Jameel Iqbal

FSH Directly Regulates Bone Mass

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.

TSH Is a Negative Regulator of Skeletal Remodeling

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.

Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice

We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense beta-gal histo- or cytostaining in adult noggin+/- mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.

Oxytocin is an anabolic bone hormone

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3, Osterix, and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-κB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca2+ release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the β-subunit of follicle-stimulating hormone (FSHβ) are protected from bone loss despite severe estrogen deficiency. Here we show that FSHβ-deficient mice have lowered TNFα levels. However, TNFα-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNFα is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNFα production from bone marrow granulocytes and macrophages. We also explore how TNFα up-regulation induces bone loss. By modeling the known actions of TNFα, we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNFα inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNFα. Likewise, ascorbic acid blunts the effects of TNFα in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNFα production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNFα.

Glucocerebrosidase gene-deficient mouse recapitulates Gaucher disease displaying cellular and molecular dysregulation beyond the macrophage

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

ACTH protects against glucocorticoid-induced osteonecrosis of bone.

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.

Physiological loading of joints prevents cartilage degradation through CITED2

Both overuse and disuse of joints up‐regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation;however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300‐interacting transacti‐vator with ED‐rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondropro‐tective effect of moderate mechanical loading. In vivo, hind‐limb immobilization of Sprague‐Dawley rats up‐regulates MMP‐1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up‐regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP‐1 expression and up‐regulates CITED2 in human chondro‐cytes, whereas high IHP (10 MPa) down‐regulates CITED2 and increases MMP‐1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP‐1 expression by competing with MMP transactivator, Ets‐1 for its coactivator p300. Furthermore, CITED2 up‐regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.—Leong, D. J., Li, Y. H., Gu, X. I., Sun, L., Zhou, Z., Nasser, P., Laudier, D. M., Iqbal, J., Majeska, R. J., Schaffler, M. B., Goldring, M. B., Cardoso, L., Zaidi, M., Sun, H. B. Physiological loading of joints prevents cartilage degradation through CITED2. FASEB J. 25, 182–191 (2011). www.fasebj.org

Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption

We have evaluated the role of the ADP‐ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+‐releasing second messenger, cyclic ADP‐ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin‐6 (IL‐6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38−/− mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38−/− mice showed a dramatic ~fourfold increase in osteoclast formation in response to incubation for 6 days with RANK‐L and M‐CSF. The osteoclasts so formed in these cultures showed a ~2.5‐fold increase in resorptive activity compared with wild‐type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase‐positive colony‐forming units (CFU‐Fs), those derived from CD38−/− mice showed a significant reduction in differentiation compared with wild‐type cells. Real‐time RT‐PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL‐6 and IL‐6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, α, β, and γ. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ “sensor,” particularly during periods of active motility and secretion.—Sun, L., Iqbal, J., Dolgilevich, S., Yuen, T., Wu, X.‐B., Moonga, B. S., Adebanjo, O. A., Bevis, P. J. R., Lund, F., Huang, C. L. H., Blair, H. C., Abe, E., Zaidi, M. Disordered osteoclast formation and function in a CD38 (ADP‐ribosyl cyclase) ‐deficient mouse establishes an essential role for CD38 in bone resorption. FASEB J. 17, 369–375 (2003)