Notch signaling suppresses glucose metabolism in mesenchymal progenitors to restrict osteoblast differentiation.

Notch signaling critically controls cell fate decisions in mammals, both during embryogenesis and in adults. In the skeleton, Notch suppresses osteoblast differentiation and sustains bone marrow mesenchymal progenitors during postnatal life. Stabilizing mutations of Notch2 cause Hajdu-Cheney syndrome, which is characterized by early-onset osteoporosis in humans, but the mechanism whereby Notch inhibits bone accretion is not fully understood. Here, we report that activation of Notch signaling by either Jagged1 or the Notch2 intracellular domain suppresses glucose metabolism and osteoblast differentiation in primary cultures of bone marrow mesenchymal progenitors. Importantly, deletion of Notch2 in the limb mesenchyme increases both glycolysis and bone formation in the long bones of postnatal mice, whereas pharmacological reduction of glycolysis abrogates excessive bone formation. Mechanistically, Notch reduces the expression of glycolytic and mitochondrial complex I genes, resulting in a decrease in mitochondrial respiration, superoxide production, and AMPK activity. Forced activation of AMPK restores glycolysis in the face of Notch signaling. Thus, suppression of glucose metabolism contributes to the mechanism, whereby Notch restricts osteoblastogenesis from bone marrow mesenchymal progenitors.

Bmp Induces Osteoblast Differentiation through both Smad4 and mTORC1 Signaling.

The bone morphogenetic protein (Bmp) family of secreted molecules has been extensively studied in the context of osteoblast differentiation. However, the intracellular signaling cascades that mediate the osteoblastogenic function of Bmp have not been fully elucidated. By profiling mRNA expression in the bone marrow mesenchymal progenitor cell line ST2, we discover that BMP2 induces not only genes commonly associated with ossification and mineralization but also genes important for general protein synthesis. We define the two groups of genes as mineralization related versus protein anabolism signatures of osteoblasts. Although it induces the expression of several Wnt genes, BMP2 activates the osteogenic program largely independently of de novo Wnt secretion. Remarkably, although Smad4 is necessary for the activation of the mineralization-related genes, it is dispensable for BMP2 to induce the protein anabolism signature, which instead critically depends on the transcription factor Atf4. Upstream of Atf4, BMP2 activates mTORC1 to stimulate protein synthesis, resulting in an endoplasmic reticulum stress response mediated by Perk. Thus, Bmp signaling induces osteoblast differentiation through both Smad4- and mTORC1-dependent mechanisms.

Rictor is required for optimal bone accrual in response to anti-sclerostin therapy in the mouse.

Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictor(f/f), hereafter RiCKO) were subjected to Scl-Ab treatment for 5weeks starting at 4months of age. In vivo micro-computed tomography (µCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-ß-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption.

PTH Promotes Bone Anabolism by Stimulating Aerobic Glycolysis via IGF Signaling.

Teriparatide, a recombinant peptide corresponding to amino acids 1-34 of human parathyroid hormone (PTH), has been an effective bone anabolic drug for over a decade. However, the mechanism whereby PTH stimulates bone formation remains incompletely understood. Here we report that in cultures of osteoblast-lineage cells, PTH stimulates glucose consumption and lactate production in the presence of oxygen, a hallmark of aerobic glycolysis, also known as Warburg effect. Experiments with radioactively labeled glucose demonstrate that PTH suppresses glucose entry into the tricarboxylic acid cycle (TCA cycle). Mechanistically, the increase in aerobic glycolysis is secondary to insulin-like growth factor (Igf) signaling induced by PTH, whereas the metabolic effect of Igf is dependent on activation of mammalian target of rapamycin complex 2 (mTORC2). Importantly, pharmacological perturbation of glycolysis suppresses the bone anabolic effect of intermittent PTH in the mouse. Thus, stimulation of aerobic glycolysis via Igf signaling contributes to bone anabolism in response to PTH.

SMILE upregulated by metformin inhibits the function of androgen receptor in prostate cancer cells.

Metformin, a diabetes drug, has been reported to inhibit the growth of prostate cancer cells. In this study, we investigated the effect and action mechanism of metformin on the function of androgen receptor (AR), a key molecule in the proliferation of prostate cancer cells. Metformin was found to reduce androgen-dependent cell growth and the expression of AR target genes by inhibiting AR function in prostate cancer cells such as LNCaP and C4-2 cells. Interestingly, metformin upregulated the protein level of small heterodimer partner-interacting leucine zipper (SMILE), a coregulator of nuclear receptors, and knockdown of SMILE expression with shRNA abolished the inhibitory effect of metformin on AR function. Further studies revealed that SMILE protein itself suppressed the transactivation of AR, and its ectopic expression resulted in the repressed expression of endogenous AR target genes, PSA and NKX3.1, in LNCaP cells. In addition, SMILE protein physically interacted with AR and competed with the AR coactivator SRC-1 to modulate AR transactivation. As expected, SMILE repressed androgen-dependent growth of LNCaP and C4-2 cells. Taken together, these results suggest that SMILE, which is induced by metformin, functions as a novel AR corepressor and may mediate the inhibitory effect of metformin on androgen-dependent growth of prostate cancer cells.

ERα/E2 signaling suppresses the expression of steroidogenic enzyme genes via cross-talk with orphan nuclear receptor Nur77 in the testes.

Estrogen receptor alpha (ERα) has been reported to affect steroidogenesis in testicular Leydig cells, but its molecular mechanism remains unclear. Here, we investigate the effect of estrogen and ERα on Nur77, a major transcription factor that regulates the expression of steroidogenic enzyme genes. In MA-10 Leydig cells, estradiol (E2) treatment, and interestingly ERα overexpression, suppressed the cAMP-induced and Nur77-activated promoter activity of steroidogenic enzyme genes via the suppression of Nur77 transactivation. ERα physically interacted with Nur77 and inhibited its DNA binding activity. In addition, ERα/E2 signaling decreased Nur77 protein levels. Consistent with the above results, the testicular testosterone level was higher in Leydig cell-specific ERα knock-out mice (ERα(flox/flox)Cyp17iCre) than in wild-type mice (ERα(flox/flox)). Taken together, these results suggest that ERα/E2 signaling controls the Nur77-mediated expression of steroidogenic enzyme genes in Leydig cells. These findings may provide a mechanistic explanation for the local regulation of testicular steroidogenesis by estrogenic compounds and ERα.

Gene expression profile of rat prostate during pubertal growth and maturation.

Temporal gene expression profiling can provide valuable insight into mechanisms of differentiation and may be helpful in laying a foundation for characterization of the molecular aspects of development. Prostate development begins in fetal life and is complete at sexual maturity, and androgen stimulation is both necessary and sufficient for development and maturity of the prostate. In this study, we investigated gene expression profiles of rat prostate at 3 different developmental stages (2 weeks, 3.5 weeks, and 8 weeks), when serum testosterone levels are low, intermediate, and high. Through this analysis, we attempted to narrow down genes whose expression is affected by androgen increase during pubertal growth and maturation of the prostate.

ROS inhibit the expression of testicular steroidogenic enzyme genes via the suppression of Nur77 transactivation.

Steroidogenesis decreases with aging in the testis, whereas the levels of reactive oxygen species (ROS) increase. In addition, ROS have been reported to inhibit testicular steroidogenesis. Here, we investigated the effects of ROS on the transcriptional activity of Nur77, one of the major transcription factors that regulate the expression of steroidogenic enzyme genes. ROS signaling inhibited Nur77 transactivation, which was diminished by either treatment with c-Jun N-terminal kinase (JNK) inhibitor or the expression of a dominant negative form of JNK. This suggests the involvement of JNK signaling, which elevates the expression of c-Jun as well as its phosphorylation in Leydig cells. In transient transfection assays, c-Jun suppressed Nur77 transactivation in a dose-dependent manner. Further studies using c-Jun mutants revealed that the protein level of c-Jun, but not phosphorylation itself, was important for the suppression of Nur77 transactivation. Nur77 directly interacted with c-Jun in vivo, which blocked the DNA binding activity of Nur77. Together, these results suggest that ROS signaling-mediated c-Jun upregulation suppresses the expression of steroidogenic enzyme genes by inhibiting Nur77 transactivation, resulting in the reduction of testicular steroidogenesis. These findings may provide a mechanistic explanation for the age-related decline in testicular steroid hormone production.

Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10 melanoma cells.

Cucurbitacins 1 and 2 were isolated from the root of Trichosanthes kirilowii by tyrosinase inhibitory activity-guided fractionation. Spectroscopic analysis revealed that compounds 1 and 2 were cucurbitacin D and 23,24-dihydro-cucurbitacin D, respectively. Compounds 1 and 2 effectively inhibited the activity of tyrosinase (IC(50) = 0.18 microM and 6.7 microM, respectively), and the synthesis of melanin (IC(50) = 0.16 microM and 7.5 microM, respectively) in B16/F10 melanoma cells.