Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

The AR in bone marrow progenitor cells protects against short-term high-caloric diet-induced weight gain in male mice.

We previously identified a novel pathway of testosterone action via the androgen receptor (AR) in bone marrow mesenchymal precursor cells (BM-PCs) to negatively regulate fat mass and improve metabolic function in male mice. This was achieved using our PC-AR Gene Replacement mouse model in which the AR is only expressed in BM-PCs and deleted in all other tissues. We hypothesise that the markedly reduced fat mass and increased insulin sensitivity of PC-AR Gene Replacements will confer protection from diet-induced overweight and obesity. To test this, 6-week-old male PC-AR Gene Replacements and controls (WT, global-AR knockouts (KOs)) were fed a chow or high-caloric diet (HCD) for 8 or 18 weeks. Following 8 weeks (short-term) of HCD, WT and Global-ARKOs had markedly increased subcutaneous white adipose tissue (WAT) and retroperitoneal visceral adipose tissue (VAT) mass compared to chow-fed controls. In contrast, PC-AR Gene Replacements were resistant to WAT and VAT accumulation following short-term HCD feeding accompanied by fewer large adipocytes and upregulation of expression of the metabolic genes Acaca and Pnlpa2. Following long-term HCD feeding for 18 weeks, the PC-AR Gene Replacements were no longer resistant to increased WAT and VAT adiposity, however, maintained their improved whole-body insulin sensitivity with an increased rate of glucose disappearance and increased glucose uptake into subcutaneous WAT. In conclusion, the action of testosterone via the AR in BM-PCs to negatively regulate fat mass and improve metabolism confers resistance from short-term diet-induced weight gain and partial protection from long-term diet-induced obesity in male mice.

The calcitonin receptor regulates osteocyte lacunae acidity during lactation in mice.

The physiological role of calcitonin, and its receptor, the CTR (or Calcr), has long been debated. We previously provided the first evidence for a physiological role of the CTR to limit maternal bone loss during lactation in mice by a direct action on osteocytes to inhibit osteocytic osteolysis. We now extend these findings to show that CTR gene expression is upregulated two- to three-fold in whole bone of control mice at the end of pregnancy (E18) and lactation (P21) compared to virgin controls. This was associated with an increase in osteoclast activity evidenced by increases in osteoclast surface/bone surface and Dcstamp gene expression. To investigate the mechanism by which the CTR inhibits osteocytic osteolysis, in vivo acidification of the osteocyte lacunae during lactation (P14 days) was assessed using a pH indicator dye. A lower pH was observed in the osteocyte lacunae of lactating Global-CTRKOs compared to controls and was associated with an increase in the gene expression of ATPase H+ transporting V0 subunit D2 (Atp6v0d2) in whole bone of Global-CTRKOs at the end of lacation (P21). To determine whether the CTR is required for the replacement of mineral within the lacunae post-lactation, lacunar area was determined 3 weeks post-weaning. Comparison of the largest 20% of lacunae by area did not differ between Global-CTRKOs and controls post-lactation. These results provide evidence for CTR activation to inhibit osteocytic osteolysis during lactation being mediated by regulating the acidity of the lacunae microenvironment, whilst the CTR is dispensable for replacement of bone mineral within lacunae by osteocytes post-lactation.

Vitamin D receptor expression in mature osteoclasts reduces bone loss due to low dietary calcium intake in male mice.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to respond to active vitamin D (1α, 25-dihydroxyvitamin D3; 1,25(OH)2D3) by regulating cell maturation and activity. However, the in vivo consequences of vitamin D signalling directly within functionally mature osteoclasts is only partially understood. To investigate the in vivo role of VDR in mature osteoclasts, conditional deletion of the VDR under control of the cathepsin K promoter (CtskCre/Vdr-/-), was assessed in 6 and 12-week-old mice, either under normal dietary conditions (NormCaP) or when fed a low calcium (0.03 %), low phosphorous (0.08 %) diet (LowCaP). Splenocytes from CtskCre/Vdr-/- mice were co-cultured with MLO-Y4 osteocyte-like cells to assess the effect on osteoclastogenesis. Six-week-old CtskCre/Vdr-/- mice demonstrated a 10 % decrease in vertebral bone volume (p < 0.05), which was associated with increased osteoclast size (p < 0.05) when compared to Vdrfl/fl control mice. Control mice fed a LowCaP diet exhibited extensive trabecular bone loss associated with increased osteoclast surface, number and size (p < 0.0001). Interestingly, CtskCre/Vdr-/- mice fed a LowCaP diet showed exacerbated loss of bone volume fraction (BV/TV%) and trabecular number (Tb.N), by a further 22 % and 21 %, respectively (p < 0.05), suggesting increased osteoclastic bone resorption activity with the loss of VDR in mature osteoclasts under these conditions. Co-culture of CtskCre/Vdr-/- splenocytes with MLO-Y4 cells increased resulting osteoclast numbers 2.5-fold, which were greater in nuclei density and exhibited increased resorption of dentine compared to osteoclasts derived from Vdrfl/fl splenocyte cultures. These data suggest that in addition to RANKL-mediated osteoclastogenesis, intact VDR signalling is required for the direct regulation of the differentiation and activity of osteoclasts in both in vivo and ex vivo settings.

The androgen receptor in the hypothalamus positively regulates hind-limb muscle mass and voluntary physical activity in adult male mice.

We have previously shown that expression of the androgen receptor (AR) in neurons within the brain positively regulates hind-limb muscle mass and physical activity in male mice. To further investigate the region of the brain responsible for mediating these effects of testosterone and to determine whether they are only important for muscle mass accrual during development or whether they are also important for the maintenance of muscle mass in the adult, we deleted the AR specifically in the hypothalamus of adult male mice (Hyp-ARKOs). Hyp-ARKO mice were generated by bilateral stereotaxic microinjection of an adeno-associated virus (AAV) expressing GFP and iCre recombinase under the control of the e-synapsin promoter into the hypothalamus of 10-week-old exon 3-AR floxed male mice. AR mRNA was deleted by 45% in the hypothalamus of Hyp-ARKOs at 5 weeks post-AAV-eSyn-iCre injection. This led to an increase in the mass of the androgen-dependent organs, seminal vesicles and kidneys, by 30% (P < 0.01) and 10% (P < 0.05) respectively, and an increase in serum luteinizing hormone (LH) by 2 fold (P < 0.05). Whilst the mean value for serum testosterone was higher in the Hyp-ARKOs, this did not reach statistical significance. Despite a phenotype consistent with increased androgen bioactivity in Hyp-ARKOs, which would be expected to increase muscle mass, the mass of the hind-limb muscles, gastrocnemius (Gast) (P = 0.001), extensor digitorum longus (EDL) (P < 0.001) and soleus (Sol) (P < 0.01) were paradoxically decreased by 12-19% compared to controls. Voluntary physical activity was reduced by 65% (P < 0.05) in Hyp-ARKO male mice and was associated with a reduction in gene expression of Drd1a and Maob (P ≤ 0.05) in the hypothalamus, suggesting involvement of the brain dopaminergic system. These data provide compelling evidence that androgen signalling via the AR in the hypothalamus acts to positively regulate the maintenance of hind-limb muscle mass and voluntary activity in adult male mice, independent of AR signalling in peripheral tissues.

The androgen receptor in bone marrow progenitor cells negatively regulates fat mass.

It is well established that testosterone negatively regulates fat mass in humans and mice; however, the mechanism by which testosterone exerts these effects is poorly understood. We and others have shown that deletion of the androgen receptor (AR) in male mice results in a phenotype that mimics the three key clinical aspects of hypogonadism in human males; increased fat mass and decreased bone and muscle mass. We now show that replacement of the Ar gene specifically in mesenchymal progenitor cells (PCs) residing in the bone marrow of Global-ARKO mice, in the absence of the AR in all other tissues (PC-AR Gene Replacements), completely attenuates their increased fat accumulation. Inguinal subcutaneous white adipose tissue and intra-abdominal retroperitoneal visceral adipose tissue depots in PC-AR Gene Replacement mice were 50-80% lower than wild-type (WT) and 75-90% lower than Global-ARKO controls at 12 weeks of age. The marked decrease in subcutaneous and visceral fat mass in PC-AR Gene Replacements was associated with an increase in the number of small adipocytes and a healthier metabolic profile compared to WT controls, characterised by normal serum leptin and elevated serum adiponectin levels. Euglycaemic/hyperinsulinaemic clamp studies reveal that the PC-AR Gene Replacement mice have improved whole-body insulin sensitivity with higher glucose infusion rates compared to WT mice and increased glucose uptake into subcutaneous and intra-abdominal fat. In conclusion, these data provide the first evidence for an action of androgens via the AR in mesenchymal bone marrow PCs to negatively regulate fat mass and improve metabolic function.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr-/- and CtskCre/Cyp27b1-/- mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr-/- mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr-/- female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1-/- mice as well as in Dmp1Cre/VDR-/- mice. When CtskCre/Vdr-/- mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr-/- was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr-/- mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr-/- mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

Androgen Action via the Androgen Receptor in Neurons Within the Brain Positively Regulates Muscle Mass in Male Mice.

Although it is well established that exogenous androgens have anabolic effects on skeletal muscle mass in humans and mice, data from muscle-specific androgen receptor (AR) knockout (ARKO) mice indicate that myocytic expression of the AR is dispensable for hind-limb muscle mass accrual in males. To identify possible indirect actions of androgens via the AR in neurons to regulate muscle, we generated neuron-ARKO mice in which the dominant DNA binding-dependent actions of the AR are deleted in neurons of the cortex, forebrain, hypothalamus, and olfactory bulb. Serum testosterone and luteinizing hormone levels were elevated twofold in neuron-ARKO males compared with wild-type littermates due to disruption of negative feedback to the hypothalamic-pituitary-gonadal axis. Despite this increase in serum testosterone levels, which was expected to increase muscle mass, the mass of the mixed-fiber gastrocnemius (Gast) and the fast-twitch fiber extensor digitorum longus hind-limb muscles was decreased by 10% in neuron-ARKOs at 12 weeks of age, whereas muscle strength and fatigue of the Gast were unaffected. The mass of the soleus muscle, however, which consists of a high proportion of slow-twitch fibers, was unaffected in neuron-ARKOs, demonstrating a stimulatory action of androgens via the AR in neurons to increase the mass of fast-twitch hind-limb muscles. Furthermore, neuron-ARKOs displayed reductions in voluntary and involuntary physical activity by up to 60%. These data provide evidence for a role of androgens via the AR in neurons to positively regulate fast-twitch hind-limb muscle mass and physical activity in male mice.

Muscle-specific androgen receptor deletion shows limited actions in myoblasts but not in myofibers in different muscles in vivo.

The aim of this study was to investigate the direct muscle cell-mediated actions of androgens by comparing two different mouse lines. The cre-loxP system was used to delete the DNA-binding activity of the androgen receptor (AR) in mature myofibers (MCK mAR(ΔZF2)) in one model and the DNA-binding activity of the AR in both proliferating myoblasts and myofibers (α-actin mAR(ΔZF2)) in another model. We found that hind-limb muscle mass was normal in MCK mAR(ΔZF2) mice and that relative mass of only some hind-limb muscles was reduced in α-actin mAR(ΔZF2) mice. This suggests that myoblasts and myofibers are not the major cellular targets mediating the anabolic actions of androgens on male muscle during growth and development. Levator ani muscle mass was decreased in both mouse lines, demonstrating that there is a myofiber-specific effect in this unique androgen-dependent muscle. We found that the pattern of expression of genes including c-myc, Fzd4 and Igf2 is associated with androgen-dependent changes in muscle mass; therefore, these genes are likely to be mediators of anabolic actions of androgens. Further research is required to identify the major targets of androgen actions in muscle, which are likely to include indirect actions via other tissues.

Androgen receptor action in osteoblasts in male mice is dependent on their stage of maturation.

Androgen action via the androgen receptor (AR) is essential for normal skeletal growth and bone maintenance post-puberty in males; however, the molecular and cellular mechanisms by which androgens exert their actions in osteoblasts remains relatively unexplored in vivo. To identify autonomous AR actions in osteoblasts independent of AR signaling in other tissues, we compared the extent to which the bone phenotype of the Global-ARKO mouse was restored by replacing the AR in osteoblasts commencing at either the (1) proliferative or (2) mineralization stage of their maturation. In trabecular bone, androgens stimulated trabecular bone accrual during growth via the AR in proliferating osteoblasts and maintained trabecular bone post-puberty via the AR in mineralizing osteoblasts, with its predominant action being to inhibit bone resorption by decreasing the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG) gene expression. During growth, replacement of the AR in proliferating but not mineralizing osteoblasts of Global-ARKOs was able to partially restore periosteal circumference, supporting the concept that androgen action in cortical bone to increase bone size during growth is mediated via the AR in proliferating osteoblasts. This study provides further significant insight into the mechanism of androgen action via the AR in osteoblasts, demonstrating that it is dependent on the stage of osteoblast maturation.

A Role for the Calcitonin Receptor to Limit Bone Loss During Lactation in Female Mice by Inhibiting Osteocytic Osteolysis.

During lactation, the large transfer of calcium from the mother to the milk is primarily sourced from the maternal skeleton. To determine whether the calcitonin receptor (CTR) plays a physiological role to protect the skeleton from excessive resorption during lactation, we assessed the maternal skeleton of global CTR knockout (CTRKO) and littermate control mice at the end of lactation (postnatal day 21). Micro-computed tomography analyses showed no effect on trabecular or cortical bone in the distal femur and L1 vertebra of maternal global CTR deletion at the end of lactation in global CTRKO mice compared with that in control mice. Bone resorption, as assessed by osteoclast number and activity at the end of lactation, was unaffected by maternal CTR deletion. Cathepsin K, carbonic anhydrase 2, matrix metalloproteinase 13, and receptor activator of nuclear factor-κB ligand mRNA levels, however, were markedly elevated by 3- to 6.5-fold in whole bone of lactating global CTRKO females. Because these genes have been shown to be up-regulated in osteocytes during lactation when osteocytes resorb their surrounding bone matrix, together with their reported expression of the CTR, we determined the osteocyte lacunar area in cortical bone. After lactation, the top 20% of osteocyte lacunar area in global CTRKO mice was 10% larger than the top 20% in control mice. These data are consistent with an increased osteocytic osteolysis in global CTRKO mice during lactation, which is further supported by the increased serum calcium observed in global CTRKO mice after lactation. These results provide evidence for a physiological role for the CTR to protect the maternal skeleton during lactation by a direct action on osteocytes to inhibit osteolysis.

Identification of gene pathways altered by deletion of the androgen receptor specifically in mineralizing osteoblasts and osteocytes in mice.

Androgens play a key role in skeletal growth and maintenance in males and can mediate their actions, at least in part, via the androgen receptor (AR) in osteoblasts. To investigate the mechanisms by which androgens exert their effects via the AR in mineralizing osteoblasts and osteocytes, we identified gene targets/pathways regulated by the AR using targeted gene expression and microarray approaches on bone isolated from mice in which the AR is specifically deleted in mineralizing osteoblasts and osteocytes (mOBL-ARKOs). Gene ontology mining indicated a number of biological processes to be affected in the bones of mOBL-ARKOs including skeletal and muscular system development and carbohydrate metabolism. All genes identified to have altered expression in the bones of mOBL-ARKOs were confirmed by Q-PCR for their androgen responsiveness in an androgen deprivation and replacement mouse model. The osteoblast genes Col1a1 and Bglap and the osteoclast genes Ctsk and RANKL (Tnfs11) were upregulated in the bones of mOBL-ARKOs, consistent with the increased matrix synthesis, mineralization, and bone resorption observed previously in these mice. Of significant interest, we identified genes involved in carbohydrate metabolism (adiponectin and Dpp4) and in growth and development (GH, Tgfb (Tgfb2), Wnt4) as potential targets of androgen action via the AR in mineralizing osteoblasts.