Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.

Effects of PTH on osteoblast bioenergetics in response to glucose.

Parathyroid hormone acts through its receptor, PTHR1, expressed on osteoblasts, to control bone remodeling. Metabolic flexibility for energy generation has been demonstrated in several cell types dependent on substrate availability. Recent studies have identified a critical role for PTH in regulating glucose, fatty acid and amino acid metabolism thus stimulating both glycolysis and oxidative phosphorylation. Therefore, we postulated that PTH stimulates increased energetic output by osteoblasts either by increasing glycolysis or oxidative phosphorylation depending on substrate availability. To test this hypothesis, undifferentiated and differentiated MC3T3E1C4 calvarial pre-osteoblasts were treated with PTH to study osteoblast bioenergetics in the presence of exogenous glucose. Significant increases in glycolysis with acute ∼1 h PTH treatment with minimal effects on oxidative phosphorylation in undifferentiated MC3T3E1C4 in the presence of exogenous glucose were observed. In differentiated cells, the increased glycolysis observed with acute PTH was completely blocked by pretreatment with a Glut1 inhibitor (BAY-876) resulting in a compensatory increase in oxidative phosphorylation. We then tested the effect of PTH on the function of complexes I and II of the mitochondrial electron transport chain in the absence of glycolysis. Utilizing a novel cell plasma membrane permeability mitochondrial (PMP) assay, in combination with complex I and II specific substrates, slight but significant increases in basal and maximal oxygen consumption rates with 24 h PTH treatment in undifferentiated MC3T3E1C4 cells were noted. Taken together, our data demonstrate for the first time that PTH stimulates both increases in glycolysis and the function of the electron transport chain, particularly complexes I and II, during high energy demands in osteoblasts.

Estrogen Stimulation of Pleiotrophin Enhances Osteoblast Differentiation and Maintains Bone Mass in IGFBP-2 Null Mice.

Insulin-like growth factor binding protein-2 (IGFBP-2) stimulates osteoblast differentiation but only male Igfbp2 null mice have a skeletal phenotype. The trophic actions of IGFBP-2 in bone are mediated through its binding to receptor tyrosine phosphatase beta (RPTPß). Another important ligand for RPTPß is pleiotrophin (PTN), which also stimulates osteoblast differentiation. We determined the change in PTN and RPTPß in Igfbp2-/- mice. Analysis of whole bone mRNA in wild-type and knockout mice revealed increased expression of Ptn. Rptpß increased in gene-deleted animals with females having greater expression than males. Knockdown of PTN expression in osteoblasts in vitro inhibited differentiation, and addition of PTN to the incubation medium rescued the response. Estradiol stimulated PTN secretion and PTN knockdown blocked estradiol-stimulated differentiation. PTN addition to IGFBP-2 silenced osteoblast stimulated differentiation, and an anti-fibronectin-3 antibody, which inhibits PTN binding to RPTPß, inhibited this response. Estrogen stimulated PTN secretion and downstream signaling in the IGFBP-2 silenced osteoblasts and these effects were inhibited with anti-fibronectin-3. Administration of estrogen to wild-type and Igfbp2-/- male mice stimulated an increase in both areal bone mineral density and trabecular bone volume fraction but the increase was significantly greater in the Igfbp2-/- animals. Estrogen also stimulated RPTPß expression in the null mice. We conclude that loss of IGFBP-2 expression is accompanied by upregulation of PTN and RPTPß expression in osteoblasts, that the degree of increase is greater in females due to estrogen secretion, and that this compensatory change may account for some component of the maintenance of normal bone mass in female mice.

Thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information.

Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology. To evaluate the potential contribution of aberrant thyroid hormone status to the epigenetic inheritance of neurological phenotypes, we examined genetically normal F2 generation descendants of mice that were developmentally overexposed to thyroid hormone due to a Dio3 mutation. Hypothalamic gene expression profiling in postnatal day 15 F2 descendants on the paternal lineage of ancestral male and female T3-overexposed mice revealed, respectively, 1089 and 1549 differentially expressed genes. A large number of them, 675 genes, were common to both sets, suggesting comparable epigenetic effects of thyroid hormone on both the male and female ancestral germ lines. Oligodendrocyte- and neuron-specific genes were strongly overrepresented among genes showing, respectively, increased and decreased expression. Altered gene expression extended to other brain regions and was associated in adulthood with decreased anxiety-like behavior, increased marble burying and reduced physical activity. The sperm of T3-overexposed male ancestors revealed significant hypomethylation of CpG islands associated with the promoters of genes involved in the early development of the central nervous system. Some of them were candidates for neurodevelopmental disorders in humans including Nrg3, Nrxn1, Gabrb3, Gabra5, Apba2, Grik3, Reln, Nsd1, Pcdh8, En1, and Elavl2. Thus, developmental levels of thyroid hormone influence the epigenetic information of the germ line, disproportionately affecting genes with critical roles in early brain development, and leading in future generations to disease-relevant alterations in postnatal brain gene expression and adult behavior.

The mitophagy receptor Bcl-2-like protein 13 stimulates adipogenesis by regulating mitochondrial oxidative phosphorylation and apoptosis in mice.

Metabolic programming of bone marrow stromal cells (BMSCs) could influence the function of progenitor osteoblasts or adipocytes and hence determine skeletal phenotypes. Adipocytes predominantly utilize oxidative phosphorylation, whereas osteoblasts use glycolysis to meet ATP demand. Here, we compared progenitor differentiation from the marrow of two inbred mouse strains, C3H/HeJ (C3H) and C57BL6J (B6). These strains differ in both skeletal mass and bone marrow adiposity. We hypothesized that genetic regulation of metabolic programs controls skeletal stem cell fate. Our experiments identified Bcl-2-like protein 13 (Bcl2l13), a mitochondrial mitophagy receptor, as being critical for adipogenic differentiation. We also found that Bcl2l13 is differentially expressed in the two mouse strains, with C3H adipocyte progenitor differentiation being accompanied by a >2-fold increase in Bcl2l13 levels relative to B6 marrow adipocytes. Bcl2l13 expression also increased during adipogenic differentiation in mouse ear mesenchymal stem cells (eMSCs) and the murine preadipocyte cell line 3T3-L1. The higher Bcl2l13 expression correlated with increased mitochondrial fusion and biogenesis. Importantly, Bcl2l13 knockdown significantly impaired adipocyte differentiation in both 3T3-L1 cells and eMSCs. Mechanistically, Bcl2l13 knockdown reprogrammed cells to rely more on glycolysis to meet ATP demand in the face of impaired oxidative phosphorylation. Bcl2l13 knockdown in eMSCs increased mitophagy. Moreover, Bcl2l13 prevented apoptosis during adipogenesis. Our findings indicate that the mitochondrial receptor Bcl2l13 promotes adipogenesis by increasing oxidative phosphorylation, suppressing apoptosis, and providing mitochondrial quality control through mitophagy. We conclude that genetic programming of metabolism may be important for lineage determination and cell function within the bone marrow.

Progenitor recruitment and adipogenic lipolysis contribute to the anabolic actions of parathyroid hormone on the skeleton.

Intermittent administration of parathyroid hormone (PTH) stimulates bone formation in vivo and also suppresses the volume of bone marrow adipose tissue (BMAT). In contrast, a calorie-restricted (CR) diet causes bone loss and induces BMAT in both mice and humans. We used the CR model to test whether PTH would reduce BMAT in mice by both altering cell fate and inducing lipolysis of marrow adipocytes. Eight-week-old mice were placed on a control (Ctrl) diet or CR diet. At 12 wk, CR and Ctrl mice were injected daily with PTH (CR/PTH or Ctrl/PTH) or vehicle for 4 wk. Two other cohorts were CR and simultaneously injected (CR + PTH or CR + Veh) for 4 wk. CR mice had low bone mass and increased BMAT in the proximal tibias. PTH significantly increased bone mass in all cohorts despite calorie restrictions. Adipocyte density and size were markedly increased with restriction of calories. PTH reduced adipocyte numbers in CR + PTH mice, whereas adipocyte size was reduced in CR/PTH-treated mice. In contrast, osteoblast number was increased 3-8-fold with PTH treatment. In vitro, bone marrow stromal cells differentiated into adipocytes and, treated with PTH, exhibited increased production of glycerol and fatty acids. Moreover, in cocultures of bone marrow adipocyte and osteoblast progenitors, PTH stimulated the transfer of fatty acids to osteoblasts. In summary, PTH administration to CR mice increased bone mass by shifting lineage allocation toward osteogenesis and inducing lipolysis of mature marrow adipocytes. The effects of PTH on bone marrow adiposity could enhance its anabolic actions by providing both more cells and more fuel for osteoblasts during bone formation.-Maridas, D. E., Rendina-Ruedy, E., Helderman, R. C., DeMambro, V. E., Brooks, D., Guntur, A. R., Lanske, B., Bouxsein, M. L., Rosen, C. J. Progenitor recruitment and adipogenic lipolysis contribute to the anabolic actions of parathyroid hormone on the skeleton.

Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research.

High fat diet attenuates hyperglycemia, body composition changes, and bone loss in male streptozotocin-induced type 1 diabetic mice.

There is a growing and alarming prevalence of obesity and the metabolic syndrome in type I diabetic patients (T1DM), particularly in adolescence. In general, low bone mass, higher fracture risk, and increased marrow adipose tissue (MAT) are features of diabetic osteopathy in insulin-deficient subjects. On the other hand, type 2 diabetes (T2DM) is associated with normal or high bone mass, a greater risk of peripheral fractures, and no change in MAT. Therefore, we sought to determine the effect of weight gain on bone turnover in insulin-deficient mice. We evaluated the impact of a 6-week high-fat (HFD) rich in medium chain fatty acids or low-fat diet (LFD) on bone mass and MAT in a streptozotocin (STZ)-induced model using male C57BL/6J mice at 8 weeks of age. Dietary intervention was initiated after diabetes confirmation. At the endpoint, lower non-fasting glucose levels were observed in diabetic mice fed with high fat diet compared to diabetic mice fed the low fat diet (STZ-LFD). Compared to euglycemic controls, the STZ-LFD had marked polydipsia and polyphagia, as well as reduced lean mass, fat mass, and bone parameters. Interestingly, STZ-HFD mice had higher bone mass, namely less cortical bone loss and more trabecular bone than STZ-LFD. Thus, we found that a HFD, rich in medium chain fatty acids, protects against bone loss in a T1DM mouse model. Whether this may also translate to T1DM patients who are overweight or obese in respect to maintenance of bone mass remains to be determined through longitudinal studies.

IGFBP4 Is Required for Adipogenesis and Influences the Distribution of Adipose Depots.

Insulinlike growth factor (IGF) I induces adipogenesis in vitro. IGF-binding protein 4 (IGFBP4) is highly expressed in adipocytes and osteoblasts and is inhibitory of IGFs in vitro. We previously reported that Igfbp4 null mice (Igfbp4-/-) had decreased fat proportions at 8 and 16 weeks of age. However, the mechanism leading to the reduced adiposity remains unknown. The purpose of this study was to elucidate how IGFBP4 mediates adipose tissue development in vivo. Our results showed that inguinal and gonadal white adipose tissue (gWAT) from Igfbp4-/- mice had decreased weights and Pparγ expression. Cultures of primary bone marrow stromal cells (BMSCs) and ear mesenchymal stem cells (eMSCs) from mutant mice showed reduced adipogenesis. Both BMSCs and eMSC had a strong induction of Igfbp4 expression during adipogenesis. Furthermore, the increase in phosphorylated Akt (p-Akt), a downstream target of IGF-I signaling, in wild-type cells, was blunted in mutant eMSCs. On a high-fat diet (HFD) there were sexual differences in adipocyte expansion of Igfbp4-/- mice. Mutant males gained weight by expanding their white fat depots. However, Igfbp4-/- female mice were protected against diet-induced obesity. Ovariectomized Igfbp4-/- female mice gained weight in a manner similar to that seen in ovariectomized controls. Thus, Igfbp4 is required for inguinal fat expansion in female mice but not in male mice. However, gWAT expansion, which is prevented by estrogen during HFD, does not require Igfbp4.

Blocking FSH induces thermogenic adipose tissue and reduces body fat.

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the ß-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the ß-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

IGFBP-4 regulates adult skeletal growth in a sex-specific manner.

Insulin-like growth factor-1 (IGF-1) and its binding proteins are critical mediators of skeletal growth. Insulin-like growth factor-binding protein 4 (IGFBP-4) is highly expressed in osteoblasts and inhibits IGF-1 actions in vitro Yet, in vivo studies suggest that it could potentiate IGF-1 and IGF-2 actions. In this study, we hypothesized that IGFBP-4 might potentiate the actions of IGF-1 on the skeleton. To test this, we comprehensively studied 8- and 16-week-old Igfbp4-/- mice. Both male and female adult Igfbp4-/- mice had marked growth retardation with reductions in body weight, body and femur lengths, fat proportion and lean mass at 8 and 16 weeks. Marked reductions in aBMD and aBMC were observed in 16-week-old Igfbp4-/- females, but not in males. Femoral trabecular BV/TV and thickness, cortical fraction and thickness in 16-week-old Igfbp4-/- females were significantly reduced. However, surprisingly, males had significantly more trabeculae with higher connectivity density than controls. Concordantly, histomorphometry revealed higher bone resorption and lower bone formation in Igfbp4-/- females. In contrast, Igfbp4-/- males had lower mineralized surface/bone surface. Femoral expression of Sost and circulating levels of sclerostin were reduced but only in Igfbp4-/- males. Bone marrow stromal cultures from mutants showed increased osteogenesis, whereas osteoclastogenesis was markedly increased in cells from Igfbp4-/- females but decreased in males. In sum, our results indicate that loss of Igfbp4 affects mesenchymal stromal cell differentiation, regulates osteoclastogenesis and influences both skeletal development and adult bone maintenance. Thus, IGFBP-4 modulates the skeleton in a gender-specific manner, acting as both a cell autonomous and cell non-autonomous factor.

Igfbp2 Deletion in Ovariectomized Mice Enhances Energy Expenditure but Accelerates Bone Loss.

Previously, we reported sexually dimorphic bone mass and body composition phenotypes in Igfbp2(-/-) mice (-/-), where male mice exhibited decreased bone and increased fat mass, whereas female mice displayed increased bone but no changes in fat mass. To investigate the interaction between IGF-binding protein (IGFBP)-2 and estrogen, we subjected Igfbp2 -/- and +/+ female mice to ovariectomy (OVX) or sham surgery at 8 weeks of age. At 20 weeks of age, mice underwent metabolic cage analysis and insulin tolerance tests before killing. At harvest, femurs were collected for microcomputed tomography, serum for protein levels, brown adipose tissue (BAT) and inguinal white adipose tissue (IWAT) adipose depots for histology, gene expression, and mitochondrial respiration analysis of whole tissue. In +/+ mice, serum IGFBP-2 dropped 30% with OVX. In the absence of IGFBP-2, OVX had no effect on preformed BAT; however, there was significant "browning" of the IWAT depot coinciding with less weight gain, increased insulin sensitivity, lower intraabdominal fat, and increased bone loss due to higher resorption and lower formation. Likewise, after OVX, energy expenditure, physical activity and BAT mitochondrial respiration were decreased less in the OVX-/- compared with OVX+/+. Mitochondrial respiration of IWAT was reduced in OVX+/+ yet remained unchanged in OVX-/- mice. These changes were associated with significant increases in Fgf21 and Foxc2 expression, 2 proteins known for their insulin sensitizing and browning of WAT effects. We conclude that estrogen deficiency has a profound effect on body and bone composition in the absence of IGFBP-2 and may be related to changes in fibroblast growth factor 21.

Propranolol Attenuates Risperidone-Induced Trabecular Bone Loss in Female Mice.

Atypical antipsychotic (AA) drugs cause significant metabolic side effects, and clinical data are emerging that demonstrate increased fracture risk and bone loss after treatment with the AA, risperidone (RIS). The pharmacology underlying the adverse effects on bone is unknown. However, RIS action in the central nervous system could be responsible because the sympathetic nervous system (SNS) is known to uncouple bone remodeling. RIS treatment in mice significantly lowered trabecular bone volume fraction (bone volume/total volume), owing to increased osteoclast-mediated erosion and reduced osteoblast-mediated bone formation. Daytime energy expenditure was also increased and was temporally associated with the plasma concentration of RIS. Even a single dose of RIS transiently elevated expression of brown adipose tissue markers of SNS activity and thermogenesis, Pgc1a and Ucp1. Rankl, an osteoclast recruitment factor regulated by the SNS, was also increased 1 hour after a single dose of RIS. Thus, we inferred that bone loss from RIS was regulated, at least in part, by the SNS. To test this, we administered RIS or vehicle to mice that were also receiving the nonselective ß-blocker propranolol. Strikingly, RIS did not cause any changes in trabecular bone volume/total volume, erosion, or formation while propranolol was present. Furthermore, ß2-adrenergic receptor null (Adrb2(-/-)) mice were also protected from RIS-induced bone loss. This is the first report to demonstrate SNS-mediated bone loss from any AA. Because AA medications are widely prescribed, especially to young adults, clinical studies are needed to assess whether ß-blockers will prevent bone loss in this vulnerable population.

Altered thermogenesis and impaired bone remodeling in Misty mice.

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a, and less sympathetic innervation compared to wild-type (+/ +)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hours), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2, and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wild-type. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wild-type and Misty mice with the ß-blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild-type. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.

A high-fat diet induces bone loss in mice lacking the Alox5 gene.

5-Lipoxygenase catalyzes leukotriene generation from arachidonic acid. The gene that encodes 5-lipoxygenase, Alox5, has been identified in genome-wide association and mouse Quantitative Trait Locus studies as a candidate gene for obesity and low bone mass. Thus, we tested the hypothesis that Alox5(-/-) mice would exhibit metabolic and skeletal changes when challenged by a high-fat diet (HFD). On a regular diet, Alox5(-/-) mice did not differ in total body weight, percent fat mass, or bone mineral density compared with wild-type (WT) controls (P < 0.05). However, when placed on a HFD, Alox5(-/-) gained more fat mass and lost greater areal bone mass vs. WT (P < 0.05). Microarchitectural analyses revealed that on a HFD, WT showed increases in cortical area (P < 0.01) and trabecular thickness (P < 0.01), whereas Alox5(-/-) showed no change in cortical parameters but a decrease in trabecular number (P < 0.05) and bone volume fraction compared with WT controls (P < 0.05). By histomorphometry, a HFD did not change bone formation rates of either strain but produced an increase in osteoclast number per bone perimeter in Alox5(-/-) mice (P < 0.03). In vitro, osteoclastogenesis of marrow stromal cells was enhanced in mutant but not WT mice fed a HFD. Gene expression for Rankl, Pparg, and Cox-2 was greater in the femur of Alox5(-/-) than WT mice on a HFD (P < 0.01), but these increases were suppressed in the Alox5(-/-) mice after 8 wk of treatment with celecoxib, a cyclooxygenase-2 inhibitor. In sum, there is a strong gene by environmental interaction for bone mass when mice lacking the Alox5 gene are fed a HFD.

Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation.

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of insulin-like growth factor-binding protein-2 (IGFBP-2) in regulating osteoclast differentiation, we cultured Igfbp2(-/-) bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full-length IGFBP-2 restored osteoclast differentiation, fusion, and resorption. To determine the molecular domains of IGFBP-2 that were required for this effect to be manifest, Igfbp2(-/-) bone marrow cells were transfected with constructs in which the heparin-binding (HBD) or the IGF-binding domains of IGFBP-2 were mutated. We found that both domains were necessary for osteoclastogenesis because expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP-2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF-I were analyzed. Igfbp2(-/-) cells had elevated levels of PTEN and phospho-PTEN compared with controls. Expression of wild-type IGFBP-2 reduced the level of PTEN to that of wild-type cells. Cells expressing the IGF-binding mutant showed suppression of PTEN and phospho-PTEN equivalent to the wild-type protein, whereas those expressing the IGFBP-2 HBD mutant showed no PTEN suppression. When the ability of IGF-I to stimulate AKT activation, measured by Thr³°8 and Ser47³ phosphorylation, was analyzed, stimulation of Ser47³ in response to IGF-I in preosteoclasts required the presence of intact IGFBP-2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts, stimulation of Thr³°8 phosphorylation required the presence of intact IGFBP-2. We conclude that IGFBP-2 is an important regulator of osteoclastogenesis and that both the heparin- and the IGF-binding domains of IGFBP-2 are essential for the formation of fully differentiated and functional osteoclasts.

An essential role for the association of CD47 to SHPS-1 in skeletal remodeling.

Integrin-associated protein (IAP/CD47) has been implicated in macrophage-macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47(-/-) mice with Cd47(+/+) controls. Cd47(-/-) mice weighed less and had decreased areal bone mineral density compared with controls. Cd47(-/-) femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone-formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47(-/-) mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47(-/-) bone marrow cells was significantly decreased compared with wild-type cultures and was associated with a decrease in bone-resorption capacity. Furthermore, by disrupting the CD47-SHPS-1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS-1 phosphorylation, SHP-1 phosphatase recruitment, and subsequent dephosphorylation of non-muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47(-/-) mice. Our finding of cell-autonomous defects in Cd47(-/-) osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47(-/-) mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption.

The heparin-binding domain of IGFBP-2 has insulin-like growth factor binding-independent biologic activity in the growing skeleton.

Insulin-like growth factor-binding protein 2 (IGFBP-2) is a member of a family of six highly conserved IGFBPs that are carriers for the insulin-like growth factors (IGFs). IGFBP-2 levels rise during rapid neonatal growth and at the time of peak bone acquisition. In contrast, Igfbp2(-/-) mice have low bone mass accompanied by reduced osteoblast numbers, low bone formation rates, and increased PTEN expression. In the current study, we postulated that IGFBP-2 increased bone mass partly through the activity of its heparin-binding domain (HBD). We synthesized a HBD peptide specific for IGFBP-2 and demonstrated in vitro that it rescued the mineralization phenotype of Igfbp2(-/-) bone marrow stromal cells and calvarial osteoblasts. Consistent with its cellular actions, the HBD peptide ex vivo stimulated metacarpal periosteal expansion. Furthermore, administration of HBD peptide to Igfbp2(-/-) mice increased osteoblast number, suppressed marrow adipogenesis, restored trabecular bone mass, and reduced bone resorption. Skeletal rescue in the Igfbp2(-/-) mice was characterized by reduced PTEN expression followed by enhanced Akt phosphorylation in response to IGF-I and increased ß-catenin signaling through two mechanisms: 1) stimulation of its cytosolic accumulation and 2) increased phosphorylation of serine 552. We conclude that the HBD peptide of IGFBP-2 has anabolic activity by activating IGF-I/Akt and ß-catenin signaling pathways. These data support a growing body of evidence that IGFBP-2 is not just a transport protein but rather that it functions coordinately with IGF-I to stimulate growth and skeletal acquisition.

BMD regulation on mouse distal chromosome 1, candidate genes, and response to ovariectomy or dietary fat.

The distal end of mouse chromosome 1 (Chr 1) harbors quantitative trait loci (QTLs) that regulate bone mineral density (BMD) and share conserved synteny with human chromosome 1q. The objective of this article was to map this mouse distal Chr 1 region and identify gene(s) responsible for BMD regulation in females. We used X-ray densitometry [ie, dual-energy X-ray Absorptiometry (DXA), micro-computed tomography (µCT), and peripheral quantitative computed tomography (pQCT)] to phenotype a set of nested congenic strains constructed from C57BL/6BmJ (B6/Bm) and C3H/HeJ (C3H) mice to map the region associated with the BMD QTL. The critical region has been reduced to an interval of 0.152 Mb that contributes to increased BMD when C3H alleles are present. Histomorphometry and osteoblast cultures indicated that increased osteoblast activity was associated with increased BMD in mouse strains with C3H alleles in this critical region. This region contains two genes, Aim2, which binds with cytoplasmic dsDNA and results in apoptosis, and AC084073.22, a predicted gene of unknown function. Ovariectomy induced bone loss in the B6/Bm progenitor and the three congenic strains regardless of the alleles present in the critical BMD region. High dietary fat treatment (thought to suppress distal Chr 1 QTL for BMD in mice) did not induce bone loss in the congenics carrying C3H alleles in the critical 0.152 Mb carrying the AIM2 and AC084073.22 genes. Gene expression studies in whole bone of key congenics showed differential expression of AC084073.22 for strains carrying B6/Bm versus C3H alleles but not for Aim2. In conclusion, our data suggest that osteoblasts are the cellular target of gene action and that AC084073.22 is the best candidate for female BMD regulation in the distal region of mouse Chr 1.

A novel spontaneous mutation of Irs1 in mice results in hyperinsulinemia, reduced growth, low bone mass and impaired adipogenesis.

A spontaneous mouse mutant, designated 'small' (sml), was recognized by reduced body size suggesting a defect in the IGF1/GH axis. The mutation was mapped to the chromosome 1 region containing Irs1, a viable candidate gene whose sequence revealed a single nucleotide deletion resulting in a premature stop codon. Despite normal mRNA levels in mutant and control littermate livers, western blot analysis revealed no detectable protein in mutant liver lysates. When compared with the control littermates, Irs1(sml)/Irs1(sml) (Irs1(sml/sml)) mice were small, lean, hearing impaired; had 20% less serum IGF1; were hyperinsulinemic; and were mildly insulin resistant. Irs1(sml/sml) mice had low bone mineral density, reduced trabecular and cortical thicknesses, and low bone formation rates, while osteoblast and osteoclast numbers were increased in the females but not different in the males compared with the Irs1(+/+) controls. In vitro, Irs1(sml/sml) bone marrow stromal cell cultures showed decreased alkaline phosphatase-positive colony forming units (pre-osteoblasts; CFU-AP+) and normal numbers of tartrate-resistant acid phosphatase-positive osteoclasts. Irs1(sml/sml) stromal cells treated with IGF1 exhibited a 50% decrease in AKT phosphorylation, indicative of defective downstream signaling. Similarities between engineered knockouts and the spontaneous mutation of Irs1(sml) were identified as well as significant differences with respect to heterozygosity and gender. In sum, we have identified a spontaneous mutation in the Irs1 gene associated with a major skeletal phenotype. Changes in the heterozygous Irs1(+)(/sml) mice raise the possibility that similar mutations in humans are associated with short stature or osteoporosis.