Yogendra P. Kharode

High bone mass in mice expressing a mutant LRP5 gene

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease.

Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.

The Pairing of a Selective Estrogen Receptor Modulator, Bazedoxifene, with Conjugated Estrogens as a New Paradigm for the Treatment of Menopausal Symptoms and Osteoporosis Prevention

The menopausal transition is associated with decreased ovarian function and concomitant decline in estrogen production, which may result in physiological effects such as hot flashes, reduced bone mass, and altered lipid profile. It is well established that these unfavorable changes are effectively offset with estrogen therapy (ET) or, in women with a uterus, estrogens in combination with a progestin (hormone therapy). Selective estrogen receptor (ER) modulators (SERMs), which exhibit both ER agonist and antagonist activities depending on the target tissue, have been regarded as offering the potential to provide the benefits of ET and hormone therapy with an improved safety and tolerability profile. To date, no SERM alone has demonstrated an ideal benefit-risk profile for menopausal therapy. The tissue-selective estrogen complex, or the pairing of a SERM with estrogens, may provide an optimal blend of ER agonist and antagonist activities. We evaluated the physiological profile of this novel therapeutic paradigm by using various in vivo models to assess uterine, vasomotor, lipid, and skeletal responses to a tissue-selective estrogen complex partnering bazedoxifene with conjugated estrogens (CE). Bazedoxifene at 3.0 mg/kg effectively antagonized CE-induced uterine stimulation without reversing the positive effects of CE on vasomotor instability. When paired with CE, bazedoxifene at 3.0 mg/kg reduced total cholesterol levels by up to 20% compared with CE alone and significantly increased total bone density relative to control. These preclinical findings showed that the appropriate dose combination of bazedoxifene/CE exhibits positive vasomotor, lipid, and skeletal responses with minimal uterine stimulation.

Systemically administered rhBMP‐2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post‐menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP‐2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra‐peritoneal (i.p.) injections of rhBMP‐2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP‐2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP‐2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP‐2. J. Cell. Biochem. 86: 461–474, 2002.

Bone anabolic effects of parathyroid hormone are blunted by deletion of the Wnt antagonist secreted frizzled-related protein-1.

Secreted frizzled‐related protein (sFRP)‐1 is a Wnt antagonist that when deleted in mice leads to increased trabecular bone formation in adult animals after 13 weeks of age. Treatment of mice with parathyroid hormone (PTH) also increases trabecular bone formation, and some of the anabolic actions of this hormone may result from altered expression of Wnt pathway components. To test this hypothesis, we treated +/+ and −/− female sFRP‐1 mice with PTH 1–34 for 30 days and measured distal femur trabecular bone parameters by peripheral quantitative computed tomography (pQCT) and high‐resolution micro‐computed tomography. During the course of the 32‐week study, volumetric bone mineral density (vBMD) declined 41% in vehicle‐treated +/+ mice, but increased 24% in vehicle‐treated −/− animals. At 8 weeks of age when vBMD was not altered by deletion of sFRP‐1, treatment of +/+ and −/− mice with PTH increased vBMD by 147 and 163%, respectively. In contrast, at 24 weeks of age when vBMD was 75% higher in −/− mice than in +/+ controls, treatment with PTH increased vBMD 164% in +/+ animals, but only 58% in −/− mice. Furthermore, at 36 weeks of age when vBMD was 117% higher in −/− mice than in +/+ controls, treatment with PTH increased vBMD 74% in +/+ animals, while no increase was observed in −/− mice. At each of these time points, PTH treatment increased vBMD to a similar level in +/+ and −/− mice, and this level declined with age. In addition, at 36 weeks of age, the vBMD level reached by PTH treatment of +/+ mice was the same as that achieved solely by deletion of sFRP‐1. These results indicate that loss of sFRP‐1 and PTH treatment increase vBMD to a similar extent. Moreover, as the effects of sFRP‐1 deletion on vBMD increase, the ability of PTH to enhance vBMD declines suggesting that there are overlapping mechanisms of action. J. Cell. Physiol. 210: 352–357, 2007.

Lack of change of cancellous bone volume with short-term use of the new immunosuppressant rapamycin in rats

SummaryImmunosuppressants have adverse effects on bone mineral metabolism in animal and human studies, with corticosteroids producing low-turnover osteopenia, and cyclosporin-A (CsA) producing high-turnover osteopenia. Rapamycin (RAPA) is a new immunosuppressant reported to be at least 10 times more potent than CsA, and acts via a different pathway to CsA and the other new immunosuppressant FK506. This study investigated the effects of RAPA on bone mineral metabolism in the rat. Forty-two, 10-week-old, male Sprague Dawley rats were divided into three groups, and treated according to the following protocol: group A (control) received RAPA vehicle by daily gavage for 14 days (n = 12); group B (high dose RAPA) received RAPA 2.5 mg/kg/day by daily gavage for 14 days (n = 15); group C (low dose RAPA) received RAPA 1.25 mg/kg/day by daily gavage for 14 days (n = 15). Rats were weighed and bled on days 0, 7, and 14 for measurement of blood ionized calcium, bone Gla protein (BGP), parathyroid hormone (PTH), and 1,25(OH)2D. Tibial bone histomorphometry was determined on day 14 after double-calcein labeling. Weight gain was similar in the two groups treated with RAPA compared with control animals. High-dose RAPA (group B) transiently depressed serum BGP levels on day 7, with elevated blood ionized calcium levels on day 7, and lowered 1,25(OH)2D levels on day 14. Serum PTH levels were unchanged. Low dose RAPA (group C) did not affect calciotropic hormones. Histomorphometric analyses of tibial metaphyses revealed that parameters of bone formation and resorption were not significantly different in the groups treated with RAPA (group B and C) compared with control animals (group A). Trabecular bone volume (BV/TV) in group B (high-dose RAPA) (15.39 ± 1.01%) and C (low-dose RAPA) (15.38 ±0.57%) was not significantly altered compared with group A (control) (16.42 ± 0.86%). Short-term treatment with RAPA, unlike CsA, does not result in excess resorption and loss of bone volume. The depressed serum 1,25(OH)2D levels seen with high-dose RAPA therapy may adversely effect bone mineral metabolism in the long term.

Semaphorin 3B is a 1,25-Dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice.

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.

Utility of the Ovariectomized Rat as a Model for Human Osteoporosis in Drug Discovery

Ovariectomy-induced osteopenia in the rat produces skeletal responses similar to that in a post-menopausal woman. In the ovariectomized (ovx) rat, high bone turnover, and subsequent bone loss, like in the human post-menopausal condition, can be prevented by estrogen replacement. Because of the striking resemblance of skeletal responses in humans and rats in the state of estrogen deficiency, the ovx rat is considered to be a gold standard model for evaluating drugs for prevention and reversal of osteoporosis. This chapter describes the procedure for performing ovariectomy on the rat and the utility of the ovx rat model we have utilized over the last two decades in our laboratory.

(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: A Wingless β-Catenin Agonist That Increases Bone Formation Rate

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.

Identification of a PTH regulated gene selectively induced in vivo during PTH-mediated bone formation.

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well‐established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH‐regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100‐fold (maximal) by a PTH 1–34 anabolic treatment regimen in a time‐dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR‐106 osteoblastic cells show that PTH 1–34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1–34, BAALC expression can also be induced by other bone forming factors including PGE2 and 1,25 dihydroxy vitamin D3. We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3‐E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1–34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function. J. Cell. Biochem. 98: 1203–1220, 2006.