Publications by authors named "Sun-Kyeong Lee"

43 Publications

Inhibition of miR-29 Activity in the Myeloid Lineage Increases Response to Calcitonin and Trabecular Bone Volume in Mice.

Endocrinology 2021 Oct;162(10)

Center on Aging, UConn Health, Farmington, CT 06030, USA.

The miR-29-3p family (miR-29a, miR-29b, miR-29c) of microRNAs is increased during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. In vivo, activation of a miR-29-3p tough decoy inhibitor in Cre recombinase under the control of the lysozyme 2 promoter-expressing cells (myeloid lineage) resulted in mice displaying enhanced trabecular and cortical bone volume because of decreased bone resorption. Calcitonin receptor (Calcr) is a miR-29 target that negatively regulates bone resorption. CALCR was significantly increased in RANKL-treated miR-29-decoy osteoclasts, and these cells were more responsive to the inhibitory effect of calcitonin on osteoclast formation. Further, cathepsin K (Ctsk), which is critical for resorption, was decreased in miR-29-decoy cells. CALCR is a Gs-coupled receptor and its activation raises cAMP levels. In turn, cAMP suppresses cathepsin K, and cAMP levels were increased in miR-29-decoy cells. siRNA-mediated knock-down of Calcr in miR-29 decoy osteoclasts allowed recovery of cathepsin K levels in these cells. Overall, using a novel knockin tough decoy mouse model, we identified a new role for miR-29-3p in bone homeostasis. In RANKL-driven osteoclastogenesis, as seen in normal bone remodeling, miR-29-3p promotes resorption. Consequently, inhibition of miR-29-3p activity in the myeloid lineage leads to increased trabecular and cortical bone. Further, this study documents an interrelationship between CALCR and CTSK in osteoclastic bone resorption, which is modulated by miR-29-3p.
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http://dx.doi.org/10.1210/endocr/bqab135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328098PMC
October 2021

Sexual Dimorphism in Differentiating Osteoclast Precursors Demonstrates Enhanced Inflammatory Pathway Activation in Female Cells.

J Bone Miner Res 2021 06 10;36(6):1104-1116. Epub 2021 Mar 10.

Arthritis and Tissue Degeneration Program, David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, USA.

Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4270DOI Listing
June 2021

Inhibition of miR-29-3p isoforms via tough decoy suppresses osteoblast function in homeostasis but promotes intermittent parathyroid hormone-induced bone anabolism.

Bone 2021 02 28;143:115779. Epub 2020 Nov 28.

Center for Molecular Oncology, UConn Health Center, Farmington, CT, United States of America. Electronic address:

miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/βcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.
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http://dx.doi.org/10.1016/j.bone.2020.115779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770763PMC
February 2021

miRNAs in osteoclast biology.

Bone 2021 02 17;143:115757. Epub 2020 Nov 17.

Department of Medicine, Division of Endocrinology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA; Robert and Arlene Kogod Center on Aging, Rochester, MN 55905, USA. Electronic address:

MicroRNAs (miRNAs) are a class of short RNA molecules that mediate the regulation of gene activity through interactions with target mRNAs and subsequent silencing of gene expression. It has become increasingly clear the miRNAs regulate many diverse aspects of bone biology, including bone formation and bone resorption processes. The role of miRNAs specifically in osteoclasts has been of recent investigation, due to clinical interest in discovering new paradigms to control excessive bone resorption, as is observed in multiple conditions including aging, estrogen deprivation, cancer metastases or glucocorticoid use. Therefore understanding the role that miRNAs play during osteoclastic differentiation is of critical importance. In this review, we highlight and discuss general aspects of miRNA function in osteoclasts, including exciting data demonstrating that miRNAs encapsulated in extracellular vesicles (EVs) either originating from osteoclasts, or signaling to osteoclast from divergent sites, have important roles in bone homeostasis.
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http://dx.doi.org/10.1016/j.bone.2020.115757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769925PMC
February 2021

Osteoclasts Derive Predominantly from Bone Marrow-Resident CXCR1 Precursor Cells in Homeostasis, whereas Circulating CXCR1 Cells Contribute to Osteoclast Development during Fracture Repair.

J Immunol 2020 02 8;204(4):868-878. Epub 2020 Jan 8.

Department of Medicine, UConn Health, Farmington, CT 06030;

Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CXCR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CXCR1-EGFP/TRAP tdTomato mice, we found CXCR1 expression in mononuclear cells, but not in multinucleated TRAP OC. However, CXCR1expressing cells generated TRAP OC on bone within 5 d in CXCR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CXCR1 cells migrated to bone, lost expression of CXCR1, and became OC. These data demonstrate that OCP, but not mature OC, express CXCR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CXCR1 cells can become OC.
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http://dx.doi.org/10.4049/jimmunol.1900665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002248PMC
February 2020

Rac1 Inhibition Via Srgap2 Restrains Inflammatory Osteoclastogenesis and Limits the Clastokine, SLIT3.

J Bone Miner Res 2020 04 9;35(4):789-800. Epub 2020 Jan 9.

Center on Aging, UConn Health, Farmington, CT, USA.

The Rac1-specific guanosine triphosphatase (GTPase)-activating protein Slit-Robo GAP2 (Srgap2) is dramatically upregulated during RANKL-induced osteoclastogenesis. Srgap2 interacts with the cell membrane to locally inhibit activity of Rac1. In this study, we determined the role of Srgap2 in the myeloid lineage on bone homeostasis and the osteoclastic response to TNFα treatment. The bone phenotype of mice specifically lacking Srgap2 in the myeloid lineage (Srgap2 :LysM-Cre; Srgap2 conditional knockout [cKO]) was investigated using histomorphometric analysis, in vitro cultures and Western blot analysis. Similar methods were used to determine the impact of TNFα challenge on osteoclast formation in Srgap2 cKO mice. Bone parameters in male Srgap2 cKO mice were unaffected. However, female cKO mice displayed higher trabecular bone volume due to increased osteoblast surface and bone formation rate, whereas osteoclastic parameters were unaltered. In vitro, cells from Srgap2 cKO had strongly enhanced Rac1 activation, but RANKL-induced osteoclast formation was unaffected. In contrast, conditioned medium from Srgap2 cKO osteoclasts promoted osteoblast differentiation and had increased levels of the bone anabolic clastokine SLIT3, providing a possible mechanism for increased bone formation in vivo. Rac1 is rapidly activated by the inflammatory cytokine TNFα. Supracalvarial injection of TNFα caused an augmented osteoclastic response in Srgap2 cKO mice. In vitro, cells from Srgap2 cKO mice displayed increased osteoclast formation in response to TNFα. We conclude that Srgap2 plays a prominent role in limiting osteoclastogenesis during inflammation through Rac1, and restricts expression of the paracrine clastokine SLIT3, a positive regulator of bone formation. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690287PMC
April 2020

CD55 Regulates Bone Mass in Mice by Modulating RANKL-Mediated Rac Signaling and Osteoclast Function.

J Bone Miner Res 2020 01 23;35(1):130-142. Epub 2019 Oct 23.

UCONN Center on Aging, University of Connecticut Health Center, Farmington, CT, USA.

CD55 is a glycosylphosphatidylinositol (GPI)-anchored protein that regulates complement-mediated and innate and adaptive immune responses. Although CD55 is expressed in various cell types in the bone marrow, its role in bone has not been investigated. In the current study, trabecular bone volume measured by μCT in the femurs of CD55KO female mice was increased compared to wild type (WT). Paradoxically, osteoclast number was increased in CD55KO with no differences in osteoblast parameters. Osteoclasts from CD55KO mice exhibited abnormal actin-ring formation and reduced bone-resorbing activity. Moreover, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) treatment failed to activate Rac guanosine triphosphatase (GTPase) in CD55KO bone marrow macrophage (BMM) cells. In addition, apoptotic caspases activity was enhanced in CD55KO, which led to the poor survival of mature osteoclasts. Our results imply that CD55KO mice have increased bone mass due to defective osteoclast resorbing activity resulting from reduced Rac activity in osteoclasts. We conclude that CD55 plays an important role in the survival and bone-resorption activity of osteoclasts through regulation of Rac activity. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3861DOI Listing
January 2020

Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism.

J Immunol 2019 07 20;203(1):105-116. Epub 2019 May 20.

Department of Medicine, UConn Health, Farmington, CT 06030;

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.
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http://dx.doi.org/10.4049/jimmunol.1801032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581625PMC
July 2019

MicroRNAs are Critical Regulators of Osteoclast Differentiation.

Curr Mol Biol Rep 2019 Feb 16;5(1):65-74. Epub 2019 Jan 16.

Center for Molecular Oncology, UConn Health, Farmington, CT 03030.

Purpose Of Review: Our goal is to comprehensively review the most recent reports of microRNA (miRNA) regulation of osteoclastogenesis. We highlight validated miRNA-target interactions and their place in the signaling networks controlling osteoclast differentiation and function.

Recent Findings: Using unbiased approaches to identify miRNAs of interest and reporter-3'UTR assays to validate interactions, recent studies have elucidated the impact of specific miRNA-mRNA interactions during in vitro osteoclastogenesis. There has been a focus on signaling mediators downstream of the RANK and CSF1R signaling, and genes essential for differentiation and function. For example, several miRNAs directly and indirectly target the master osteoclast transcription factor, Nfatc1 (e.g. miR-124 and miR-214) and Rho-GTPases, Cdc42 and Rac1 (e.g. miR-29 family).

Summary: Validating miRNA expression patterns, targets, and impact in osteoclasts and other skeletal cells is critical for understanding basic bone biology and for fulfilling the therapeutic potential of miRNA-based strategies in the treatment bone diseases.
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http://dx.doi.org/10.1007/s40610-019-0116-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380495PMC
February 2019

Blackcurrant Supplementation Improves Trabecular Bone Mass in Young but Not Aged Mice.

Nutrients 2018 Nov 5;10(11). Epub 2018 Nov 5.

Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA.

Due to deleterious side effects of currently available medications, the search for novel, safe, and effective preventive agents for improving bone health in aging continues and is urgently needed. This study aimed to determine whether dietary blackcurrants (BC), an anthocyanin-rich berry, can improve bone mass in a mouse model of age-related bone loss. Thirty-five female C57BL/6J mice, 3 months old ( = 20) and 18 months old ( = 15), were randomized to consume either a standard chow diet or a standard chow diet with 1% (/) BC for four months. Dual-energy X-ray absorptiometry, Micro computed tomography (µCT), and histomorphometric analyses were conducted to assess bone parameters on femurs. Biochemical assays were conducted to determine bone resorption, antioxidant activity, and inflammation in humerus homogenates. Trabecular bone volume (BV/TV) was significantly lower in aged mice compared to young mice (young control, 3.7 ± 0.4% vs aged control, 1.5 ± 0.5%, mean ± SEM (standard error of mean), < 0.01; young BC, 5.3 ± 0.6% vs aged BC, 1.1 ± 0.3%, < 0.001). µCT analysis revealed that BC supplementation increased trabecular BV/TV in young mice by 43.2% ( < 0.05) compared to controls. Histomorphometric analysis revealed a 50% increase, though this effect was not statistically significant ( = 0.07). The osteoblast surface increased by 82.5% in aged mice with BC compared to controls ( < 0.01). In humerus homogenates of young mice, BC consumption reduced C-telopeptide of type I collagen by 12.4% ( < 0.05) and increased glutathione peroxidase by 96.4% ( < 0.05). In humerus homogenates of aged mice, BC consumption increased catalase by 12% ( = 0.09). Aged mice had significantly elevated concentrations of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine contributing to bone resorption, which was reduced by 43.3% with BC consumption ( = 0.06). These results suggest that early consumption of BC may protect from aging-associated bone loss.
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http://dx.doi.org/10.3390/nu10111671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266496PMC
November 2018

Osteoclast-secreted SLIT3 coordinates bone resorption and formation.

J Clin Invest 2018 04 5;128(4):1429-1441. Epub 2018 Mar 5.

Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.

Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
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http://dx.doi.org/10.1172/JCI91086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873876PMC
April 2018

DJ-1 controls bone homeostasis through the regulation of osteoclast differentiation.

Nat Commun 2017 11 15;8(1):1519. Epub 2017 Nov 15.

Department of Immunology and Physiology, School of Medicine, Konkuk University, Chungju, 380-701, Republic of Korea.

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.
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http://dx.doi.org/10.1038/s41467-017-01527-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688089PMC
November 2017

Splenomegaly, myeloid lineage expansion and increased osteoclastogenesis in osteogenesis imperfecta murine.

Bone 2017 Oct 7;103:1-11. Epub 2017 Jun 7.

Department of Reconstructive Sciences, University of Connecticut, Farmington, CT 06030, USA. Electronic address:

Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters.
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http://dx.doi.org/10.1016/j.bone.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764163PMC
October 2017

Free Fatty Acid Receptor 4 (GPR120) Stimulates Bone Formation and Suppresses Bone Resorption in the Presence of Elevated n-3 Fatty Acid Levels.

Endocrinology 2016 07 4;157(7):2621-35. Epub 2016 May 4.

Department of Endocrinology and Metabolism (S.H.A.), Inha University Hospital, Inha University School of Medicine, Incheon 402-751, South Korea; Asan Institute for Life Sciences (S.-Y.P., J.-E.B., S.-Youn.L., W.-.Y.B., S.-Young.L., Y.-S.L.) and Biomedical Research Center (H.J.Y.) and Division of Endocrinology and Metabolism (H.K., S.H.L., B.-J.K., J.-M.K.), Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea; Molecular Inflammation Research Center for Aging Intervention and College of Pharmacy (D.-S.I.), Pusan National University, Busan 609-735, South Korea; and UConn Center on Aging (S.-K.L.), University of Connecticut Health Center, Farmington, Connecticut 06030-1601.

Free fatty acid receptor 4 (FFA4) has been reported to be a receptor for n-3 fatty acids (FAs). Although n-3 FAs are beneficial for bone health, a role of FFA4 in bone metabolism has been rarely investigated. We noted that FFA4 was more abundantly expressed in both mature osteoclasts and osteoblasts than their respective precursors and that it was activated by docosahexaenoic acid. FFA4 knockout (Ffar4(-/-)) and wild-type mice exhibited similar bone masses when fed a normal diet. Because fat-1 transgenic (fat-1(Tg+)) mice endogenously converting n-6 to n-3 FAs contain high n-3 FA levels, we crossed Ffar4(-/-) and fat-1(Tg+) mice over two generations to generate four genotypes of mice littermates: Ffar4(+/+);fat-1(Tg-), Ffar4(+/+);fat-1(Tg+), Ffar4(-/-);fat-1(Tg-), and Ffar4(-/-);fat-1(Tg+). Female and male littermates were included in ovariectomy- and high-fat diet-induced bone loss models, respectively. Female fat-1(Tg+) mice decreased bone loss after ovariectomy both by promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption than their wild-type littermates, only when they had the Ffar4(+/+) background, but not the Ffar4(-/-) background. In a high-fat diet-fed model, male fat-1(Tg+) mice had higher bone mass resulting from stimulated bone formation and reduced bone resorption than their wild-type littermates, only when they had the Ffar4(+/+) background, but not the Ffar4(-/-) background. In vitro studies supported the role of FFA4 as n-3 FA receptor in bone metabolism. In conclusion, FFA4 is a dual-acting factor that increases osteoblastic bone formation and decreases osteoclastic bone resorption, suggesting that it may be an ideal target for modulating metabolic bone diseases.
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http://dx.doi.org/10.1210/en.2015-1855DOI Listing
July 2016

Anthocyanin-Rich Blackcurrant Extract Attenuates Ovariectomy-Induced Bone Loss in Mice.

J Med Food 2016 Apr;19(4):390-7

1 Department of Nutritional Sciences, University of Connecticut , Storrs, Connecticut, USA .

Although several animal and cell studies have indicated that blackcurrant anthocyanins exert antioxidative and anti-inflammatory properties, which could potentially improve bone mass, the effect of blackcurrant on bone health has not been reported yet. Thus, this study was aimed to evaluate the effect of blackcurrant anthocyanins on bone mass in an estrogen deficiency mouse model. Fourteen-week-old C57BL/6J mice (n = 54) were ovariectomized or sham operated. The ovariectomized mice were divided into two groups, basal diet (OVX) or basal diet containing 1% anthocyanin-rich blackcurrant extract (OVX+BC), and sacrificed at 4, 8, and 12 weeks. Femoral bone mineral density (BMD) and trabecular bone volume by dual-energy X-ray absorptiometry and micro-computed tomography, respectively, and serum bone markers were measured. Ovariectomy significantly reduced BMD and trabecular bone volume at all time points (P < .05). Blackcurrant supplementation attenuated ovariectomy-induced bone loss measured by BMD and trabecular bone volume at 8 weeks (P = .055 and P = .057) and the effect was more pronounced at 12 weeks (P = .053 and P < .05). Ovariectomy and blackcurrant treatment did not alter serum biomarkers of bone formation and resorption. Bone marrow cells extracted from OVX mice significantly induced osteoclast-like (OCL) cell formation compared with cells from sham controls (P < .05). Blackcurrant treatment decreased the number of TRAP(+) OCL compared with OVX mice at 8 and 12 weeks (P < .05). Furthermore, blackcurrant supplementation reduced bone resorption activity when measured by resorption pit assay, compared with OVX group (P < .05). These results demonstrate that blackcurrant may be effective in mitigating osteoclast-induced postmenopausal bone loss.
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http://dx.doi.org/10.1089/jmf.2015.0148DOI Listing
April 2016

Soy Isoflavones and Osteoporotic Bone Loss: A Review with an Emphasis on Modulation of Bone Remodeling.

J Med Food 2016 Jan 15;19(1):1-14. Epub 2015 Dec 15.

1 Department of Nutritional Sciences, University of Connecticut , Storrs, Connecticut, USA.

Osteoporosis is an age-related disorder that affects both women and men, although estrogen deficiency induced by menopause accelerates bone loss in older women. As the demographic shifts to a more aged population, a growing number of men and women will be afflicted with osteoporosis. Since the current drug therapies available have multiple side effects, including increased risk of developing certain types of cancer or complications, a search for potential nonpharmacologic alternative therapies for osteoporosis is of prime interest. Soy isoflavones (SI) have demonstrated potential bone-specific effects in a number of studies. This article provides a systematic review of studies on osteoporotic bone loss in relation to SI intake from diet or supplements to comprehensively explain how SI affect the modulation of bone remodeling. Evidence from epidemiologic studies supports that dietary SI attenuate menopause-induced osteoporotic bone loss by decreasing bone resorption and stimulating bone formation. Other studies have also illustrated that bone site-specific trophic and synergistic effects combined with exercise intervention might contribute to improve the bioavailability of SI or strengthen the bone-specific effects. To date, however, the effects of dietary SI on osteoporotic bone loss remain inconclusive, and study results vary from study to study. The current review will discuss the potential factors that result in the conflicting outcomes of these studies, including dosages, intervention materials, study duration, race, and genetic differences. Further well-designed studies are needed to fully understand the underlying mechanism and evaluate the effects of SI on osteoporosis in humans.
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http://dx.doi.org/10.1089/jmf.2015.0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717511PMC
January 2016

Contradictory Role of CD97 in Basal and Tumor Necrosis Factor-Induced Osteoclastogenesis In Vivo.

Arthritis Rheumatol 2016 05;68(5):1301-13

University of Connecticut Health Center, Farmington.

Objective: CD97, a member of the 7-transmembrane epidermal growth factor family of adhesion G protein-coupled receptors, is expressed on various cell types. This study was undertaken to elucidate the functions of CD97 in bone and inflammation in an experimental mouse model, by examining the effect of CD97 on osteoclastogenesis in vitro, characterizing the skeletal phenotype of CD97-deficient (CD97-knockout [KO]) mice, and assessing the responses to tumor necrosis factor (TNF) treatment.

Methods: Femoral tissue and bone marrow (BM)-derived cells from CD97-KO and wild-type (WT) mice were assessed using histomorphometric analyses, in vitro cultures, and reverse transcription-polymerase chain reaction. Serum cytokine and chemokine levels in the presence or absence of TNF challenge were analyzed by multiplex assay.

Results: In cultures of mouse BM-derived macrophages in vitro, RANKL induced the expression of CD97. In vivo, the trabecular bone volume of the femurs of female CD97-KO mice was increased, and this was associated with a decrease in the number of osteoclasts. Compared to WT mice, CD97-KO mice had a reduced potential to form osteoclast-like cells in vitro. Furthermore, TNF treatment augmented the formation of osteoclasts in the calvaria of CD97-KO mice in vivo, by increasing the production of RANKL and other cytokines and chemokines and by reducing the production of osteoprotegerin by calvarial cells.

Conclusion: These findings demonstrate that CD97 is a positive regulator of osteoclast-like cell differentiation, a mechanism that influences bone homeostasis. However, the presence of CD97 may be essential to suppress the initial osteoclastogenesis that occurs in response to acute and local inflammatory stimuli.
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http://dx.doi.org/10.1002/art.39538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514326PMC
May 2016

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.

J Biol Chem 2016 Jan 1;291(4):1538-1551. Epub 2015 Dec 1.

From the Departments of Orthopaedic Surgery,; Medicine.

Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.
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http://dx.doi.org/10.1074/jbc.M115.685453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722436PMC
January 2016

Pathway analysis of microRNA expression profile during murine osteoclastogenesis.

PLoS One 2014 15;9(9):e107262. Epub 2014 Sep 15.

Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling osteoclast proliferation, differentiation, and apoptosis. Disruption of miRNA-mediated regulation alters osteoclast formation and bone resorption. Prior studies profiled miRNA expression in murine osteoclast precursors treated with RANKL for 24 hours. However, a more complete miRNA signature, encompassing early, mid and late stages of osteoclastogenesis, is wanting. An Agilent microarray platform was used to analyze expression of mature miRNAs in an enriched population of murine bone marrow osteoclast precursors (depleted of B220+ and CD3+ cells) undergoing 1, 3, or 5 days of RANKL-driven differentiation. Expression of 93 miRNAs, changed by >2 fold during early, mid, and late stages of osteoclastogenesis, were identified and sorted into 7 clusters. We validated the function and expression of miR-365, miR-451, and miR-99b, which were found in distinct clusters. Inhibition of miR-365 increased osteoclast number but decreased osteoclast size, while miR-99b inhibition decreased both osteoclast number and size. In contrast, overexpression of miR-451 had no effect. Computational analyses predicted mTOR, PI3 kinase/AKT, cell-matrix interactions, actin cytoskeleton organization, focal adhesion, and axon guidance pathways to be top targets of several miRNA clusters. This suggests that many miRNA clusters differentially expressed during osteoclastogenesis converge on some key functional pathways. Overall, our study is unique in that we identified miRNAs differentially expressed during early, mid, and late osteoclastogenesis in a population of primary mouse bone marrow cells enriched for osteoclast progenitors. This novel data set contributes to our understanding of the molecular mechanisms regulating the complex process of osteoclast differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107262PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164525PMC
January 2016

Macrophage migration inhibitory factor down-regulates the RANKL-RANK signaling pathway by activating Lyn tyrosine kinase in mouse models.

Arthritis Rheumatol 2014 Sep;66(9):2482-93

University of Connecticut Health Center, Farmington.

Objective: Macrophage migration inhibitory factor (MIF) is an important modulator of innate and adaptive immunity as well as local inflammatory responses. We previously reported that MIF down-regulated osteoclastogenesis through a mechanism that requires CD74. The aim of the current study was to examine whether MIF modulates osteoclastogenesis through Lyn phosphorylation, and whether down-regulation of RANKL-mediated signaling requires the association of CD74, CD44, and Lyn.

Methods: CD74-knockout (CD74-KO), CD44-KO, and Lyn-KO mouse models were used to investigate whether Lyn requires these receptors and coreceptors. The effects of MIF on osteoclastogenesis were assessed using Western blot analysis, small interfering RNA (siRNA)-targeted down-regulation of Lyn, Lyn-KO mice, and real-time imaging of Lyn molecules to surface proteins.

Results: MIF treatment induced Lyn expression, and MIF down-regulated RANKL-induced activator protein 1 (AP-1) and the Syk/phospholipase Cγ cascade during osteoclastogenesis through activated Lyn tyrosine kinase. The results of immunoprecipitation studies revealed that MIF receptors associated with Lyn in response to MIF treatment. Studies using Lyn-specific siRNA and Lyn-KO mice confirmed our findings.

Conclusion: Our findings indicate that the tyrosine kinase Lyn is activated when MIF binds to its receptor CD74 and its coreceptor CD44 and, in turn, down-regulates the RANKL-mediated signaling cascade by suppressing NF-ATc1 protein expression through down-regulation of AP-1 and calcium signaling components.
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http://dx.doi.org/10.1002/art.38723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146704PMC
September 2014

Dietary Polyphenols, Berries, and Age-Related Bone Loss: A Review Based on Human, Animal, and Cell Studies.

Antioxidants (Basel) 2014 Mar 11;3(1):144-58. Epub 2014 Mar 11.

Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269-4017, USA.

Bone loss during aging has become an increasing public health concern as average life expectancy has increased. One of the most prevalent forms of age-related bone disease today is osteoporosis in which the body slows down bone formation and existing bone is increasingly being resorbed by the body to maintain the calcium balance. Some causes of this bone loss can be attributed to dysregulation of osteoblast and osteoclast activity mediated by increased oxidative stress through the aging process. Due to certain serious adverse effects of the currently available therapeutic agents that limit their efficacy, complementary and alternative medicine (CAM) has garnered interest as a natural means for the prevention of this debilitating disease. Natural antioxidant supplementation, a type of CAM, has been researched to aid in reducing bone loss caused by oxidative stress. Naturally occurring polyphenols, such as anthocyanins rich in berries, are known to have anti-oxidative properties. Several studies have been reviewed to determine the impact polyphenol intake-particularly that of berries-has on bone health. Studies reveal a positive association of high berry intake and higher bone mass, implicating berries as possible inexpensive alternatives in reducing the risk of age related bone loss.
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http://dx.doi.org/10.3390/antiox3010144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665444PMC
March 2014

miR-29 promotes murine osteoclastogenesis by regulating osteoclast commitment and migration.

J Biol Chem 2013 Nov 1;288(46):33347-60. Epub 2013 Oct 1.

From the Center for Molecular Medicine and.

Osteoclast differentiation is regulated by transcriptional, post-transcriptional, and post-translational mechanisms. MicroRNAs are fundamental post-transcriptional regulators of gene expression. The function of the miR-29 (a/b/c) family in cells of the osteoclast lineage is not well understood. In primary cultures of mouse bone marrow-derived macrophages, inhibition of miR-29a, -29b, or -29c diminished formation of TRAP (tartrate-resistant acid phosphatase-positive) multinucleated osteoclasts, and the osteoclasts were smaller. Quantitative RT-PCR showed that all miR-29 family members increased during osteoclast differentiation, in concert with mRNAs for the osteoclast markers Trap (Acp5) and cathepsin K. Similar regulation was observed in the monocytic cell line RAW264.7. In stably transduced RAW264.7 cell lines expressing an inducible miR-29 competitive inhibitor (sponge construct), miR-29 knockdown impaired osteoclastic commitment and migration of pre-osteoclasts. However, miR-29 knockdown did not affect cell viability, actin ring formation, or apoptosis in mature osteoclasts. To better understand how miR-29 regulates osteoclast function, we validated miR-29 target genes using Luciferase 3'-UTR reporter assays and specific miR-29 inhibitors. We demonstrated that miR-29 negatively regulates RNAs critical for cytoskeletal organization, including Cdc42 (cell division control protein 42) and Srgap2 (SLIT-ROBO Rho GTPase-activating protein 2). Moreover, miR-29 targets RNAs associated with the macrophage lineage: Gpr85 (G protein-coupled receptor 85), Nfia (nuclear factor I/A), and Cd93. In addition, Calcr (calcitonin receptor), which regulates osteoclast survival and resorption, is a novel miR-29 target. Thus, miR-29 is a positive regulator of osteoclast formation and targets RNAs important for cytoskeletal organization, commitment, and osteoclast function. We hypothesize that miR-29 controls the tempo and amplitude of osteoclast differentiation.
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http://dx.doi.org/10.1074/jbc.M113.484568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829182PMC
November 2013

Identification, characterization, and isolation of a common progenitor for osteoclasts, macrophages, and dendritic cells from murine bone marrow and periphery.

J Bone Miner Res 2013 May;28(5):1203-13

Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030-3710, USA.

Osteoclasts are specialized bone-resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype B220(-) CD3(-) CD11b(-/low) CD115(+) and either CD117(hi), CD117(intermediate), or CD117(low). We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single-cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages, and antigen-presenting dendritic cells in vitro with efficiencies of more than 90%, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages, and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined.
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http://dx.doi.org/10.1002/jbmr.1822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625454PMC
May 2013

Deletion of CD74, a putative MIF receptor, in mice enhances osteoclastogenesis and decreases bone mass.

J Bone Miner Res 2013 Apr;28(4):948-59

UCONN Center on Aging, University of Connecticut Health Center, Farmington, CT 06030-1835, USA.

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF-regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild-type (WT) and CD74-deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8-week-old mice was measured by micro-computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast-like cells (OCLs) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate-resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL-induced-signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL-induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c-Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL-induced activator protein 1 (AP-1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis.
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http://dx.doi.org/10.1002/jbmr.1787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563845PMC
April 2013

Changes in bone sclerostin levels in mice after ovariectomy vary independently of changes in serum sclerostin levels.

J Bone Miner Res 2013 Mar;28(3):618-26

Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030-5456, USA.

We examined the effects that ovariectomy had on sclerostin mRNA and protein levels in the bones of 8-week-old mice that were either sham-operated (SHAM) or ovariectomized (OVX) and then euthanized 3 or 6 weeks later. In this model, bone loss occurred between 3 and 5 weeks postsurgery. In calvaria, ovariectomy significantly decreased sclerostin mRNA levels at 6 weeks postsurgery (by 52%) but had no significant effect at 3 weeks. In contrast, sclerostin mRNA levels were significantly lower in OVX femurs at 3 weeks postsurgery (by 53%) but equal to that of SHAM at 6 weeks. The effects of ovariectomy on sclerostin were not a global response of osteocytes because they were not mimicked by changes in the mRNA levels for two other relatively osteocyte-specific genes: DMP-1 and FGF-23. Sclerostin protein decreased by 83% and 60%, at 3 and 6 weeks postsurgery in calvaria, respectively, and by 38% in lumbar vertebrae at 6 weeks. We also detected decreases in sclerostin by immunohistochemistry in cortical osteocytes of the humerus at 3 weeks postsurgery. However, there were no significant effects of ovariectomy on sclerostin protein in femurs or on serum sclerostin at 3 and 6 weeks postsurgery. These results demonstrate that ovariectomy has variable effects on sclerostin mRNA and protein in mice, which are dependent on the bones examined and the time after surgery. Given the discrepancy between the effects of ovariectomy on serum sclerostin levels and sclerostin mRNA and protein levels in various bones, these results argue that, at least in mice, serum sclerostin levels may not accurately reflect changes in the local production of sclerostin in bones. Additional studies are needed to evaluate whether this is also the case in humans.
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http://dx.doi.org/10.1002/jbmr.1773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554870PMC
March 2013

Negative regulation of osteoclast precursor differentiation by CD11b and β2 integrin-B-cell lymphoma 6 signaling.

J Bone Miner Res 2013 Jan;28(1):135-49

Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY, USA.

Negative regulation of osteoclastogenesis is important for bone homeostasis and prevention of excessive bone resorption in inflammatory and other diseases. Mechanisms that directly suppress osteoclastogenesis are not well understood. In this study we investigated regulation of osteoclast differentiation by the β2 integrin CD11b/CD18 that is expressed on myeloid lineage osteoclast precursors. CD11b-deficient mice exhibited decreased bone mass that was associated with increased osteoclast numbers and decreased bone formation. Accordingly, CD11b and β2 integrin signaling suppressed osteoclast differentiation by preventing receptor activator of NF-κB ligand (RANKL)-induced induction of the master regulator of osteoclastogenesis nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and of downstream osteoclast-related NFATc1 target genes. CD11b suppressed induction of NFATc1 by the complementary mechanisms of downregulation of RANK expression and induction of recruitment of the transcriptional repressor B-cell lymphoma 6 (BCL6) to the NFATC1 gene. These findings identify CD11b as a negative regulator of the earliest stages of osteoclast differentiation, and provide an inducible mechanism by which environmental cues suppress osteoclastogenesis by activating a transcriptional repressor that makes genes refractory to osteoclastogenic signaling.
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http://dx.doi.org/10.1002/jbmr.1739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522783PMC
January 2013

Osteoblast-specific overexpression of human interleukin-7 rescues the bone mass phenotype of interleukin-7-deficient female mice.

J Bone Miner Res 2012 May;27(5):1030-42

Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030-1601, USA.

Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3-kb rat collagen 1α1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition, we crossed these mice with IL-7-deficient mice to determine if the alterations in lymphopoiesis, bone mass, and osteoclast formation observed in the IL-7 knockout (KO) mice could be rescued by osteoblast-specific overexpression of IL-7. Here, we show that mice overexpressing human IL-7 in the osteoblast lineage showed increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B-cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7-overexpressing mouse models were observed only in females. These results likely reflect both direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and sex-specific differences in responses to IL-7.
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http://dx.doi.org/10.1002/jbmr.1553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361560PMC
May 2012

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow.

J Bone Miner Res 2011 Jun;26(6):1207-16

Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030, USA.

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1-34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115(+) CD117(high) and TN/CD115(+) CD117(int) cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115(+) CD117(low) cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF-κB ligand (RANKL)- and macrophage colony-stimulating factor (M-CSF)-stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115(+) cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin(-) CD117(+) Sca-1(+) ) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M-CSF and RANKL.
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http://dx.doi.org/10.1002/jbmr.324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312755PMC
June 2011

Macrophage migration inhibitory factor inhibits osteoclastogenesis.

Bone 2009 Oct 7;45(4):640-9. Epub 2009 Jul 7.

Center for Immunotherapy, University of Connecticut Health Center, Farmington, CT, USA.

MIF is an important regulator of innate and adaptive immunity, which is produced by a variety of cell types including activated T cells and macrophages. We examined the effects of MIF on osteoclastogenesis in bone marrow (BM) cultures from WT and MIF-deficient (KO) mice as well as the bone mass of MIF KO mice. Exogenous MIF inhibited osteoclast formation in BM cultures by decreasing fusion in cells that were treated with M-CSF and RANKL. However, inhibition of OCL formation by MIF treatment was not mediated by fusion-related molecules in heterogeneous bone marrow cultures. BM cultures from MIF KO mice that were treated with M-CSF and RANKL, PTH or vitamin D had significantly increased OCL number compared to cells from WT mice. MIF also significantly inhibited OCL formation in cultures of RAW 264.7 cells that were treated with RANKL. In addition, the number of CFU-GM and Mac-1(+) cells in the BM of MIF KO mice was greater than in WT controls. Trabecular bone volume (TBV) in the femurs and vertebrae of MIF KO mice was decreased compared to WT mice. In addition, serum bone resorption and formation markers were decreased in MIF KO mice compared to WT mice. These results demonstrate that MIF has inhibitory effects on OCL formation in vitro. We also found that BM cell cultures from MIF KO mice had an increased capacity to form osteoclasts. Furthermore, MIF KO animals had significantly decreased TBV with low turnover. We conclude that MIF is an inhibitor of osteoclastogenesis in vitro, which may regulate bone turnover via indirect mechanism in vivo.
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http://dx.doi.org/10.1016/j.bone.2009.06.028DOI Listing
October 2009

CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent.

J Immunol 2007 Aug;179(4):2203-14

Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030, USA.

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.
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http://dx.doi.org/10.4049/jimmunol.179.4.2203DOI Listing
August 2007
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