Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand.

The bone loss induced by ovariectomy (ovx) has been linked to increased production of osteoclastogenic cytokines by bone marrow cells, including T cells and stromal cells (SCs). It is presently unknown whether regulatory interactions between these lineages contribute to the effects of ovx in bone, however. Here, we show that the T-cell costimulatory molecule CD40 ligand (CD40L) is required for ovx to expand SCs; promote osteoblast proliferation and differentiation; regulate the SC production of the osteoclastogenic factors macrophage colony-stimulating factor, receptor activator of nuclear factor-κB ligand, and osteoprotegerin; and up-regulate osteoclast formation. CD40L is also required for ovx to activate T cells and stimulate their production of TNF. Accordingly, ovx fails to promote bone loss and increase bone resorption in mice depleted of T cells or lacking CD40L. Therefore, cross-talk between T cells and SCs mediated by CD40L plays a pivotal role in the disregulation of osteoblastogenesis and osteoclastogenesis induced by ovx.

NFAT signaling in osteoblasts regulates the hematopoietic niche in the bone microenvironment.

Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic development in vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFAT(OB)). Bone histomorphometry showed that dnNFAT(OB) mice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFAT(OB) mice displayed a significant increase (87%) in Lineage(-)cKit(+)Sca-1(+) (LSK) cells and significant decreases in B220(+)CD19(-)IgM(-) pre-pro-B cells (41%) and B220(+)CD19(+)IgM(+) immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220(+) cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFAT(OB) mice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFAT(OB) mice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.

Alterations in the immuno-skeletal interface drive bone destruction in HIV-1 transgenic rats.

Osteoporosis and bone fractures are increasingly recognized complications of HIV-1 infection. Although antiretroviral therapy itself has complex effects on bone turnover, it is now evident that the majority of HIV-infected individuals already exhibit reduced bone mineral density before therapy. The mechanisms responsible are likely multifactorial and have been difficult to delineate in humans. The HIV-1 transgenic rat recapitulates many key features of human AIDS. We now demonstrate that, like their human counterparts, HIV-1 transgenic rats undergo severe osteoclastic bone resorption, a consequence of an imbalance in the ratio of receptor activator of NF-kappaB ligand, the key osteoclastogenic cytokine, to that of its physiological decoy receptor osteoprotegerin. This imbalance stemmed from a switch in production of osteoprotegerin to that of receptor activator of NF-kappaB ligand by B cells, and was further compounded by a significantly elevated number of osteoclast precursors. With the advancing age of individuals living with HIV/AIDS, low bone mineral density associated with HIV infection is likely to collide with the pathophysiology of skeletal aging, leading to increased fracture risk. Understanding the mechanisms driving bone loss in HIV-infected individuals will be critical to developing effective therapeutic strategies.

Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-ß activation.

Transforming growth factor-ß (TGF-ß) is a critical regulator of bone development and remodeling. TGF-ß must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-ß activation and TSP1 control of TGF-ß activation is critical for regulation of TGF-ß activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-ß inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-ß activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-ß. Cultured MSCs express TSP1 and both TSP1 expression and TGF-ß activity decrease during osteoblast differentiation. TSP1 and active TGF-ß block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-ß, since a peptide which blocks TSP1 TGF-ß activation reduced TGF-ß activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-ß neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-ß activity is a critical determinant of osteoblast differentiation.

Snail-mediated regulation of reactive oxygen species in ARCaP human prostate cancer cells.

Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial-mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real Time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldehyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7 days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3 days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.

Ex vivo transfer of the Hoxc-8-interacting domain of Smad1 by a tropism-modified adenoviral vector results in efficient bone formation in a rabbit model of spinal fusion.

STUDY DESIGN: Ex vivo gene transfer for spinal fusion. OBJECTIVE: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion. SUMMARY OF BACKGROUND DATA: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer. METHODS: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to alphav integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing beta-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software. RESULTS: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector. CONCLUSIONS: Delivery of the Smad1C gene construct to BMSCs by an alphav integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

Mitochondrial DNA mutation stimulates prostate cancer growth in bone stromal environment.

BACKGROUND AND OBJECTIVES: Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment. METHODS: The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wild-type and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry. RESULTS: Cybrids with the 8,993 mtDNA mutation grew faster than wild-type cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A 37 gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present. CONCLUSIONS: The ATP6 np 8,993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.

BKM1740, an acyl-tyrosine bisphosphonate amide derivative, inhibits the bone metastatic growth of human prostate cancer cells by inducing apoptosis.

PURPOSE: Survivin overexpression has been associated with an unfavorable outcome in human PCa; however, its role in metastasis remains elusive. We aim to (a) evaluate the clinical implications of survivin expression in PCa bone metastasis; (b) determine in vivo efficacy of BKM1740, a small-molecule compound, against PCa skeletal growth and survival; and (c) investigate molecular mechanism by which BKM1740 augments apoptosis in bone metastatic PCa cells. EXPERIMENTAL DESIGN: Survivin expression was analyzed in PCa specimens and experimental models. Bone metastatic C4-2 and ARCaP(M) cell lines were used to evaluate the in vitro effects of BKM1740 and molecular mechanism for the induction of apoptosis. C4-2 cells were grown intratibially in athymic nude mice to evaluate the in vivo efficacy of BKM1740. Tumor growth in mouse bone was assessed by serum prostate-specific antigen and radiography and confirmed by immunohistochemical analyses. RESULTS: Survivin expression is positively associated with clinical PCa bone metastasis. BKM1740 induced apoptosis in PCa cells by repressing survivin. Mice with established C4-2 tumors in tibia showed a marked decrease in serum prostate-specific antigen and much improved bone architecture radiographically after treatment with BKM1740. Immunohistochemical assays of mouse tumor samples confirmed that the in vivo effects were mediated by inhibition of survivin and induction of apoptosis. CONCLUSIONS: Survivin expression is associated with PCa bone metastasis. BKM1740 treatment specifically inhibited survivin and induced apoptosis in vitro and was efficacious in retarding PCa skeletal growth in a mouse model. BKM1740 is a promising small-molecule compound that could be used to treat PCa bone metastasis.

beta2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis.

The protein factor beta2-microglobulin (beta2M), purified from the conditioned medium of human prostate cancer cell lines, stimulated growth and enhanced osteocalcin (OC) and bone sialoprotein (BSP) gene expression in human prostate cancer cells by activating a cyclic AMP (cAMP)-dependent protein kinase A signaling pathway. When beta2M was overexpressed in prostate cancer cells, it induced explosive tumor growth in mouse bone through increased phosphorylated cAMP-responsive element binding protein (CREB) and activated CREB target gene expression, including OC, BSP, cyclin A, cyclin D1, and vascular endothelial growth factor. Interrupting the beta2M downstream signaling pathway by injection of the beta2M small interfering RNA liposome complex produced an effective regression of previously established prostate tumors in mouse bone through increased apoptosis as shown by immunohistochemistry and activation of caspase-9, caspase-3, and cleavage of poly(ADP-ribose) polymerase. These results suggest that beta2M signaling is an attractive new therapeutic target for the treatment of lethal prostate cancer bone metastasis.

Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation.

Cyclosporin A (CsA) is thought to prevent immune reactions after organ transplantation by inhibiting calcineurin (Cn) and its substrate, the Nuclear Factor of Activated T Cells (NFAT). A dichotomy exists in describing the effects of CsA on bone formation. The concept that the suppression of Cn/NFAT signaling by CsA inhibits bone formation is not entirely supported by many clinical reports and laboratory animal studies. Gender, dosage and basal inflammatory activity have all been suggested as explanations for these seemingly contradictory reports. Here we examine the effects of varying concentrations of CsA on bone formation and osteoblast differentiation and elucidate the role of NFATc1 in this response. We show that low concentrations of CsA (<1 microM in vitro and 35.5 nM in vivo) are anabolic as they increase bone formation, osteoblast differentiation, and bone mass, while high concentrations (>1 microM in vitro and in vivo) elicit an opposite and catabolic response. The overexpression of constitutively active NFATc1 inhibits osteoblast differentiation, and treatment with low concentrations of CsA does not ameliorate this inhibition. Treating osteoblasts with low concentrations of CsA (<1 microM) increases fra-2 gene expression and protein levels in a dose-dependent manner as well as AP-1 DNA-binding activity. Finally, NFATc1 silencing with siRNA increases Fra-2 expression, whereas NFATc1 overexpression inhibits Fra-2 expression. Therefore, NFATc1 negatively regulates osteoblast differentiation, and its specific inhibition may represent a viable anabolic therapy for osteoporosis.

Beta2-microglobulin promotes the growth of human renal cell carcinoma through the activation of the protein kinase A, cyclic AMP-responsive element-binding protein, and vascular endothelial growth factor axis.

PURPOSE: Beta(2)-microglobulin (beta2M), a soluble protein secreted by cancer and host inflammatory cells, has various biological functions, including antigen presentation. Because aberrant expression of beta2M has been reported in human renal cell carcinoma, we investigated the effects of beta2M overexpression on cancer cell growth and analyzed its molecular signaling pathway. EXPERIMENTAL DESIGN: We established clonal cell lines that overexpressed beta2M in human renal cell carcinoma (SN12C) cells and then examined cell growth in vitro and in vivo and studied the beta2M-mediated downstream cell signaling pathway. RESULTS: Our results showed that beta2M expression positively correlates with (a) in vitro growth on plastic dishes and as Matrigel colonies, (b) cell invasion and migration in Boyden chambers, and (c) vascular endothelial growth factor (VEGF) expression and secretion by cells. We found, in addition, that beta2M mediates its action through increased phosphorylation of cyclic AMP-responsive element-binding protein (CREB) via the protein kinase A-CREB axis, resulting in increased VEGF expression and secretion. In convergence with this signal axis, beta2M overexpression also activated both phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Beta2M overexpression induced accelerated growth of SN12C in mouse subcutis and bone. Interrupting the beta2M signaling pathway using small interfering RNA led to apoptosis with increased activation of caspase-3 and caspase-9 and cleaved poly(ADP-ribose) polymerase. CONCLUSIONS: Our results showed for the first time that the beta2M-protein kinase A-CREB-VEGF signaling axis plays a crucial role in support of renal cell carcinoma growth and progression and reveals a novel therapeutic target.

Bone Metastasis of Prostate Cancer Can Be Therapeutically Targeted at the TBX2-WNT Signaling Axis.

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331-44. ©2017 AACR.

NFATc1: a novel anabolic therapeutic target for osteoporosis.

Bone loss and osteoporosis are major public health problems in the elderly. With increasing life expectancy in the United States, the number of people that will develop age-related bone loss and osteoporosis is expected to rise to over 61 million by 2020. Osteoblast differentiation is a crucial aspect of bone formation and remodeling, a process severely compromised in osteoporosis. Almost all the FDA-approved treatments for building healthier bones, excluding parathyroid hormone (PTH), do not address the decrease in osteoblast differentiation seen in osteoporosis and rather are designed to target osteoclasts and bone resorption. The purpose of this study is to examine the effects of NFAT inhibition on osteoblast differentiation and to elucidate the mechanism of its action. Here we demonstrate that the inhibition of calcineurin (Cn) by using cyclosporine A (CsA) increases osteoblast differentiation, both in vivo and in vitro. Furthermore, the specific inhibition of NFATc1 by siRNA increased Fra-2 expression in osteoblasts. Taken together, our results point the way to a novel mechanism to aid in the development of anabolic treatment for osteoporosis.

Perlecan/HSPG2 and matrilysin/MMP-7 as indices of tissue invasion: tissue localization and circulating perlecan fragments in a cohort of 288 radical prostatectomy patients.

Prostate cancer (PCa) cells use matrix metalloproteinases (MMPs) to degrade tissue during invasion. Perlecan/HSPG2 is degraded at basement membranes, in reactive stroma and in bone marrow during metastasis. We previously showed MMP-7 efficiently degrades perlecan. We now analyzed PCa tissue and serum from 288 prostatectomy patients of various Gleason grades to decipher the relationship between perlecan and MMP-7 in invasive PCa. In 157 prostatectomy specimens examined by tissue microarray, perlecan levels were 18% higher than their normal counterparts. In Gleason grade 4 tissues, MMP-7 and perlecan immunostaining levels were highly correlated with each other (average correlation coefficient of 0.52) in PCa tissue, regardless of grade. Serial sections showed intense, but non-overlapping, immunostaining for MMP-7 and perlecan at adjacent borders, reflecting the protease-substrate relationship. Using a capture assay, analysis of 288 PCa sera collected at prostatectomy showed elevated levels of perlecan fragments, with most derived from domain IV. Perlecan fragments in PCa sera were associated with overall MMP-7 staining levels in PCa tissues. Domain IV perlecan fragments were present in stage IV, but absent in normal, sera, suggesting perlecan degradation during metastasis. Together, perlecan fragments in sera and MMP-7 in tissues of PCa patients are measures of invasive PCa.

RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity.

UNLABELLED: Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity. INTRODUCTION: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects. MATERIALS AND METHODS: hMSCs were seeded onto plastic microcarrier beads at a density of 10(6) and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM beta-glycerol phosphate, and 50 muM ascorbic acid 2-phosphate. RESULTS: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88+/-2% and 77+/-9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG. CONCLUSION: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.

RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.

UNLABELLED: Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown. INTRODUCTION: Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts. MATERIALS AND METHODS: RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA. RESULTS: Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated. CONCLUSION: RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.

The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss.

HIV infection causes bone loss. We previously reported that immunosuppression-mediated B-cell production of receptor activator of NF-κB ligand (RANKL) coupled with decline in osteoprotegerin correlate with decreased bone mineral density (BMD) in untreated HIV infection. Paradoxically, antiretroviral therapy (ART) worsens bone loss although existing data suggest that such loss is largely independent of specific antiretroviral regimen. This led us to hypothesize that skeletal deterioration following HIV disease reversal with ART may be related to T-cell repopulation and/or immune reconstitution. Here we transplant T cells into immunocompromised mice to mimic ART-induced T-cell expansion. T-cell reconstitution elicits RANKL and TNFα production by B cells and/or T cells, accompanied by enhanced bone resorption and BMD loss. Reconstitution of TNFα- or RANKL-null T-cells and pharmacological TNFα antagonist all protect cortical, but not trabecular bone, revealing complex effects of T-cell reconstitution on bone turnover. These findings suggest T-cell repopulation and/or immune reconstitution as putative mechanisms for bone loss following ART initiation.

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis.

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

CTLA-4Ig-induced T cell anergy promotes Wnt-10b production and bone formation in a mouse model.

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by severe joint erosion and systemic osteoporosis. Chronic T cell activation is a hallmark of RA, and agents that target the CD28 receptor on T cells, which is required for T cell activation, are being increasingly used as therapies for RA and other inflammatory diseases. Lymphocytes play complex roles in the regulation of the skeleton, and although activated T cells and B cells secrete cytokines that promote skeletal decline, under physiologic conditions lymphocytes also have key protective roles in the stabilization of skeletal mass. Consequently, disruption of T cell costimulation may have unforeseen consequences for physiologic bone turnover. This study was undertaken to investigate the impact of pharmacologic CD28 T cell costimulation blockade on physiologic bone turnover and structure. METHODS: C57BL6 mice were treated with CTLA-4Ig, a pharmacologic CD28 antagonist or with irrelevant control antibody (Ig), and serum biochemical markers of bone turnover were quantified by enzyme-linked immunosorbent assay. Bone mineral density and indices of bone structure were further measured by dual x-ray absorptiometry and micro-computed tomography, respectively, and static and dynamic indices of bone formation were quantified using bone histomorphometry. RESULTS: Pharmacologic disruption of CD28 T cell costimulation in mice significantly increased bone mass and enhanced indices of bone structure, a consequence of enhanced bone formation, concurrent with enhanced secretion of the bone anabolic factor Wnt-10b by T cells. CONCLUSION: Inhibition of CD28 costimulation by CTLA-4Ig promotes T cell Wnt-10b production and bone formation and may represent a novel anabolic strategy for increasing bone mass in osteoporotic conditions.