with bisphosphate, such as alendronate(27). Based on these evidences, decreasing the capability of adipogenesis in bMSCs may an efficient way to increase the amount of bone.
BMPs is a serial of secretary cytokines, and the osteogenic potential of BMP was first found and demonstrated by Marshall Urist in 1965(28). Because the excellent osteoblastic property of some BMPs, such as BMP2 and BMP7, it has been used for the treatment of bone defect, non-uion, and spinal fusion(29, 30). BMP9 is another member of BMPs, which has been demonstrated processing stronger osteogenic potential than that of BMP2 or BMP7 (2). For this reason, BMP9 maybe an excellent alternate for BMP2 or BMP7 to treat the related bone diseases. Although PPARγ is a classic adipogenic factor, it was still up-regulated by BMP9 and great amount of adipocytes were occurred in the BMP9-induced bone masses. However, PPARγ knockdown decreased the osteogenic ability of BMP9, instead of potentiating this effect(6). This evidence suggested that PPARγ is critical not only for adipogenesis, but also for osteogenesis; the osteogenic potential may be enhanced only if the adipogenic process was inhibited specifically.
As above mentioned, BMP9 up-regulated the well-known adipogenic marker, such as PPARγ. However, the osteoblastic ability of BMP9 was also reduced when PPARγ knockdown. This evidence suggested that PPARγ may also play an important role in osteogenesis. The adipogenic commitment of progenitor cells are controlled by various factors, such as PPARγ, TRAF4 and Leptin(31–33). To date, it remains unclear whether there exist any regulators which are exclusive for adipogenesis, no any effect on osteogenesis. Lox, also named as protein lysine 6-oxidase, can converse lysine or lysine residue to aldehyde and promote the physicological cross-link process of the matrix protein, which is very important for the mature of connective tissue(10, 11). It was reported that Lox plays an important role in the lineage commitment of adipocyte during the development(34). Besides, the abnormal level of Lox is associated with the biological behavior of cancer cells, such as metastasis(14). For this reason, Lox may affect the osteogenesis process of progenitor cells. It was reported that Lox can promote adipogenesis through inhibiting the signal of FGF-2. Lox promoted the adipogenic transcriptional factors, such as PPARγ and CCAAT enhancer binding protein (C/EBP) α in 3T3-L1 cells(34). Usually, the commitment of progenitor cells between osteogenesis and adipogenesis is mutual exclusive. Hence, Lox may also participate in the regulation of osteoblastic differentiation, but its role in the osteoblastic differentiation keeps controversial. It was reported that over-expression of Lox promote the calcification in vascular smooth muscle cells, and inhibition or knockdown of Lox almost diminished the calcification(35). Conversely, it was reported that inhibition of Lox greatly enhances the osteoblastic differentiation induced by BMP4 in MSCs(36). The converse effects of Lox on calcification or mineralization may contribute to the different of cell types, and context. In this study, we found that inhibition or knockdown of Lox promoted the osteoblastic markers or bone masses induced by BMP9 in 3T3-L1 cells. These evidences supported that Lox may function as a switcher between adipogenesis and osteogenesis, and its function may different greatly from other factors greatly, such as PPARγ. However, the exact mechanism through which Lox control the osteogenic and adipogenic commitment keeps unclear in MSCs.
The osteogenic process duing development is well-regulated by various cytokines or signals harmoniously, such as Wnt, FGF, IGF, and TGF-β (37). An expanding evidences support that moderately activate Wnt/β-catenin signal can promote the development of bone or keep the normal bone density from osteoporosis(38). Tang et al reported Wnt/β-catenin can enhance the osteogenic potential of BMP9, and knockdown of β–catenin obviously reduce this function of BMP9 in MSCs. Accordingly, BMP9 increased the activity of Wnt/β-catenin signal apparently(39). Our previous study demonstrated that all-trans retinoic acid can strengthen the osteogenesis induced by BMP9 through increasing the activation of Wnt/β-catenin signal(40). Therefore, Wnt/β-catenin is very important for osteoblastic differentiation. However, how the activity of Wnt/β-catenin signal is regulated by BMP9 or other osteogenic factors remains unclear and need to be uncovered further. Our data showed that either inhibition or knockdown of Lox can increase the BMP9-induced osteoblastic markers, which suggested that Lox may also be involved in the function of BMP9 to regulate the activity of Wnt/β-catenin signal. Our data showed that inhibition of Lox potentiates the effect of BMP9 on increasing the level of β-catenin in nucleus. BMP9 increases the protein level of sclerostin, and this effect of BMP9 was reduced by combining with Lox specific inhibitor. Herein, the effect of BMP9 on the activity of Wnt/β-catenin signal may be mediated by Lox partly.
It was reported that hypoxia-inducible factor-1α (HIF-1α) can promote the epithelial-to-mesenchymal transition through activating Lox in paraquat-induced pulmonary fibrosis(41), and HIF-1α and Lox can be regulated mutually to promote the growth of tumor cells(42). Meanwhile, Hu et al reported that BMP9 can up-regulate HIF-1α to increasing the angiogenic signal which can synergistically enhance the osteogenic capability of BMP9(43). These evidences suggested that HIF-1α may be involved in the regulatory effect of Lox on the osteoblastic inducing function of BMP9. We found that HIF-1α can be increased by BMP9 in 3T3-L1 cells, which was enhanced by the inhibition or knockdown of Lox but reduced apparently by over-expressing Lox. Based on other reports, our data also suggested that there may exist a regulatory loop between Lox and HIF-1α, and HIF-1α may be involved in mediating the regulatory effect of Lox on the osteoblastic induction ability of BMP9. It was reported that HIF-1α can activate Wnt/β-catenin signal pathway through regulating BCL9 expression in hepatocellular carcinoma(44). Hence, the effect of Lox on BMP9-increased Wnt/β-catenin signal may be carried out through regulating HIF-1α. We found that the protein level of SOST can be increased by BMP9, but reduced by combining the inhibitor of Lox. Correspondingly, the total protein level of β-catenin was elevated by BMP9. The effect of BMP9 on β-catenin was enhanced by inhibiting Lox, which was reduced notably by silencing HIF-1α. These data suggested that the effect of Lox on Wnt/β-catenin signal may be mediated partially through regulating the expression of HIF-1α during the osteoblastic commitment process induced by BMP9 in MSCs.
Summary, our findings indicated that the effect of Lox on BMP9’s osteogenic potential may be mediated through regulating Wnt/β-catenin signal via sclerostin negatively. Meanwhile, our finding also suggested that disturbing the function of Lox may be another strategy to treat the bone related diseases, such as osteoporosis. However, the exact molecular mechanism through which Lox affects Wnt/β-catenin signal need to be further analyzed.
BMP: Bone morphogenetic protein; Runx2: Runt-related transcription factor 2;OPN: Osteopotin; GAPDH: glyceraldehyde phosphate dehydrogenase; HIF-1α: Hypoxia-inducible factor 1α; bMSCs: Bone mesenchymal stem cells; µ-CT: micro-Computed Tomography; RIPA: Radio immunoprecipitation assay; cDNA: Complementary deoxyribonucleic acid ; NBT: Nitro Blue tetrazolium; BCIP: 5-Brom-4-chlor-3-indoxylphosphat; SOST: sclerostin; TGF-β: Transforming growth factor β; ECL: Enhanced chemiluminescence; SDS-PAGE: sodium dodecyl sulphate–polyacrylamide gel electrophoresis.