Xiao Wei Zhang

Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Targeting of androgen receptor in bone reveals a lack of androgen anabolic action and inhibition of osteogenesis A model for compartment-specific androgen action in the skeleton

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation.

Androgens regulate body composition in youth and declining testosterone that occurs with aging is associated with muscle wasting, increased fat mass and osteopenia. Transgenic mice with targeted androgen receptor (AR) over‐expression in mesenchymal stem cells (MSC) were generated to explore the role of androgen signaling in the regulation of body composition. Transgenic males, but not females, were shorter and have reduced body weight and visceral fat accumulation. Dual‐energy X‐ray absorptiometry (DXA) revealed significant reductions in fat mass with a reciprocal increase in lean mass, yet no difference in food consumption or locomotor activity was observed. Adipose tissue weight was normal in brown fat but reduced in both gonadal and perirenal depots, and reduced hyperplasia was observed with smaller adipocyte size in visceral and subcutaneous white adipose tissue. Although serum leptin, adiponectin, triglyceride, and insulin levels were no different between the genotypes, intraperitoneal glucose tolerance testing (IPGTT) showed improved glucose clearance in transgenic males. High levels of the AR transgene are detected in MSCs but not in mature fat tissue. Reduced fibroblast colony forming units indicate fewer progenitor cells resident in the marrow in vivo. Precocious expression of glucose transporter 4 (GLUT4), peroxisome proliferator‐activated receptor γ (PPARγ), and CCAAT enhancer‐binding protein α (C/EBPα) was observed in proliferating precursor cultures from transgenic mice compared to controls. In more mature cultures, there was little difference between the genotypes. We propose a mechanism where enhanced androgen sensitivity can alter lineage commitment in vivo to reduce progenitor number and fat development, while increasing the expression of key factors to promote smaller adipocytes with improved glucose clearance. J. Cell. Biochem. 112: 1773–1786, 2011.

Androgen prevents hypogonadal bone loss via inhibition of resorption mediated by mature osteoblasts/osteocytes

Androgen receptor (AR) is expressed throughout the osteoblast lineage. Two different AR transgenic families (AR2.3-tg and AR3.6-tg mice) demonstrating overlapping and distinct expression profiles were employed to assess the effects of enhanced androgen sensitivity to ameliorate hypogonadal loss. Two different paradigms of steroid replacement following orchidectomy (ORX) were used as either preventative or therapeutic therapy. ORX was performed in male wild-type (WT), AR2.3-tg and AR3.6-tg mice at 5 months with immediate DHT replacement (prevention, higher turnover) or at 3 months with DHT treatment delayed for 2 months (therapeutic, lower turnover), both with treatment for the last 6 weeks. Dual energy X-ray absorptiometry (DXA), micro-computed tomography (μCT), and histomorphometry were performed. In the prevention model, ORX significantly reduced BMD and BMC in all genotypes compared to sham and DHT was effective at prevention of osteopenia. In the therapeutic model, all genotypes became osteopenic compared to sham, but after a prolonged hypogonadal period, delayed DHT treatment provided little benefit. μCT analysis of mid-shaft total bone in all genotypes generally showed reductions after ORX. Delayed DHT was ineffective at restoring bone volume in any genotype whereas immediate treatment prevented loss only in AR transgenic mice. Cortical thickness also decreased with ORX but immediate DHT treatment was effective to increase thickness only in WT mice, likely due to expansion of marrow volume in both AR-tg lines. In metabolically highly active cancellous bone, ORX resulted in lower bone volume/tissue volume (BV/TV) in all genotypes, consistent among 3 sites measured. Again with delayed treatment, there was little effect of DHT to restore BV/TV, but when administered at the time of ORX, DHT completely prevented the decrease in cancellous bone in all genotypes. Improvement in cancellous bone architecture was seen with immediate DHT replacement that was enhanced in AR transgenic lines compared to WT. In contrast, there were only modest changes in all genotypes using the delayed treatment paradigm. With ORX in both paradigms, trabecular number was decreased while spacing increased. Thus, androgen therapy is effective for the prevention of endosteal and cancellous osteopenia primarily through its anti-resorptive properties, but shows little anabolic action as a therapeutic strategy to restore bone. Given the similarity in response to androgen treatment in both AR transgenic lines, overlapping expression profiles suggest that the target cells mediating androgen action in vivo are mature osteoblast/osteocytes. Combined, these results demonstrate that in the adult mouse, androgen treatment can reduce bone resorption but has little overall anabolic activity.

Signaling pathways implicated in androgen regulation of endocortical bone

Periosteal expansion is a recognized response to androgen exposure during bone development and in profoundly hypogonadal adults. However, androgen also suppresses endocortical bone formation, indicating that its effects on bone are dichotomous and envelope-specific. In fact, enhanced androgen signaling has been shown to have dramatic detrimental effects on whole bone biomechanical properties in two different transgenic models with skeletally targeted androgen receptor (AR) overexpression. As the mechanisms underlying this response are uncharacterized, we compared patterns of gene expression in periosteum-free cortical bone samples derived from AR-overexpressing transgenic male mice and their wild-type counterparts. We then assessed direct androgen effects in both wild-type and AR-overexpressing osteoblasts in primary culture. Among major signaling pathways associated with bone formation, focused quantitative RT-PCR (qPCR) array-based analysis of endocortical bone gene expression from wild-type vs. transgenic males identified the transforming growth factor-beta (TGF-beta) superfamily and bone morphogenetic protein (BMP) signaling as significantly altered by androgen in vivo. Bioinformatic analyses indicated proliferation, osteoblast differentiation and mineralization as major biological processes affected. Consistent with the in vivo array data and bioinformatic analyses, inhibition of differentiation observed with androgen exposure was reduced by exogenous BMP2 treatment of AR-overexpressing cultures to stimulate BMP signaling, confirming array pathway analysis. In addition, nonaromatizable dihydrotestosterone (DHT) inhibited osteoblast proliferation, differentiation and several indices of mineralization, including mineral accumulation and mineralized nodule formation in primary cultures from both wild-type and AR-transgenic mice. These findings identify a molecular mechanism based on altered BMP signaling that contributes to androgen inhibition of osteoblast differentiation and mineralization. Such detrimental effects of androgen on osteoblast function may underlie the generally disappointing results of androgen therapy.

Reduced G-protein-coupled-receptor kinase 2 activity results in impairment of osteoblast function

Rapid phosphorylation of many G-protein-coupled receptors (GPCRs) by G-protein-coupled receptor kinases (GRKs) accompanies stimulus-driven desensitization. Recent evidence suggests that GRKs and their associated arresting proteins, beta-arrestins, function as essential elements in the GPCR-mediated mitogen-activated protein (MAP) kinase signaling cascade. We investigated the interaction between GRKs and MAP kinase activation by growth factors in UMR 106-H5 osteoblastic cells stably expressing a dominant negative mutant of GRK2 (K220R). Expression of K220R in osteoblastic cells results in reduced cellular proliferation, both basally and in response to insulin-like growth factor-1 (IGF-1), and blunting of IGF-1- and EGF-induced MAP kinase activation. Reduced MAP kinase activation is not associated with alterations in IGF-1-receptor autophosphorylation. Both a constitutively active Ras mutant and PMA fully activate MAP kinase in K220R cells. We found that disruption of the GRK2 gene results in: (1) reduced osteoblast proliferation in response to growth factors, and (2) impaired receptor tyrosine kinase activation of mitogenic signaling pathways. Thus, GRK2 may regulate growth factor responsiveness in osteoblasts by modulating multiprotein complex formation following receptor tyrosine kinase activation.

Bone vs. fat: Embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male and not in female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal and in not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSC profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest.

Stem cell activation in adults can reverse detrimental changes in body composition to reduce fat and increase lean mass in both sexes

Detrimental changes in body composition are often associated with declining levels of testosterone. Here, we evaluated the notion that multipotent mesenchymal stem cells, that give rise to both fat and muscle tissue, can play a significant role to alter existing body composition in the adult. Transgenic mice with targeted androgen receptor (AR) overexpression in stem cells were employed. Wild‐type littermate and AR‐transgenic male and female mice were gonadectomized and left untreated for 2 months. After the hypogonadal period, mice were then treated with 5α‐dihydrotestosterone (DHT) for 6 weeks. After orchidectomy (ORX), wild‐type males have reduced lean mass and increased fat mass compared to shams. DHT treatment was beneficial to partially restore body composition. In wild‐type females, ovariectomy (OVX) produced a similar change but there was no improvement with DHT. In targeted AR transgenic mice, DHT treatment increased lean and reduced fat mass to sham levels. In contrast to wild‐type females, DHT treatment in female transgenic mice significantly ameliorated the increased fat and decreased lean mass changes that result after OVX. Our results show that DHT administration reduces fat mass and increases lean mass in wild‐type males but not females, indicating that wild‐type females are not as sensitive to androgen treatment. Because both male and female transgenic mice are more responsive than wild‐type, results suggest that body composition remains linked to stem cell fate in the adult and that targeted androgen signaling in stem cells can play a significant role to reverse detrimental changes in body composition in both sexes. J. Cell. Biochem. 112: 3638–3647, 2011.