JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Low-intensity pulsed ultrasound promotes bone morphogenic protein 9-induced osteogenesis and suppresses inhibitory effects of inflammatory cytokines on cellular responses via Rho-associated kinase 1 in human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low-intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9-induced osteogenic differentiation of PDLFs. In contrast, BMP2-induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS-induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS-PG), we also simultaneously treated PDLFs with LIPUS and LPS-PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK-binding kinase 1, and interferon regulatory factor 3 in LPS-PG-stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa-B ligand, and also diminished IL-1ß and tumor necrosis factor a (TNFa)-induced inflammatory reactions. Phosphorylation of Rho-associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1-specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS-PG-stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS-PG and IL-1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS-PG, IL-1ß, and TNFa and also promotes BMP9-induced osteogenesis through ROCK1 in PDLFs.

Maternal Exercise-Induced SOD3 Reverses the Deleterious Effects of Maternal High-Fat Diet on Offspring Metabolism Through Stabilization of H3K4me3 and Protection Against WDR82 Carbonylation.

Preclinical studies reveal maternal exercise as a promising intervention to reduce the transmission of multigenerational metabolic dysfunction caused by maternal obesity. The benefits of maternal exercise on offspring health may arise from multiple factors and have recently been shown to involve DNA demethylation of critical hepatic genes leading to enhanced glucose metabolism in offspring. Histone modification is another epigenetic regulator, yet the effects of maternal obesity and exercise on histone methylation in offspring are not known. Here, we find that maternal high-fat diet (HFD; 60% kcal from fat) induced dysregulation of offspring liver glucose metabolism in C57BL/6 mice through a mechanism involving increased reactive oxygen species, WD repeat-containing 82 (WDR82) carbonylation, and inactivation of histone H3 lysine 4 (H3K4) methyltransferase leading to decreased H3K4me3 at the promoters of glucose metabolic genes. Remarkably, the entire signal was restored if the HFD-fed dams had exercised during pregnancy. WDR82 overexpression in hepatoblasts mimicked the effects of maternal exercise on H3K4me3 levels. Placental superoxide dismutase 3 (SOD3), but not antioxidant treatment with N-acetylcysteine was necessary for the regulation of H3K4me3, gene expression, and glucose metabolism. Maternal exercise regulates a multicomponent epigenetic system in the fetal liver resulting in the transmission of the benefits of exercise to offspring.

Exercise Training Promotes Sex-Specific Adaptations in Mouse Inguinal White Adipose Tissue.

Recent studies demonstrate that adaptations to white adipose tissue (WAT) are important components of the beneficial effects of exercise training on metabolic health. Exercise training favorably alters the phenotype of subcutaneous inguinal WAT (iWAT) in male mice, including decreasing fat mass, improving mitochondrial function, inducing beiging, and stimulating the secretion of adipokines. In this study, we find that despite performing more voluntary wheel running compared with males, these adaptations do not occur in the iWAT of female mice. Consistent with sex-specific adaptations, we report that mRNA expression of androgen receptor coactivators is upregulated in iWAT from trained male mice and that testosterone treatment of primary adipocytes derived from the iWAT of male, but not female mice, phenocopies exercise-induced metabolic adaptations. Sex specificity also occurs in the secretome profile, as we identify cysteine-rich secretory protein 1 (Crisp1) as a novel adipokine that is only secreted from male iWAT in response to exercise. Crisp1 expression is upregulated by testosterone and functions to increase glucose and fatty acid uptake. Our finding that adaptations to iWAT with exercise training are dramatically greater in male mice has potential clinical implications for understanding the different metabolic response to exercise training in males and females and demonstrates the importance of investigating both sexes in studies of adipose tissue biology.

Placental superoxide dismutase 3 mediates benefits of maternal exercise on offspring health.

Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that maternal exercise improves the metabolic health of offspring, and here, we demonstrate that this occurs through a vitamin D receptor-mediated increase in placental superoxide dismutase 3 (SOD3) expression and secretion. SOD3 activates an AMPK/TET signaling axis in fetal offspring liver, resulting in DNA demethylation at the promoters of glucose metabolic genes, enhancing liver function, and improving glucose tolerance. In humans, SOD3 is upregulated in serum and placenta from physically active pregnant women. The discovery of maternal exercise-induced cross talk between placenta-derived SOD3 and offspring liver provides a central mechanism for improved offspring metabolic health. These findings may lead to novel therapeutic approaches to limit the transmission of metabolic disease to the next generation.