Rachel A. Davey

Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo

Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic β cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/− mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/− mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/− and Amylin +/− mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.

Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice

To identify mechanisms of anabolic androgen action in muscle, we generated male and female genomic androgen receptor (AR) knockout (ARKO) mice, and characterized muscle mass, contractile function, and gene expression. Muscle mass is decreased in ARKO males, but normal in ARKO females. The levator ani muscle, which fails to develop in normal females, is also absent in ARKO males. Force production is decreased from fast‐twitch ARKO male muscle, and slow‐twitch muscle has increased fatigue resistance. Microarray analysis shows up‐regulation of genes encoding slow‐twitch muscle contractile proteins. Realtime PCR confirms that expression of genes encoding polyamine biosynthetic enzymes, ornithine decarboxylase (Odc1), and S‐adenosylmethionine decarboxylase (Amd1), is reduced in ARKO muscle, suggesting androgens act through regulation of polyamine biosynthesis. Altered expression of regulators of myoblast progression from proliferation to terminal differentiation suggests androgens also promote muscle growth by maintaining myoblasts in the proliferate state and delaying differentiation (increased Cdkn1c and Igf2, decreased Itg1bp3). A similar pattern of gene expression is observed in orchidectomized male mice, during androgen withdrawal‐dependent muscle atrophy. In conclusion, androgens are not required for peak muscle mass in females. In males, androgens act through the AR to regulate multiple gene pathways that control muscle mass, strength, and fatigue resistance.—MacLean, H. E., Maria Chiu, W. S., Notini, A. J., Axell, A.‐M., Davey, R. A., McManus, J. F., Ma, C., Plant, D. R., Lynch, G. S., Zajac, J. D. Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB J. 22, 2676–2689 (2008)

Osteoblast deletion of exon 3 of the androgen receptor gene results in trabecular bone loss in adult male mice.

The mechanism of androgen action on bone was studied in male mice with the AR deleted in mature osteoblasts. These mice had decreased trabecular bone volume associated with a decrease in trabecular number, suggesting that androgens may act directly on osteoblasts to maintain trabecular bone.

Mineralization and bone resorption are regulated by the androgen receptor in male mice.

Androgens play a key role in skeletal growth and bone maintenance; however, their mechanism of action remains unclear. To address this, we selectively deleted the androgen receptor (AR) in terminally differentiated, mineralizing osteoblasts using the Cre/loxP system in mice (osteocalcin‐Cre AR knockouts [mOBL‐ARKOs]). Male mOBL‐ARKOs had decreased femoral trabecular bone volume compared with littermate controls because of a reduction in trabecular number at 6, 12, and 24 wk of age, indicative of increased bone resorption. The effects of AR inactivation in mineralizing osteoblasts was most marked in the young mutant mice at 6 wk of age when rates of bone turnover are high, with a 35% reduction in trabecular bone volume, decreased cortical thickness, and abnormalities in the mineralization of bone matrix, characterized by increased unmineralized bone matrix and a decrease in the amount of mineralizing surface. This impairment in bone architecture in the mOBL‐ARKOs persisted throughout adulthood despite an unexpected compensatory increase in osteoblast activity. Our findings show that androgens act through the AR in mineralizing osteoblasts to maintain bone by regulating bone resorption and the coordination of bone matrix synthesis and mineralization, and that this action is most important during times of bone accrual and high rates of bone remodeling.

Calcitonin Receptor Plays a Physiological Role to Protect Against Hypercalcemia in Mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]‐induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

Osteoblast Gene Expression in Rat Long Bones: Effects of Ovariectomy and Dihydrotestosterone on mRNA Levels

Abstract. The steroid sex hormones exert major effects on bone formation although the molecular events associated with their activity remain unclear. We have investigated the effects of ovariectomy and dihydrotestosterone (DHT) administration to both sham-operated and ovariectomized (ovx) rats on the bone mRNA levels of osteoblast genes. Rats were randomly allocated to either sham or ovariectomy operations and were administered either vehicle or 40 mg/kg body weight DHT by silastic tube implants at the time of operation for 8 weeks, at which time they were killed and total RNA was extracted from the long bones. Northern blot analysis indicated that the mRNA levels of the bone cell genes α1(I) collagen, alkaline phosphatase, osteocalcin, and osteopontin were markedly increased in ovx rats between 6- and 30-fold. DHT administration to ovary-intact, estrogen-sufficient rats increased the mRNA levels of α1(I) collagen, alkaline phosphatase, osteopontin, and osteocalcin between 3- and 9-fold. In contrast, DHT did not alter levels of these mRNA species in ovx rats. The data demonstrate that estrogen deficiency increased mRNA levels of genes expressed during osteoblast development and suggest an interplay between estrogen and androgen action in regulating the expression of a number of bone cell genes.

Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone‐marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β‐catenin, confirming activation of canonical Wnt signaling. Reducing low‐density lipoprotein receptor‐related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a‐induced inactivation of NFATc1; however, deletion of β‐catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a‐induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A‐kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β‐catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β‐catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data suggest that Rank promoter Cre‐mediated deletion of Lrp5/6 may similarly impair osteoclast progenitor proliferation.

Genetically modified animal models as tools for studying bone and mineral metabolism

Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone‐related research and the contributions these studies have made to bone biology.

Calcitonin: Physiology or fantasy?

Calcitonin, a potent hypocalcemic hormone produced by the C‐cells of the thyroid, was first discovered by Harold Copp in 1962. The physiological significance of calcitonin has been questioned, but recent studies using genetically modified mouse models have uncovered additional actions of calcitonin acting through its receptor (CTR) that are of particular significance to the regulation of bone and calcium homeostasis. Mice in which the CTR is deleted in osteoclasts are more susceptible to induced hypercalcemia and exogenous calcitonin is able to lower serum calcium in younger animals. These data are consistent with the hypothesis that calcitonin can regulate serum calcium by inhibiting the efflux of calcium from bone, and that this action is most important when bone turnover is high. Calcitonin has also been implicated in protecting the skeleton from excessive loss of bone mineral during times of high calcium demand, such as lactation. This action may be linked to an intriguing and as yet unexplained observation that calcitonin inhibits bone formation, because deletion of the CTR leads to increased bone formation. We propose several mechanisms by which calcitonin could protect the skeleton by regulating bone turnover, acting within the bone and/or centrally. A new more holistic notion of the physiological role of calcitonin in bone and calcium homeostasis is required and we have highlighted some important knowledge gaps so that future calcitonin research will help to achieve such an understanding.

A floxed allele of the androgen receptor gene causes hyperandrogenization in male mice

We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor (AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (AR(lox)) have the reverse phenotype, termed hyperandrogenization. AR(lox) mice have increased mass of androgen-dependent tissues, including kidney, (P < 0.001), seminal vesicle (P < 0.001), levator ani muscle (P = 0.001), and heart (P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and AR(lox) males are fertile. AR(lox) males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.