A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor ß-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as ß-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because ß-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

Cobinamide is a strong and versatile antioxidant that overcomes oxidative stress in cells, flies, and diabetic mice.

Increased oxidative stress underlies a variety of diseases, including diabetes. Here, we show that the cobalamin/vitamin B12 analog cobinamide is a strong and multifaceted antioxidant, neutralizing superoxide, hydrogen peroxide, and peroxynitrite, with apparent rate constants of 1.9 × 108, 3.7 × 104, and 6.3 × 106 M-1 s-1, respectively, for cobinamide with the cobalt in the +2 oxidation state. Cobinamide with the cobalt in the +3 oxidation state yielded apparent rate constants of 1.1 × 108 and 8.0 × 102 M-1 s-1 for superoxide and hydrogen peroxide, respectively. In mammalian cells and Drosophila melanogaster, cobinamide outperformed cobalamin and two well-known antioxidants, imisopasem manganese and manganese(III)tetrakis(4-benzoic acid)porphyrin, in reducing oxidative stress as evidenced by: (i) decreased mitochondrial superoxide and return of the mitochondrial membrane potential in rotenone- and antimycin A-exposed H9c2 rat cardiomyocytes; (ii) reduced JNK phosphorylation in hydrogen-peroxide-treated H9c2 cells; (iii) increased growth in paraquat-exposed COS-7 fibroblasts; and (iv) improved survival in paraquat-treated flies. In diabetic mice, cobinamide administered in the animals' drinking water completely prevented an increase in lipid and protein oxidation, DNA damage, and fibrosis in the heart. Cobinamide is a promising new antioxidant that has potential use in diseases with heightened oxidative stress.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression.

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.

Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing.

Bone fractures are a major cause of morbidity and mortality, particularly in patients with diabetes, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway's role in bone regeneration is unknown. Here, we used a mouse cortical-defect model to simulate bone fractures and studied osteoblast-specific PKG1-knockout and diabetic mice. The knockout mice had normal bone microarchitecture but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knockout mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.

Long acting GLP-1 analog liraglutide ameliorates skeletal muscle atrophy in rodents.

BACKGROUND: Skeletal muscle atrophy is characterized by muscle wasting with partial or complete functional loss. Skeletal muscle atrophy severely affects the quality of life and currently, there is no available therapy except for spinal muscular atrophy. OBJECTIVE: Drug repositioning is a promising strategy that reduces cost and time due to prior availability of safety and toxicity details. Here we investigated myogenic and anti-atrophy effects of glucagon-like peptide-1 (GLP-1) analog liraglutide. METHODS: We used several in vitro atrophy models in C2C12 cells and in vivo models in Sprague Dawley rats to study Liraglutide's efficacy. Western blotting was used to assess cAMP-dependent signaling pathways specifically activated by liraglutide. Therapeutic efficacy of liraglutide was investigated by histological analysis of transverse muscle sections followed by morphometry. Myogenic capacity was investigated by immunoblotting for myogenic factors. RESULTS: Liraglutide induced myogenesis in C2C12 myoblasts through GLP-1 receptor via a cAMP-dependent complex network of signaling events involving protein kinase A, phosphoinositide 3-kinase/protein kinase B, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Liraglutide imparted protection against freeze injury, denervation, and dexamethasone -induced skeletal muscle atrophy and improved muscular function in all these models. In a therapeutic model, liraglutide restored myofibrillar architecture in ovariectomy-induced atrophy. Anti-atrophy actions of liraglutide involved suppression of atrogene expression and enhancement in expression of myogenic factors. CONCLUSION: Liraglutide imparted protection and restored myofibrillar architecture in diverse models of muscle atrophy. Given its potent anti-atrophy, and recently reported osteoanabolic effects, we propose liraglutide's clinical evaluation in skeletal muscle atrophy and musculoskeletal disorders associated with diverse pathologies.

Stimulation of liver IGF-1 expression promotes peak bone mass achievement in growing rats: a study with pomegranate seed oil.

Peak bone mass (PBM) achieved at adulthood is a strong determinant of future onset of osteoporosis, and maximizing it is one of the strategies to combat the disease. Recently, pomegranate seed oil (PSO) has been shown to have bone-sparing effect in ovariectomized mice. However, its effect on growing skeleton and its molecular mechanism remain unclear. In the present study, we evaluated the effect of PSO on PBM in growing rats and associated mechanism of action. PSO was given at various doses to 21-day-old growing rats for 90 days by oral gavage. The changes in bone parameters were assessed by micro-computed tomography and histology. Enzyme-linked immunosorbent assay was performed to analyze the levels of serum insulin-like growth factor type 1 (IGF-1). Western blotting from bone and liver tissues was done. Chromatin immunoprecipitation assay was performed to study the histone acetylation levels at IGF-1 gene. The results of the study show that PSO treatment significantly increases bone length, bone formation rate, biomechanical parameters, bone mineral density and bone microarchitecture along with enhancing muscle and brown fat mass. This effect was due to the increased serum levels of IGF-1 and stimulation of its signaling in the bones. Studies focusing on acetylation of histones in the liver, the major site of IGF-1 synthesis, showed enrichment of acetylated H3K9 and H3K14 at IGF-1 gene promoter and body. Further, the increased acetylation at H3K9 and H3K14 was associated with a reduced HDAC1 protein level. Together, our data suggest that PSO promotes the PBM achievement via increased IGF-1 expression in liver and IGF-1 signaling in bone.

Globular adiponectin reverses osteo-sarcopenia and altered body composition in ovariectomized rats.

Adiponectin regulates various metabolic processes including glucose flux, lipid breakdown and insulin response. We recently reported that adiponectin receptor1 (adipoR1) activation by a small molecule reverses osteopenia in leptin receptor deficient db/db (diabetic) mice. However, the role of adiponectin in bone metabolism under the setting of post-menopausal (estrogen-deficiency) osteopenia and associated metabolic derangements has not been studied. Here, we studied the therapeutic effect of the globular form of adiponectin (gAd), which is predominantly an adipoR1 agonist, in aged ovariectomized (OVX) rats and compared it with standard-of-care anti-osteoporosis drugs. In OVX rats with established osteopenia, gAd completely restored BMD and load bearing capacity and improved bone quality. Skeletal effects of gAd were comparable to PTH (osteoanabolic) but better than alendronate (anti-catabolic). Both osteoanabolic and anti-catabolic mechanisms led to the anti-osteoporosis effect of gAd. In cultured osteoblasts and bones, gAd increased a) adipoR1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression to promote mitochondrial respiration, which likely fueled osteoblast differentiation, b) suppressed sclerostin (a wnt antagonist) in a sirtuin1-dependent manner and c) decreased receptor-activator of nuclear factor κB ligand (RANKL) to achieve its anti-catabolic effect. The OVX-induced sarcopenia and insulin resistance were also improved by gAd. We conclude that gAd has therapeutic efficacy in estrogen deficiency-induced osteoporosis, sarcopenia and insulin resistance and hold metabolic disease modifying potential in postmenopausal women.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/ß-catenin signaling.

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/ß-catenin pathway assessed by phosphorylation of GSK-3ß. Over-expressing of constitutively active GSK-3ß (caGSK-3ß) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3ß over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.

Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect.

Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress.

BMP signaling is required for adult skeletal homeostasis and mediates bone anabolic action of parathyroid hormone.

Bmp2 and Bmp4 genes were ablated in adult mice (KO) using a conditional gene knockout technology. Bones were evaluated by microcomputed tomography (µCT), bone strength tester, histomorphometry and serum biochemical markers of bone turnover. Drill-hole was made at femur metaphysis and bone regeneration in the hole site was measured by calcein binding and µCT. Mice were either sham operated (ovary intact) or ovariectomized (OVX), and treated with human parathyroid hormone (PTH), 17ß-estradiol (E2) or vehicle. KO mice displayed trabecular bone loss, diminished osteoid formation and reduced biomechanical strength compared with control (expressing Bmp2 and Bmp4). Both osteoblast and osteoclast functions were impaired in KO mice. Bone histomorphomtery and serum parameters established a low turnover bone loss in KO mice. Bone regeneration at the drill-hole site in KO mice was lower than control. However, deletion of Bmp2 gene alone had no effect on skeleton, an outcome similar to that reported previously for deletion of Bmp4 gene. Both PTH and E2 resulted in skeletal preservation in control-OVX, whereas in KO-OVX, E2 but not PTH was effective which suggested that the skeletal action of PTH required Bmp ligands but E2 did not. To determine cellular effects of Bmp2 and Bmp4, we used bone marrow stromal cells in which PTH but not E2 stimulated both Bmp2 and Bmp4 synthesis leading to increased Smad1/5 phosphorylation. Taken together, we conclude that Bmp2 and Bmp4 are essential for maintaining adult skeletal homeostasis and mediating the anabolic action of PTH.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats.

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.

The thiocarbamate disulphide drug, disulfiram induces osteopenia in rats by inhibition of osteoblast function due to suppression of acetaldehyde dehydrogenase activity.

Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function.