Anna Idelevich

Bone Gla Protein Increases HIF-1α–Dependent Glucose Metabolism and Induces Cartilage and Vascular Calcification

Objective— Bone Gla Protein (BGP, osteocalcin) is commonly present in the calcified vasculature and was recently shown as energy metabolism-regulating hormone. This study investigates the role of BGP in cartilage and vasculature mineralization. Methods and Results— We established an in vitro BGP-overexpression model in chondrocytes (ATDC5) and vascular smooth muscle cells (MOVAS). BGP overexpression upregulated markers of chondrogenic differentiation and intensified staining for minerals. BGP overexpression enhanced glucose uptake and increased expression of glucose transporters and glycolysis enzymes while decreasing gluconeogenesis enzymes. Treatment with purified BGP activated insulin signaling pathway and upregulated genes of glucose transport and utilization. Both BGP overexpression and treatment with purified BGP resulted in stabilization of hypoxia-inducible factor 1&agr; (HIF-1&agr;) in chondrocytes and vascular smooth muscle cells, shown essential in mediating the direct metabolic effect of BGP. The in vivo model of 1,25(OH)2D3-induced vascular calcification in rats revealed a correlation between calcification, elevated BGP levels, and increased HIF-1&agr; expression in aortas and bone growth plates. The in vivo introduction of BGP siRNA, coadministered with 1,25(OH)2D3, prevented 1,25(OH)2D3-induced HIF-1&agr; stabilization, and diminished osteochondrogenic differentiation and mineralization of aortas. Conclusion— This study demonstrates novel mechanism by which BGP locally shifts cells toward glycolytic breakdown of glucose, in a HIF-1&agr;–dependent manner, and stimulates calcification of cartilage and vasculature.

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

Background The topoisomerases Top1, Top2α and Top2β are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2β are subject to proteasomal degradation, this phenomena was not demonstrated for Top2α. Methodology/Principal Findings We show here that Top2α is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2β. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2α degradation, increases the persistence of Top2α-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2α in-vitro and cellular overexpression of Bmi1 increases drug induced Top2α ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2α ubiquitination and drug-induced Top2α degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner. Conclusions/Significance The discovery that poisoned Top2α is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.

What are the effects of leptin on bone and where are they exerted

In this issue of the Journal of Bone and Mineral Research, an article by Turner and colleagues addresses the important topic of the relationship between the adipocyte-derived hormone leptin, bone, and energy homeostasis. They report that the leptin-deficient and obese ob/ob mice and the leptin receptor–deficient db/db mice display an osteopetrotic-like skeletal phenotype at 15 weeks of age with high vertebral trabecular bone volume (BV/TV) despite a markedly decreased bone formation rate (BFR), as a result of decreased bone resorption. They also show that these phenotypes (low BFR and low resorption) can be corrected both by subcutaneous injections of leptin and by leptin hypothalamic gene therapy. Because this suggested that leptin can act both at the periphery and in the central nervous system to affect bone, they then performed marrow transplantation experiments in lethally irradiated mice. They report that transplantation of leptin receptor–deficient db/db bone marrow into lethally irradiated wild-type mice drastically reduces bone formation to levels indistinguishable from the db/db mice. Furthermore, mice engrafted with db/db marrow did not show overt alterations in energy homeostasis, suggesting that energy homeostasis is not affected by the changes in bone. They conclude that leptin acts on bone primarily through peripheral pathways and that its effects are to increase bone formation, but even more bone resorption. These results are in strong opposition with prevailing theories and call into question what leptin does in bone; ie, increasing or decreasing bone formation, and how it does it; ie, centrally through a hypothalamic relay or directly at the periphery. These findings raise several puzzling questions. First, the ob/ob and db/db skeletal phenotypes reported here are radically different from a previous report showing a 45% to 70% increase in BFR in ob/ob and db/db mice, respectively, compared to wildtype littermates. Second, the ability of peripheral leptin to affect bone, albeit supported by in vitro data published by others is in sharp contradiction with some in-depth studies from other laboratories. Third, peripheral leptin corrects the bone phenotype, although no significant alterations in energy homeostasis are noted, suggesting no direct link between bone and energy regulation in these leptin-deficient or leptin receptor–deficient mouse models. The original concept and the prevailing school of thought concerning the effects of the fat-derived hormone leptin on bone rely mainly on a series of pioneering and robust in vivo studies conducted by the Ducy and Karsenty groups (initially at the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; then at the Department of Pathology and Cell Biology [Ducy] and the Department of Genetics and Development [Karsenty], College of Physicians and Surgeons, Columbia University, New York, NY, USA). In 2000, these investigators presented their first in vivo evidence to the fact that bone gain in the ob/ob or db/db mice occurred despite gonadal failure, resulting from the absence of leptin signaling. Both ob/ob and db/dbmice exhibited high trabecular bone mass in the proximal tibiae and vertebrae, whereas intracerebroventricular (ICV) infusion of leptin into ovariectomized mice corrected this phenotype and resulted in a decreased BV/TV in both mutant and control groups. Interestingly, direct in vitro leptin treatment of primary ob/ob osteoblasts had no effect on collagen synthesis or mineralization, whereas db/db osteoblasts were indistinguishable from wild-type in culture, suggesting that leptin acted on bone solely via hypothalamic pathways. Moreover, the central action of leptin was proposed to target osteoblasts and not osteoclasts, because these authors found normal osteoclast function in the absence of leptin signaling. Subsequent work using b2-adrenergic receptor Adrb2-null mice or pharmacological treatment with b-blockers further supported the concept of a central inhibitory role of leptin on the skeleton, and proposed a model whereby activation of leptin receptors in the brain activates the sympathetic nervous system, which in COMMENTARY JBMR

1,25(OH)2D3 Alters Growth Plate Maturation and Bone Architecture in Young Rats with Normal Renal Function

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)2D3, the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)2D3 is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)2D3 treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 for one week, or intermittent 3 µg/kg 1,25(OH)2D3 for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)2D3-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)2D3 increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)2D3 lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)2D3-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)2D3 on intracortical bone formation. This study shows negative effects of 1,25(OH)2D3 on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.

The growth plate’s response to load is partially mediated by mechano-sensing via the chondrocytic primary cilium

Mechanical load plays a significant role in bone and growth-plate development. Chondrocytes sense and respond to mechanical stimulation; however, the mechanisms by which those signals exert their effects are not fully understood. The primary cilium has been identified as a mechano-sensor in several cell types, including renal epithelial cells and endothelium, and accumulating evidence connects it to mechano-transduction in chondrocytes. In the growth plate, the primary cilium is involved in several regulatory pathways, such as the non-canonical Wnt and Indian Hedgehog. Moreover, it mediates cell shape, orientation, growth, and differentiation in the growth plate. In this work, we show that mechanical load enhances ciliogenesis in the growth plate. This leads to alterations in the expression and localization of key members of the Ihh-PTHrP loop resulting in decreased proliferation and an abnormal switch from proliferation to differentiation, together with abnormal chondrocyte morphology and organization. Moreover, we use the chondrogenic cell line ATDC5, a model for growth-plate chondrocytes, to understand the mechanisms mediating the participation of the primary cilium, and in particular KIF3A, in the cell’s response to mechanical stimulation. We show that this key component of the cilium mediates gene expression in response to mechanical stimulation.

Bone quality is affected by food restriction and by nutrition-induced catch-up growth

Growth stunting constitutes the most common effect of malnutrition. When the primary cause of malnutrition is resolved, catch-up (CU) growth usually occurs. In this study, we have explored the effect of food restriction (RES) and refeeding on bone structure and mechanical properties. Sprague-Dawley male rats aged 24 days were subjected to 10 days of 40% RES, followed by refeeding for 1 (CU) or 26 days long-term CU (LTCU). The rats fed ad libitum served as controls. The growth plates were measured, osteoclasts were identified using tartrate-resistant acid phosphatase staining, and micro-computed tomography (CT) scanning and mechanical testing were used to study structure and mechanical properties. Micro-CT analysis showed that RES led to a significant reduction in trabecular BV/TV and trabecular number (Tb.N), concomitant with an increase in trabecular separation (Tb.Sp). Trabecular BV/TV and Tb.N were significantly greater in the CU group than in the RES in both short- and long-term experiments. Mechanical testing showed that RES led to weaker and less compliant bones; interestingly, bones of the CU group were also more fragile after 1 day of CU. Longer term of refeeding enabled correction of the bone parameters; however, LTCU did not achieve full recovery. These results suggest that RES in young rats attenuated growth and reduced trabecular bone parameters. While nutrition-induced CU growth led to an immediate increase in epiphyseal growth plate height and active bone modeling, it was also associated with a transient reduction in bone quality. This should be taken into consideration when treating children undergoing CU growth.

Neuronal hypothalamic regulation of body metabolism and bone density is galanin-dependent

In the brain, the ventral hypothalamus (VHT) regulates energy and bone metabolism. Whether this regulation uses the same or different neuronal circuits is unknown. Alteration of AP1 signaling in the VHT increases energy expenditure, glucose utilization, and bone density, yet the specific neurons responsible for each or all of these phenotypes are not identified. Using neuron-specific, genetically targeted AP1 alterations as a tool in adult mice, we found that agouti-related peptide–expressing (AgRP-expressing) or proopiomelanocortin-expressing (POMC-expressing) neurons, predominantly present in the arcuate nucleus (ARC) within the VHT, stimulate whole-body energy expenditure, glucose utilization, and bone formation and density, although their effects on bone resorption differed. In contrast, AP1 alterations in steroidogenic factor 1–expressing (SF1-expressing) neurons, present in the ventromedial hypothalamus (VMH), increase energy but decrease bone density, suggesting that these effects are independent. Altered AP1 signaling also increased the level of the neuromediator galanin in the hypothalamus. Global galanin deletion (VHT galanin silencing using shRNA) or pharmacological galanin receptor blockade counteracted the observed effects on energy and bone. Thus, AP1 antagonism reveals that AgRP- and POMC-expressing neurons can stimulate body metabolism and increase bone density, with galanin acting as a central downstream effector. The results obtained with SF1-expressing neurons, however, indicate that bone homeostasis is not always dictated by the global energy status, and vice versa.

Hypothalamic ΔFosB prevents age-related metabolic decline and functions via SNS

The ventral hypothalamus (VHT) integrates several physiological cues to maintain glucose homeostasis and energy balance. Aging is associated with increased glucose intolerance but the underlying mechanisms responsible for age-related metabolic decline, including neuronal signaling in the VHT, remain elusive. We have shown that mice with VHT-targeted overexpression of ΔFosB, a splice variant of the AP1 transcription factor FosB, exhibit increased energy expenditure, leading to decreased adiposity. Here, we show that VHT-targeted overexpression of ΔFosB also improves glucose tolerance, increases insulin sensitivity in target organs and thereby suppresses insulin secretion. These effects are also observed by the overexpression of dominant negative JunD, demonstrating that they occur via AP1 antagonism within the VHT. Furthermore, the improved glucose tolerance and insulin sensitivity persisted in aged animals overexpressing ΔFosB in the VHT. These beneficial effects on glucose metabolism were abolished by peripheral sympathectomy and α-adrenergic, but not β-adrenergic, blockade. Taken together, our results show that antagonizing AP1 transcription activity in the VHT leads to a marked improvement in whole body glucose homeostasis via activation of the SNS, conferring protection against age-related impairment in glucose metabolism. These findings may open novel avenues for therapeutic intervention in diabetes and age-related glucose intolerance.