Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1-ablated mice. Accordingly, Gja1-mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) "beiging" in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter leads to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high fat diet (HFD) in both sexes. These protective effects from obesogenic diet are related to increased locomotion, fuel utilization, energy expenditure, non-shivering thermogenesis, and better glucose tolerance in conditionally Gja1 ablated mice. Accordingly, Gja1 mutant mice exhibit reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis and decreased whitening under HFD. This metabolic phenotype is not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 has a hitherto unknown function in adipocyte progenitors or other targeted cells, resulting in restrained energy expenditures and fat accumulation. These results disclose an hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Gain-of-Function Lrp5 Mutation Improves Bone Mass and Strength and Delays Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes.

High fracture rate and high circulating levels of the Wnt inhibitor, sclerostin, have been reported in diabetic patients. We studied the effects of Wnt signaling activation on bone health in a mouse model of insulin-deficient diabetes. We introduced the sclerostin-resistant Lrp5A214V mutation, associated with high bone mass, in mice carrying the Ins2Akita mutation (Akita), which results in loss of beta cells, insulin deficiency, and diabetes in males. Akita mice accrue less trabecular bone mass with age relative to wild type (WT). Double heterozygous Lrp5A214V /Akita mutants have high trabecular bone mass and cortical thickness relative to WT animals, as do Lrp5A214V single mutants. Likewise, the Lrp5A214V mutation prevents deterioration of biomechanical properties occurring in Akita mice. Notably, Lrp5A214V /Akita mice develop fasting hyperglycemia and glucose intolerance with a delay relative to Akita mice (7 to 8 vs. 5 to 6 weeks, respectively), despite lack of insulin production in both groups by 6 weeks of age. Although insulin sensitivity is partially preserved in double heterozygous Lrp5A214V /Akita relative to Akita mutants up to 30 weeks of age, insulin-dependent phosphorylated protein kinase B (pAKT) activation in vitro is not altered by the Lrp5A214V mutation. Although white adipose tissue depots are equally reduced in both compound and Akita mice, the Lrp5A214V mutation prevents brown adipose tissue whitening that occurs in Akita mice. Thus, hyperactivation of Lrp5-dependent signaling fully protects bone mass and strength in prolonged hyperglycemia and improves peripheral glucose metabolism in an insulin independent manner. Wnt signaling activation represents an ideal therapeutic approach for diabetic patients at high risk of fracture. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Glucose metabolism induced by Bmp signaling is essential for murine skeletal development.

Much of the mammalian skeleton originates from a cartilage template eventually replaced by bone via endochondral ossification. Despite much knowledge about growth factors and nuclear proteins in skeletal development, little is understood about the role of metabolic regulation. Here we report that genetic deletion of the glucose transporter Glut1 (Slc2a1), either before or after the onset of chondrogenesis in the limb, severely impairs chondrocyte proliferation and hypertrophy, resulting in dramatic shortening of the limbs. The cartilage defects are reminiscent to those caused by deficiency in Bmp signaling. Importantly, deletion of Bmpr1a in chondrocytes markedly reduces Glut1 levels in vivo, whereas recombinant BMP2 increases Glut1 mRNA and protein levels, boosting glucose metabolism in primary chondrocytes. Biochemical studies identify a Bmp-mTORC1-Hif1a signaling cascade resulting in upregulation of Glut1 in chondrocytes. The results therefore uncover a hitherto unknown connection between Bmp signaling and glucose metabolism in the regulation of cartilage development.

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.

Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and stimulating osteoblast activity in mouse.

Exogenous bone morphogenetic proteins (Bmp) are well known to induce ectopic bone formation, but the physiological effect of Bmp signaling on normal bone is not completely understood. By deleting the receptor Bmpr1a in osteoblast lineage cells with Dmp1-Cre, we observed a dramatic increase in trabecular bone mass in postnatal mice, which was due to a marked increase in osteoblast number that was likely to be driven by hyperproliferation of Sp7(+) preosteoblasts. Similarly, inducible deletion of Bmpr1a in Sp7(+) cells specifically in postnatal mice increased trabecular bone mass. However, deletion of Smad4 by the same approaches had only a minor effect, indicating that Bmpr1a signaling suppresses trabecular bone formation through effectors beyond Smad4. Besides increasing osteoblast number in the trabecular bone, deletion of Bmpr1a by Dmp1-Cre also notably reduced osteoblast activity, resulting in attenuation of periosteal bone growth. The impairment in osteoblast activity correlated with reduced mTORC1 signaling in vivo, whereas inhibition of mTORC1 activity abolished the induction of protein anabolism genes by BMP2 treatment in vitro. Thus, physiological Bmpr1a signaling in bone exerts a dual function in both restricting preosteoblast proliferation and promoting osteoblast activity.

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.

Physiological notch signaling maintains bone homeostasis via RBPjk and Hey upstream of NFATc1.

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.

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.

Synthesis and antifungal evaluation of 6-hydroxy-1H-carbazole-1,4(9H)-diones.

6-Hydroxy-1H-carbazole-1,4(9H)-diones were synthesized and tested for in vitro antifungal activity against two pathogenic strains of fungi. Among them tested, many compounds showed good antifungal activity. The results suggest that 6-hydroxy-1H-carbazole-1,4(9H)-diones would be potent antifungal agents.

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.

3D-QSAR studies of heterocyclic quinones with inhibitory activity on vascular smooth muscle cell proliferation using pharmacophore-based alignment.

The abnormal proliferation and migration of vascular smooth muscle cells (SMCs) play an important role in the pathology of coronary artery atherosclerosis and restenosis following angioplasty. It was reported that some heterocyclic quinone derivatives such as 6-arylamino-quinoxaline-5,8-diones and 6-arylamino-1H-benzo[d]imidazole-4,7-diones have inhibitory activity on rat aortic smooth muscle cell (RAoSMC) proliferation. To understand the structural basis for antiproliferative activity to design more potent agents, we generated pharmacophore models of representative molecules with high activity using Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database (GALAHAD) and aligned a series of compounds to the selected pharmacophore model, then performed three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). Good cross-validated correlations were obtained with CoMFA (resulting in q(2) of 0.734 and r(2) of 0.947) and CoMSIA (resulting in q(2) of 0.736 and r(2) of 0.913). The IC(50) values of the heterocyclic quinone derivatives on RAoSMC exhibited a strong correlation with steric and hydrophobic fields of the 3D structure of the molecules, resulting in the reliable prediction of inhibitory activity of the series of compounds.

Design, synthesis and evaluation of 2-phenyl-1H-benzo[d]imidazole-4,7-diones as vascular smooth muscle cell proliferation inhibitors.

A series of 2-phenyl-1H-benzo[d]imidazole-4,7-diones were synthesized and tested for their inhibitory activity on the PDGF-stimulated proliferation of rat aortic vascular smooth muscle cells. Among the tested compounds, 6-arylthio-5-chloro-2-phenyl-1H-benzo[d]imidazole-4,7-diones exhibited an potent antiproliferative activity.

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.

Effects of the aqueous extract of Epimedii Herba on the induction of oral tolerance in mice.

We investigated the effects of the aqueous extract of Epimedii Herba (AEEH) on the induction of oral tolerance. Oral tolerance was induced in mice by giving an oral administration of 20 mg ovalbumin (OVA) 7 d before immunization with the antigen. AEEH at 40 mg/kg was given orally daily for 6 d from 24 h after the feeding of OVA. The results showed that oral administration of OVA greatly suppressed total serum and antigen-specific immunoglobulin (Ig) levels, phagocytic activity and delayed-type hypersensitivity (DTH) reaction to the antigen. The suppression of these immune responses to OVA by the oral antigen was associated with a marked reduction of the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) from spleen cells. However, AEEH treatment significantly blocked the suppression of total serum and antigen-specific IgG2a antibodies, phagocytic activity and DTH response by the oral OVA. The suppression of IFN-y production by the oral antigen was also greatly decreased by AEEH treatment. Therefore, AEEH appears to be effective in preventing the induction of oral tolerance to OVA.