NAM stimulated osteoblast differentiation and mitochondrial respiration in osteoblasts
To investigate whether NAM promotes osteoblast differentiation, MC3T3-E1 osteoblasts were treated with NAM in osteogenic medium. As shown in Fig. 1A, ALP and ARS staining showed that ALP activity and mineralization were enhanced in an NAM dose-dependent manner, and 10 µM NAM had the greatest effect on osteoblast differentiation (Fig. 1B, C). The expression levels of marker genes of osteoblast differentiation including Runx2, Osx, Dlx5, Bsp, Mepe, Opn and Ocn, were significantly increased by NAM treatment (Fig. 1D). In particular, late osteoblast differentiation markers, Ocn, Opn, and Mepe, were very highly stimulated by NAM treatment. To understand the mechanism by which NAM-stimulated osteoblast differentiation, MC3T3-E1 cells were treated with or without NAM during osteogenic differentiation for 4 or 10 days, followed by RNA sequencing analysis (RNA-seq). In total, the number of differentially expressed genes (DEGs) was higher on day 10 (3,858 genes) than on day 4 (2,246 genes). MA plots showed differential gene expression and the genes congregated around log2 fold change 0.5 (approximately 1.4 times) on both days (Supplementary Fig. 1A, B). Consistent with the results shown in Fig. 1A and D, Gene Ontology (GO) analysis on the DEGs upregulated by NAM treatment showed the enrichment of GO terms regarding osteoblast differentiation and regulation of ossification on both day 4 and day 10 (Supplementary Fig. 1C). Given that gene expression correlation analysis can be used to identify functional correlations between genes 29,30, correlation analysis of DEGs upregulated by NAM was performed here to identify genes associated with NAM-induced osteoblast differentiation (Supplementary Fig. 2A and 3A). On day 4, osteoblast differentiation and response to oxygen level showed correlation in cluster 4 (Fig. 1E). Furthermore, genes related to the cell cycle were highly enriched in all clusters (Supplementary Fig. 2B-G). On day 10, oxidative stress-related genes were included in all clusters (Supplementary Fig. 3B-I), and we found that osteoblast differentiation and response to oxidative stress were associated together in cluster 4 (Fig. 1F). The genes affiliated with cluster 4 were shown using a heatmap (Fig. 1G). These genes included Ndufa6, Gpx8, and Foxo3a, which are involved in regulating oxidative stress as well as Sod2, which encodes a mitochondrial antioxidant enzyme. These results suggested that the enhancement of osteoblast differentiation by NAM might be related to the regulation of oxidative stress.
NAM relieves mitochondrial ROS by enhancing mitochondrial antioxidant enzymes
To determine the effect of NAM on the cellular oxidative stress level, MC3T3-E1 cells were stained with a fluorogenic probe, CellROX™, which measures the ROS level in the cytosol, and MitoSOX™, which measures superoxide production in mitochondria. We observed that NAM treatment significantly reduced the accumulation of ROS in the cytosol (Fig. 2A-B) and superoxide production in the mitochondria (Fig. 2C and D).
PGC1-α is a transcriptional coactivator that plays a central role in the expression of genes acting against oxidative stress in combination with FOXO3a 31. PGC1α is also known to promote mitochondrial biogenesis and enhances the capacity of cells to detoxify ROS, allowing cells to efficiently produce ATP and respire with less oxidative stress 32. For this reason, we measured the mRNA levels of Pgc1-α and its downstream detoxifying genes in MC3T3-E1 cells treated with NAM for 4 and 7 days. NAM increased the expression levels of Pgc1-α, Ucp2, Trx2, Sod1 and Sod2 in differentiating MC3T3-E1 cells (Fig. 2E-I). NAM also significantly increased the enzymatic activity of SOD2, a key mitochondrial antioxidant enzyme, in differentiating MC3T3-E1 osteoblast cells (Fig. 2J).
To investigate whether NAM upregulates the expression of ROS-detoxifying enzymes through PGC1-α, MC3T3-E1 cells were treated with small interfering RNAs (siRNAs) targeting Pgc1-α were treated in MC3T3-E1 cells. siPgc1α efficiently knocked down the expression of Pgc1-α and NAM increased Pgc1-α expression in both siCtrl- and siPgc1-α-transfected MC3T3-E1 cells (Fig. 2K). As previously reported, Pgc1-α knockdown significantly decreased the mRNA levels of Ucp2, Trx2, Sod1 and Sod2 (Fig. 2L-O). The increase of the expression of mRNAs encoding ROS-detoxifying enzyme by NAM in siPgc1α-transfected cells was much lower than that in siCtrl-transfected cells. Consistent with this, the protein levels of ROS-detoxifying enzymes were increased by NAM treatment in MC3T3-E1 osteoblast cells (Fig. 2P). These results suggest that NAM relieves mitochondrial oxidative stress by inducing the expression of antioxidant enzymes.
NAM induces antioxidant enzymes expression through SIRT3/FOXO3a axis.
Treatment with nicotinamide riboside (NR), one of the NAD+ precursors, is known to increase NAD+ levels and activate SIRT3, an NAD+-dependent protein deacetylase 33. NAM was also found to activate SIRT1, resulting in improved liver function 34. Because SIRT3 is located in the mitochondria and regulates mitochondrial functions 35, we tested whether NAM treatment also promotes SIRT3 activity in osteoblast cells. MC3T3-E1 osteoblast cells were treated with NAM and the mitochondrial fraction was isolated to determine mitochondrial SIRT3 activity. The results showed that such activity was increased by NAM treatment in a dose-dependent manner in MC3T3-E1 cells (Fig. 3A). The mRNA level of Sirt3 was also significantly upregulated by treatment with 10 µM NAM in MC3T3-E1 cells (Fig. 3B). SIRT3 promotes the transcription-activating activity of FOXO3a, by decreasing the latter’s acetylation and phosphorylation, which in turn induces the expression of genes encoding anti-oxidant enzymes 36. We investigated whether NAM regulates the activity of FOXO3a by using FOXO3a reporter, FHRE-Luc plasmids. NAM significantly increased both the endogenous FOXO3a transactivation activity (Fig. 3C), and the activity of exogenous transfected FOXO3a transcription-activating activity in MC3T3-E1 osteoblast cells (Fig. 3D). SIRT3 physically interacts with FOXO3 in mitochondria and promotes the transcriptional activity of FOXO3a 37. SIRT3 regulates the deacetylation of FOXO3a, followed by the reduction of its phosphorylation, ubiquitination, and degradation 38. Moreover, the FOXO3a-dependent mitochondrial enzymes, such as SOD2, PRX3, PRX5 and TRX2, are involved in ROS detoxification. The acetylation of FOXO3a was diminished by NAM treatment in MC3T3-E1 cells (Fig. 3E). The de-phosphorylation of FOXO3a at S253 was also reported to stimulate the translocation of FOXO3a from the cytoplasm to the nucleus 39. The phosphorylation level of FOXO3a (S253) was decreased by NAM in a dose-dependent manner without affecting the FOXO3a protein level (Fig. 3F). Next, we examined whether NAM regulates the subcellular localization of FOXO3a. The NAM treatment increased the translocation of FOXO3a from the cytoplasm to the nucleus in MC3T3-E1 cells (Fig. 3G). Taking these findings together, NAM activates SIRT3 and FOXO3a, which in turn promotes the expression of mitochondrial antioxidant enzymes to facilitate ROS detoxification in osteoblasts.
NAM improved mitochondrial function during osteoblast differentiation.
Excessive oxidative stress reduces mitochondrial function in osteoblasts 40. Therefore, we tested whether NAM also regulates mitochondrial function in osteoblasts. First, to investigate the effect of osteoblast differentiation on mitochondrial respiration, we measured the oxygen consumption rate (OCR) before differentiation (day 0) and on days 4, 7, and 14 of differentiation of MC3T3-E1 cells. OCR significantly increased during the differentiation period (Supplementary Fig. 4A-H). As NAM improved mitochondrial respiration in undifferentiated osteoblasts (Fig. 4A-D and Supplementary Fig. 4I-L), we investigated whether it can reinforce it in differentiating osteoblast cells. NAM was applied to MC3T3-E1 cells during osteogenic differentiation. NAM increased the OCR in differentiating MC3T3-E1 cells cultured in differentiation medium for 7 days (Fig. 4E-H and Supplementary Fig. 4M-P). Because the mitochondrial OCR was increased by NAM treatment, we investigated the changes in expression of mitochondrial biogenesis-related marker proteins. The protein levels of cytochrome c and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were increased by treatment with 10 µM NAM during the differentiation of MC3T3-E1 cells for 7 days (Fig. 4I). In addition, the levels of other oxidative-phosphorylation-related proteins were examined using an antibody cocktail that recognizes subunits of each mitochondrial respiratory chain complex, Complex I subunit NDUF88, Complex II SDHB, Complex III UQCRC2, Complex IV MTCO1, and Complex V ATP5a. NAM increased the protein levels of all subunits of complexes I-V compared with the findings from vehicle-treated cells (Fig. 4J). We further quantified the ratio of mtDNA:nDNA after amplification of the mitochondrial and nuclear genomes, as another representative marker of mitochondrial biogenesis 41,42. The results showed that NAM significantly increased this ratio (Fig. 4K). Consistent with this, NAM treatment also significantly increased the ATP level in MC3T3-E1 osteoblast cells (Fig. 4L). These results indicate that NAM enhances mitochondrial function, which plays an important role in supplying the energy necessary for osteogenic differentiation.
NAM prevents osteoblast damage induced by H 2 O 2 oxidative stress.
The accumulation of oxidative stress is strongly related to reduced bone mineral density and impaired osteoblast differentiation 15,43. Because we observed that NAM induced the expression of mitochondrial antioxidant enzymes in osteoblasts, we tested whether NAM treatment relieves the ROS-damaged osteoblasts. MC3T3-E1 cells were treated with NAM in the presence or absence of 100 µM H2O2 during osteogenic differentiation for 4 or 10 days. ALP and ARS staining results showed that NAM prevented the impairment of osteoblast differentiation caused by chronic exposure to 100 µM H2O2 (Fig. 5A-C). RNA-seq analysis was performed to identify the effect of NAM on osteoblasts-damaged by H2O2. When the cells were treated with only H2O2 for 4 days, 405 genes were upregulated and 228 genes were downregulated (Supplementary Fig. 5A, B). Meanwhile, on day 10, 247 genes were upregulated and 298 genes were downregulated by H2O2 treatment (Supplementary Fig. 5C, D). The genes downregulated by H2O2 were associated with GO terms related to osteoblast differentiation on both day 4 and day 10, particularly on day 10 (Supplementary Fig. 5B, D). To determine whether NAM enables osteoblasts to recover from ROS-induced damage, GO analysis was performed on genes whose expression was decreased by H2O2 but restored by NAM (Fig. 5D). On day 4, the expression of only six genes was restored by NAM (Supplementary Fig. 6A). However, on day 10, the expression of 114 genes was significantly restored by NAM, these genes were particularly associated with GO terms related to osteoblast differentiation or bone formation (Fig. 5D). A correlation test was performed on the DEGs whose expression was decreased by H2O2 but restored by NAM, included in the top 20 GOs (Supplementary Fig. 6B). Next, we listed the top 10 ranked GO in each cluster (Fig. 5E, F, and Supplementary Fig. 6C). Interestingly, cellular response to reactive oxygen species and bone mineralization were related in the first cluster (Fig. 5E). Overall, the expression of 80% of genes involved in cellular response to reactive oxygen species was decreased by H2O2 but restored by co-treatment with H2O2 and NAM (Fig. 5E). In addition, cluster 1 contained Runx2 and Wnt10b, which are crucial factors in osteoblast differentiation (Supplementary Table 2). Cluster 2, included genes associated with many GO categories related to bone development and cartilage development (Fig. 5F), such as Mmp13, Vdr and Dlx5 (Supplementary Table 3). Genes in cluster 3 were related to GO categories, including cartilage development, bone development, and extracellular matrix organization (Supplementary Fig. 6C). Our RNA-seq results, showed that the expression of Col1A1, Col1A2, and Vdr genes associated with osteoporosis (as revealed by OMIM disease enrichment analysis) was decreased by H2O2 (Table 1). Interestingly, NAM restored the H2O2-decreased expression of Col1A1 and Vdr (Table 2). These results suggest that NAM can prevent osteoblast dysfunction caused by oxidative stress.
ROS are known to impede mitochondrial function 44. Therefore, we determined whether NAM could regulate ROS-damaged mitochondrial function. We observed that NAM prevented the reductions of mitochondrial respiration and oxygen consumption rate caused by treating MC3T3-E1 osteoblast cells with H2O2 (Fig. 5G-J and Supplementary Fig. 6D-G).
The accumulation of γH2AX foci, involving histone H2A.X phosphorylation, is a biomarker for DNA damage and genotoxicity accompanying the DNA damage response (DDR) 45. As previously reported 15, H2O2 treatment increased the number of MC3T3-E1 osteoblast cells with γH2AX foci (Fig. 5K-L). NAM significantly ameliorated the accumulation of γH2AX foci that occurred at the basal level as well as that induced by H2O2 treatment. Excessive ROS can result in cellular apoptosis accompanied by the release of cytochrome c from damaged mitochondria, which in turn disrupts redox homeostasis in tissues 46,47. We investigated whether NAM alleviates the apoptosis of osteoblasts damaged by acute exposure to excessive H2O2. Treatment with 300 µM H2O2 caused acute oxidative damages that significantly decreased the population of Annexin V- and propidium iodide (PI)-negative healthy live cells, but significantly increased both Annexin V- and PI-positive late apoptotic cells, and PI-positive and Annexin V-negative necrotic cells (Fig. 5M-Q). Co-treatment with H2O2 and NAM significantly increased the population of live cells compared with that upon H2O2–treatment alone. Taking these findings together, NAM prevents osteoblasts from suffering H2O2-induced acute or chronic oxidative damage to mitochondria and DNA.