Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHbeta nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHbeta+/- mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G(i2alpha)-coupled FSHRs that activate MEK/Erk, NF-kappaB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.

Recent epidemiological evidence indicates that insulin resistance, a proximal cause of Type II diabetes [a non-insulin dependent form of diabetes mellitus (NIDDM)], is associated with an increased relative risk for Alzheimer's disease (AD). In this study we examined the role of dietary conditions leading to NIDDM-like insulin resistance on amyloidosis in Tg2576 mice, which model AD-like neuropathology. We found that diet-induced insulin resistance promoted amyloidogenic beta-amyloid (Abeta) Abeta1-40 and Abeta1-42 peptide generation in the brain that corresponded with increased gamma-secretase activities and decreased insulin degrading enzyme (IDE) activities. Moreover, increased Abeta production also coincided with increased AD-type amyloid plaque burden in the brain and impaired performance in a spatial water maze task. Further exploration of the apparent interrelationship of insulin resistance to brain amyloidosis revealed a functional decrease in insulin receptor (IR)-mediated signal transduction in the brain, as suggested by decreased IR beta-subunit (IRbeta) Y1162/1163 autophosphorylation and reduced phosphatidylinositol 3 (PI3)-kinase/pS473-AKT/Protein kinase (PK)-B in these same brain regions. This latter finding is of particular interest given the known inhibitory role of AKT/PKB on glycogen synthase kinase (GSK)-3alpha activity, which has previously been shown to promote Abeta peptide generation. Most interestingly, we found that decreased pS21-GSK-3alpha and pS9-GSK-3beta phosphorylation, which is an index of GSK activation, positively correlated with the generation of brain C-terminal fragment (CTF)-gamma cleavage product of amyloid precursor protein, an index of gamma-secretase activity, in the brain of insulin-resistant relative to normoglycemic Tg2576 mice. Our study is consistent with the hypothesis that insulin resistance may be an underlying mechanism responsible for the observed increased relative risk for AD neuropathology, and presents the first evidence to suggest that IR signaling can influence Abeta production in the brain.

Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication.

We report that adrenocorticotropic hormone (ACTH) protects against osteonecrosis of the femoral head induced by depot methylprednisolone acetate (depomedrol). This therapeutic response likely arises from enhanced osteoblastic support and the stimulation of VEGF by ACTH; the latter is largely responsible for maintaining the fine vascular network that surrounds highly remodeling bone. We suggest examining the efficacy of ACTH in preventing human osteonecrosis, a devastating complication of glucocorticoid therapy.osteoporosis | osteoclast | osteoblast T he use of glucocorticoids for medical conditions as diverse as asthma, ulcerative colitis, kidney diseases, and rheumatologic disorders causes not only a variety of metabolic and medical complications, including diabetes and osteoporosis, but also a painful debilitating condition, osteonecrosis, usually affecting the femoral head (1). Osteonecrosis almost invariably requires surgical debridement of dead bone and contributes to approximately 10% of the more than 500,000 hip replacements annually in the United States (2). In addition, 30-50% patients on long-term glucocorticoids sustain a hip fracture with a 2-to 2.5-fold increased risk (3).Osteocyte apoptosis is thought to be the key determinant of glucocorticoid-induced cortical bone loss (4). Reduced osteoblast function manifesting in attenuated bone formation has also been documented in trabecular bone in rodents and humans (5). In contrast to glucocorticoid-induced osteoporosis, the pathogenesis of glucocorticoid-induced osteonecrosis is unclear (6). It resembles the osteonecrosis caused by traumatic damage to the artery that supplies the femoral head, hence the name, avascular necrosis (3), but the necrosis actually begins as regional trabecular death (6), likely from osteoblast and osteocyte apoptosis. However, there is strong evidence for an ischemic component. For example, studies using a rat model of Legg-Calve-Perthe's disease suggest that the intracortical blockade of lateral epiphyseal arteries that supply approximately 80% of the femoral head (7) can, in part, be attributed to their anatomical predisposition. It is nonetheless unclear whether ischemia is the initiating event or is secondary to local cellular or vascular bed damage (8).It is further surprising that osteonecrosis is not a cardinal feature of adrenocorticotropic hormone (ACTH)-producing adenomas (9), where glucocorticoid excess is profound. A question therefore arises-does ACTH protect against glucocorticoid-induced osteonecrosis? Indeed, one of our groups has documented functional ACTH receptors (MC2Rs) on osteoblasts; their activation enhances cell proliferation (10). These data are consistent with the presence of receptors for other anterior pituitary hormones, FSH and TSH, on bone cells, as well as with the description of another pituitary-bone axis, in which these hormones bypass traditional endocrine targets to regulate bone mass directly (11-13).We were thus prompted to investigate whether glucocorticoidinduced osteonecrosis could, i...

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca 2؉ ͞calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin A␣ into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin A␣ ؊/؊ mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain-and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.FK506 ͉ osteoporosis ͉ runx-2

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNF␣, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR ؊/؊ and TSHR ؉/؊ mice is abrogated in compound TSHR ؊͞؊ ͞TNF␣ ؊/؊ and TSHR ؉/؊ ͞ TNF␣ ؉/؊ mice, respectively. In parallel studies, we find that TSH directly inhibits TNF␣ production, reduces the number of TNF␣-producing osteoclast precursors, and attenuates the induction of bone remodeling ͉ osteoclast ͉ macrophage ͉ cytokine A nterior pituitary hormones have long been thought just to stimulate the secretion of master hormones from target endocrine glands, except for our recent demonstration of direct effects of thyroid-stimulating hormone (TSH) and folliclestimulating hormone on the skeleton (1). Thus, until recently, TSH was considered solely to regulate thyroid follicular cell differentiation and thyroid hormone secretion by binding to a seven transmembrane, glycosylated G protein-coupled receptor, the TSH receptor (TSHR). Previous studies had identified TSHRs in other tissues and cells, including the pituitary, thymus, testes, kidney, brain, lymphocytes, adipocytes, and fibroblasts (2, 3), but their functional significance has remained uncertain.Gene ablation studies in mice revealed that TSHR haploinsufficiency did not affect thyroid gland development or function, whereas the total absence of the TSHR expectedly disrupted thyroid follicular structure (4). However, bone mass was reduced not only in homozygote mice but also in the haploinsufficient heterozygotes (5). That TSHR haploinsufficiency, in the absence of a thyroid defect, resulted in osteoporosis established a primary role for the TSHR in bone metabolism. Furthermore, supplementation of TSHR Ϫ/Ϫ mice with thyroid extract to render them euthyroid corrected all hypothyroid abnormalities, including runting, but not reductions in bone mass (5) or sodium-iodide symporter expression (4). The latter observation confirmed that the osteoporosis arose from TSHR deficiency rather than altered thyroid hormone levels. Consistent with this notion, the genetic ablation of the ␣1͞␤ thyroid hormone receptor has been shown not to result in a bone remodeling defect (6).We found that the osteoporosis in TSHR knockout mice was the result of an enhancement in osteoclast differentiation. Consistent with the low bone mass, ex vivo cultures of bone marrow cell precursors from both heterozygote and homozygote mice showed increased osteoclast formation and the enhanced expression of an osteoclast marker tartrate-resistant acid phosphatase (TRAP) (5). This enhanced osteoclast formation was not associated with increased receptor activator of NF-B ligand (RANKL) production but instead with a several-fold increase in the synthesis and release of TNF␣, another...

In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.

Unloading, neural lesions, and hormonal disorders after acute motor-complete spinal cord injury (SCI) cause one of the most severe forms of bone loss, a condition that has been refractory to available interventions tested to date. Thus, these features related to acute SCI provide a unique opportunity to study complex bone problems, potential efficacious interventions, and mechanisms of action that are associated with these dramatic pathological changes. This study was designed to explore the therapeutic potential of sclerostin antibody (Scl-Ab) in a rat model of bone loss after motor-complete SCI, and to investigate mechanisms underlying bone loss and Scl-Ab action. SCI rats were administered Scl-Ab (25 mg/kg/week) or vehicle beginning 7 days after injury then weekly for 7 weeks. SCI resulted in significant decreases in bone mineral density (-25%) and trabecular bone volume (-67%) at the distal femur; Scl-Ab completely prevented these deteriorations of bone in SCI rats, concurrent with markedly increased bone formation. Scanning electron microscopy revealed that SCI reduced numbers of osteocytes and dendrites concomitant with a morphology change from a spindle to round shape; Scl-Ab corrected these abnormalities in osteocytes. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increases in mRNA levels of LRP5, osteoprotegerin (OPG), and the OPG/RANKL ratio, and a decrease in DKK1 mRNA. Our findings provide the first evidence that robust bone loss after acute motor-complete SCI can be blocked by Scl-Ab, at least in part, through the preservation of osteocyte morphology and structure and related bone remodeling. Our findings support the inhibition of sclerostin as a promising approach to mitigate the striking bone loss that ensues after acute motor-complete SCI, and perhaps other conditions associated with disuse osteoporosis as a consequence of neurological disorders.