Oxidative stress plays an important role in the pathogenesis of aging-related osteoporosis through the increased bone resorption or reduced bone formation. Melatonin, which can exert beneficial actions through antioxidant, anti-inflammatory, and bone-preserving effects, shows promise in preventing oxidative stress-inhibited osteogenesis. However, specific mechanisms by which melatonin rescues oxidative stress-inhibited osteogenesis of human mesenchymal stem cells (MSCs) have not been fully elucidated yet. We therefore investigated whether activation of AMPK by melatonin regulates the antagonistic crosstalk between oxidative stress and osteogenic differentiation in human MSCs. Melatonin treatment significantly enhanced osteogenic differentiation of human MSCs through activation of AMPK and upregulation of FOXO3a and RUNX2 which were known as master transcription factors responsible for the mechanistic link between oxidative stress and osteogenic phenotype. Osteogenic differentiation determined by calcium deposition was significantly increased by melatonin treatment against oxidative stress. In addition, melatonin treatment reconstituted activation of AMPK and expression of FOXO3a and RUNX2 inhibited by oxidative stress. Overall, these results demonstrate that melatonin enhances osteogenic differentiation of human MSCs and restores oxidative stress-inhibited osteogenesis through AMPK activation in human MSCs, suggesting that activation of AMPK by melatonin may represent a promising new therapeutic strategy for treating metabolic bone diseases such as osteoporosis.

In the Wnt canonical pathway, Wnt3A has been known to stabilize β-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of β-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3β at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to β-catenin via the N-terminal domain of β-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/β-catenin complex. Notably, p-Tyr42 RhoA as well as β-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

Mesenchymal stem cells (MSCs) have the potential to differentiate into specialized cell lineages such as osteoblasts and adipocytes in vitro. There exists a reciprocal relationship between osteogenic and adipogenic differentiation of MSCs that an osteogenic phenotype occurs at the expense of an adipogenic phenotype and vice versa, which in turn influence one another's phenotype through negative feedback loops. Thus, it is important to understand what signaling molecules modulate the lineage commitment of MSCs. Protein kinase C (PKC) plays a central role in cellular signal transduction for mediating diverse biological functions, and dysregulation of PKC activity is involved in various metabolic diseases including cancer, diabetes, and heart disease. Although the role of individual PKC isoforms has been investigated in various fields, the potential role of PKC in bone metabolism is not completely understood. In this study, we investigated the potential role of PKCδ in osteogenic lineage commitment of human bone marrow-derived mesenchymal stem cells (hBMSCs). We observed that expression and phosphorylation of PKCδ were increased during osteogenic differentiation of hBMSCs. Pharmacological inhibition and genetic ablation of PKCδ in hBMSCs resulted in a significant attenuation of osteogenic differentiation as evidenced by reduced ALP activity and ECM mineralization, as well as down-regulation of the expression of osteoblast-specific genes. These effects were also accompanied by induction of adipogenic differentiation and up-regulation of the expression of adipocyte-specific genes involved in lipid synthesis in osteogenic induction of hBMSCs. Additionally, the activation of AMPK, which is a key cellular energy sensor, induced osteogenesis of hBMSCs. However, the inhibition of AMPK activity by compound C did not affect the activation of PKCδ at all, indicating that there is no direct correlation between AMPK and PKCδ in osteogenesis of hBMSCs. These results suggest that PKCδ is a critical regulator for the balance between osteogenesis and adipogenesis of hBMSCs and thus has a potential novel therapeutic target for the treatment of metabolic bone diseases.

Upregulation of the expression of Delta/notch-like epidermal growth factor-related receptor (DNER) and its oncogenic role have been reported in several cancers, including gastric, breast, and prostate cancers. This study aimed to investigate the oncogenic role of DNER and the mechanisms behind its oncogenic role in gastric cancer. Analysis of the RNASeq data of gastric cancer tissues obtained from the TCGA database revealed that the expression of DNER was associated with the pathology of advanced gastric cancer and the prognosis of patients. DNER expression was increased upon stem cell-enriching cancer spheroid culture. Knockdown of DNER expression inhibited cell proliferation and invasion, induced apoptosis, enhanced chemosensitivity, and decreased spheroid formation of SNU-638 gastric cancer cells. DNER silencing elevated the expression of p53, p21^cip/waf, and p27, and increased G₁ phase cells at the expense of S phase cells. Knockdown of p21^cip/waf expression in the DNER-silenced cells partially restored cell viability and S phase progression. DNER silencing also induced the apoptosis of SNU-638 cells. While both cleaved caspases-8 and 9 were detected in adherent cells, only cleaved caspase-8 was found to have increased in spheroid-cultured cells, suggesting a distinct activation pattern of caspase activation depending on the growth condition. Knockdown of p53 expression rescued the DNER-silenced cells from apoptosis and partially restored cell viability. In contrast, overexpression of the Notch intracellular domain (NICD) decreased the expression of p53, p21^cip/waf, and cleaved caspase-3 in DNER-silenced cells. Moreover, NICD expression fully reverted the cell viability reduction, arrest in the G₁ phase, and elevated apoptosis caused by DNER silencing, thereby suggesting activation of Notch signaling by DNER. Expression of a membrane-unbound mutant of mDNER also decreased cell viability and induced apoptosis. On the other hand, TGF-β signals were found to be involved in DNER expression in both adherent and spheroid-cultured cells. DNER could therefore be a link connecting TGF-β signaling to Notch signaling. Taken together, DNER regulates cell proliferation, survival, and invasive capacity of the gastric cancer cells through the activation of Notch signaling, which may facilitate tumor progression into an advanced stage. This study provides evidences suggesting that DNER could be a potential prognostic marker, a therapeutic target, and a drug candidate in the form of a cell-free mutant.