The utility of surrogate markers in predicting HLA alleles associated with adverse drug reactions in Vietnamese.

BACKGROUND: Screening for HLA-A*31:01/HLA-B*15:02, HLA-B*57:01 and HLA-B*58:01 is recommended in selected populations for prevention of carbamazepine, abacavir, and allopurinol-induced severe cutaneous adverse reactions (SCARs). Compared to conventional methods for detection of HLA alleles, PCR using a tag single nucleotide polymorphism (SNP) can be cost-effective, particularly where the surrogate marker SNP is in absolute linkage disequilibrium with the relevant HLA allele. OBJECTIVE: To determine guidelines for prevention of SCARs though predictive screening for the Australian Vietnamese population, the prevalence of four HLA alleles (HLA-A*31:01, HLA-B*15:02, HLA-B*57:01 and HLA-B*58:01) was examined. The utility of surrogate markers, rs2395029 and rs9263726, was investigated to predict for the presence of HLA-B*57:01 and HLA-B*58:01, respectively. METHODS: Genotyping for specific HLA alleles was performed in 152 healthy Vietnamese living in Sydney using validated and established PCR-based methods. SNP genotyping was conducted using restriction-fragment-length-polymorphism analysis. RESULTS: rs2395029 and rs9263726 strongly correlated with HLA-B*57:01 (κ = 1, p < 0.001) and HLA-B*58:01 (Κ = 0.9, p < 0.001) with 100% sensitivity and 100% negative predictive value for predicting the HLA-B*57:01 and HLA-B*58:01 carriers, respectively. A high prevalence of carriers of HLA-A*31:01 (3.29%), HLA-B*15:02 (14.47%), HLA-B*57:01 (6.58%) and HLA-B*58:01 (9.21%) was revealed. Conclusions: Screening is recommended for these alleles in Australian Vietnamese prior to introducing relevant therapies. SNPs, rs2395029 and rs9263726, can be successfully used as surrogate markers for HLA-B*57:01 and HLA-B*58:01 in this population.

The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation.

Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro-osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic differentiation were performed. MAN1 knockdown increased osteogenesis and mineralization. In contrast, osteogenesis remained stable upon MAN1 overexpression. Regarding a mechanism, we found that low levels of MAN1 facilitated the nuclear accumulation of regulatory smads and smads-related complexes, with a concurrently high expression of nuclear ß-Catenin. In addition, we found adipogenesis to be decreased in both conditions, although predominantly affected by MAN1 overexpression. Finally, lamin A, a protein of the nuclear envelope that regulates MSC differentiation, was unaffected by changes in MAN1. In conclusion, our studies demonstrated that lower levels of MAN1 in differentiating MSC are associated with higher osteogenesis and lower adipogenesis. High levels of MAN1 only affected adipogenesis. These effects could have an important role in the understanding of the role of the proteins of the nuclear envelope in bone formation. J. Cell. Biochem. 118: 4425-4435, 2017. © 2017 Wiley Periodicals, Inc.

The kynurenine pathway of tryptophan degradation is activated during osteoblastogenesis.

The mechanisms involved in the anabolic effect of interferon gamma (IFNγ) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNγ upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNγ. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNγ in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1(-/-)) mice. Compared to their wild-type littermates, Ido1(-/-) mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNγ. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo.

Lamin A/C Acts as an Essential Factor in Mesenchymal Stem Cell Differentiation Through the Regulation of the Dynamics of the Wnt/ß-Catenin Pathway.

Changes in the expression of lamin A/C, a fibrilar protein of the nuclear envelope, are associated with the cellular features of age-related bone loss. Reduced expression of lamin A/C inhibits osteoblastogenesis while facilitating adipogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. In this study we investigated the regulatory role that lamin A/C plays on the essential elements of the Wnt/ß-catenin pathway, which are pivotal in MSC differentiation. Initially, we assessed the effect of lamin A/C gene (LMNA) overexpression on MSC differentiation while compared it to lamin A/C depleted MSC. Osteogenesis and gene expression of osteogenic factors were higher in LMNA-transfected MSC as compared to control. Conversely, adipogenesis and expression of adipogenic factors were significantly lower in LMNA transfected cells. Nuclear ß-catenin was significantly higher (∼two fold) in MSC expressing higher levels of LMNA as compared to control with nuclear ß-catenin levels being significantly lower (∼ -42%) in siRNA-treated MSC. Luciferase activity for ß-catenin-mediated transcriptional activation was significantly higher in cells overexpressing LMNA. These data indicate that MSC overexpressing LMNA have higher osteogenic and lower adipogenic differentiation potential. In conclusion, our studies demonstrate that lamin A/C plays a significant role in the differentiation of both osteoblasts and adipocytes by regulating some of the elements of Wnt/ß-catenin signaling during early MSC differentiation.

Interferon gamma inhibits adipogenesis in vitro and prevents marrow fat infiltration in oophorectomized mice.

Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSCs) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin, we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulating osteoblastogenesis in human MSC. To test this hypothesis, human MSCs were induced to differentiate into adipocytes in the presence or absence of osteogenic doses of IFNγ (1, 10, and 100 ng/ml). IFNγ-treated MSC showed a decrease in adipocyte differentiation and lipid deposition when compared with vehicle-treated controls. Additionally, adipogenic markers were significantly decreased by IFNγ treatment at the same doses that have been reported to have a strong osteogenic effect in vitro. Furthermore, DNA binding of peroxisome proliferator-activated receptor gamma was significantly lower in IFNγ-treated differentiating MSC. Subsequently, ovariectomized C57BL6 mice were treated with osteogenic doses of IFNγ three times a week for 6 weeks. In distal femur, treated mice showed significantly higher hematopoiesis concomitant with lower levels of fat volume/total volume, adipocyte number, and expression of adipogenic markers when compared with the vehicle-treated mice. Together, these findings demonstrate that, at osteogenic doses, IFNγ also acts as an inhibitor of adipogenesis in vitro and prevents marrow fat infiltration while favors hematopoiesis in ovariectomized mice.

Genetic susceptibilities and prediction modeling of carbamazepine and allopurinol-induced severe cutaneous adverse reactions in Vietnamese.

Aims: To determine genetic susceptibility markers for carbamazepine (CBZ) and allopurinol-induced severe cutaneous adverse reactions (SCARs) in Vietnamese. Methods: A case-control study was performed involving 122 patients with CBZ or allopurinol-induced SCARs and 120 drug tolerant controls. Results:HLA-B*58:01 was strongly associated with allopurinol-induced SCARs and strongly correlated with SNP rs9263726. HLA-B*15:02 was associated with CBZ-induced Stevens-Johnson syndrome/toxic epidermal necrolysis but not with drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms. No association was found between HLA-A*31:01 and CBZ-induced SCARs. HLA-B*58:01 and rs3909184 allele A with renal insufficiency were shown to increase the risk of allopurinol-induced SCARs. Conclusion:HLA-B*58:01 and HLA-B*15:02 confer susceptibility to allopurinol-induced SCARs and CBZ-induced SJS/TEN in Vietnamese. SNP rs9263726 can be used as a surrogate marker in identifying HLA-B*58:01.

Picolinic Acid, a Catabolite of Tryptophan, Has an Anabolic Effect on Bone In Vivo.

Fractures attributable to osteoporosis have a severe impact on our older population. Reports of side effects with commonly prescribed osteoporosis drugs have led to the investigation of new and safer treatments with novel mechanisms of action. Picolinic acid (PIC), a catabolite of tryptophan, induces in vitro osteogenic differentiation of mesenchymal stem cells. Here we demonstrate that PIC has an anabolic effect on bone in vivo by increasing bone formation, bone mass, and bone strength in normal and ovariectomized C57BL/6 mice. Activation of the osteogenic pathways triggered this osteoanabolic response without any cross-related effects on mineral absorption or calciotropic hormones. Because PIC was also well tolerated and absorbed with no side effects, it is an ideal potential candidate for the treatment of osteoporosis. © 2020 American Society for Bone and Mineral Research.

Gene expression profiling in allopurinol-induced severe cutaneous adverse reactions in Vietnamese.

Aim: To examine gene expression in different clinical phenotypes of allopurinol-induced severe cutaneous adverse reactions (SCARs). Materials & methods: Gene expression profiling was performed using microarray on 11 RNA samples (four controls, three hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms, four Stevens-Johnson syndrome/toxic epidermal necrolysis) followed by quantitative real-time PCR in a total of 11 SCARs patients and 11 controls. Results: The biological pathways which were significantly enriched in differentially expressed genes in Stevens-Johnson syndrome/toxic epidermal necrolysis compared with hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms patients included; cell surface interactions at the vascular wall, immunoregulatory interactions at the immunological synapse and MyD88 signaling pathways. Overexpression of miR146a occurred in allopurinol-tolerant HLA-B*58:01 carriers. Conclusion: Biological pathways are identified which appear to be implicated in determining clinical phenotypes in allopurinol-induced SCARs. Overexpression of miR146a is potentially important for allopurinol tolerance in HLA-B*58:01 carriers.

Human leukocyte antigen-associated severe cutaneous adverse drug reactions: from bedside to bench and beyond.

Despite their being uncommon, severe cutaneous adverse drug reactions (SCARs) result in a very great burden of disease. These reactions not only carry with them a high mortality (10%-50%) and high morbidity (60%) with severe ocular complications, alopecia, oral and dental complications and development of autoimmune diseases, but also create a substantial economic burden for patients' families and society. SCARs are, therefore, an important medical problem needing a solution in many countries, especially in Asia. The clinical spectrum of SCARs comprises Stevens-Johnson syndrome, toxic epidermal necrolysis, DRESS (drug rash with eosinophilia and systemic symptoms) (also known as drug hypersensitivity syndrome or drug-induced hypersensitivity syndrome) and acute generalised exanthematous pustulosis. Recent crucial advances in determining genetic susceptibility and understanding how T cells recognise certain medications or their metabolites via the major histocompatibility complex and the effects of cofactors, have led to the implementation of cost-effective screening programs enabling prevention in a number of countries, and to further understanding of the patho-mechanisms involved in SCARs and their significance. In this review, we document comprehensively the journey of SCARs from bedside to bench and outline future perspectives in SCARs research.

Developing pharmacogenetic screening methods for an emergent country: Vietnam.

BACKGROUND: The finding of strong associations between certain human leukocyte antigen (HLA) genotypes and the development of severe cutaneous adverse drug reactions (SCARs), [for example, HLA-B*57:01 and abacavir (ABC), HLA-B*15:02 and carbamazepine (CBZ) and HLA-B*58:01 and allopurinol], has led to HLA screening being used to prevent SCARs. Screening has been shown to be of great benefit in a number of studies. Clinical translation from bench to bedside, however, depends upon the development of simple, rapid and cost-effective assays to detect these risk alleles. In highly populated developing countries such as Vietnam, where there is a high prevalence of HLA-B*15:02 and HLA-B*58:01 correlating with a high incidence of CBZ- and allopurinol-induced SCARs, the crucial factor in the implementation of comprehensive screening programs to detect these major risk HLA alleles is the availability of suitable assays. BODY: We have summarized the role and economic benefits of HLA screening, reviewed published HLA screening methods used currently in pharmacogenetic screening and examined the advantages and disadvantages of assays developed specifically for use in screening for risk alleles in the prevention of HLA-associated SCARs in Vietnam. CONCLUSION: The optimal approach we propose may serve as a template for the development of screening programs in other emergent countries.

Treatment with an inhibitor of fatty acid synthase attenuates bone loss in ovariectomized mice.

Bone and fat cells have an antagonistic relationship. Adipocytes exert a toxic effect on bone cells in vitro through the secretion of fatty acids, which are synthesized by fatty acid synthase (FAS). Inhibition of FAS in vitro rescues osteoblasts from fat-induced toxicity and cell death. In this study, we hypothesized that FAS inhibition would mitigate the loss of bone mass in ovariectomized (OVX) mice. We treated OVX C57BL/6 mice with cerulenin (a known inhibitor of FAS) for 6 weeks and compared their bone phenotype with vehicle-treated controls. Cerulenin-treated mice exhibited a significant decrease in body weight, triglycerides, leptin, and marrow and subcutaneous fat without changes in serum glucose or calciotropic hormones. These effects were associated with attenuation of bone loss and normalization of the bone phenotype in the cerulenin-treated OVX group compared to the vehicle-treated OVX group. Our results demonstrate that inhibition of FAS enhances bone formation, induces uncoupling between osteoblasts and osteoclasts, and favors mineralization, thus providing evidence that inhibition of FAS could constitute a new anabolic therapy for osteoporosis.

Refined mapping of X-linked reticulate pigmentary disorder and sequencing of candidate genes.

X-linked reticulate pigmentary disorder with systemic manifestations in males (PDR) is very rare. Affected males are characterized by cutaneous and visceral symptoms suggestive of abnormally regulated inflammation. A genetic linkage study of a large Canadian kindred previously mapped the PDR gene to a greater than 40 Mb interval of Xp22-p21. The aim of this study was to identify the causative gene for PDR. The Canadian pedigree was expanded and additional PDR families recruited. Genetic linkage was performed using newer microsatellite markers. Positional and functional candidate genes were screened by PCR and sequencing of coding exons in affected males. The location of the PDR gene was narrowed to a approximately 4.9 Mb interval of Xp22.11-p21.3 between markers DXS1052 and DXS1061. All annotated coding exons within this interval were sequenced in one affected male from each of the three multiplex families as well as one singleton, but no causative mutation was identified. Sequencing of other X-linked genes outside of the linked interval also failed to identify the cause of PDR but revealed a novel nonsynonymous cSNP in the GRPR gene in the Maltese population. PDR is most likely due to a mutation within the linked interval not affecting currently annotated coding exons.

Linkage to chromosome 11p12 in two Maltese families with a highly penetrant form of osteoporosis.

Osteoporosis is a metabolic bone disease with a strong genetic component. Family-based linkage studies were performed by a number of investigators to try to identify loci that might contain genes responsible for an increased susceptibility to osteoporosis. A whole-genome linkage scan using 400 microsatellite markers was performed in 27 members from two Maltese families with a highly penetrant form of osteoporosis. The phenotype was defined by lumbar and femoral z-scores calculated after measurement of bone mineral density by DEXA. Both males and females were among the affected individuals. Multipoint parametric and non-parametric linkage analyses were performed by EasyLinkage v4.01 using GENEHUNTER v2.1, assuming dominant and recessive modes of inheritance with variable penetrance. Evidence of linkage was observed to a marker at 11p12 where a non-parametric LOD score of 5.77 (P=0.0006) was obtained. A maximum heterogeneity LOD score of 2.55 for this region was obtained for the dominant mode of inheritance with 90% penetrance and a phenocopy rate of 1%. Following fine mapping, the critical interval was narrowed to a region that is 52.94 cM from 11p-telomere. In this region, the gene for tumour necrosis factor receptor-associated factor 6 (TRAF6) is located approximately 1 cM away from the indicated marker. Sequencing of the promoter region and exons of the TRAF6 gene revealed three sequence variants, one of which was found in three affected members within one family.

Validation of a rapid test for HLA-B*58:01/57:01 allele screening to detect individuals at risk for drug-induced hypersensitivity.

AIM: In prevention of allopurinol and abacavir hypersensitivity, screening HLA-B*58:01/57:01 has been highly recommended prior to commencing these therapies. Therefore, we aimed at developing and validating a rapid and robust screening method for HLA-B*58:01/57:01. MATERIALS & METHODS: Real-time polymerase chain reaction with TaqMan probes was employed to detect HLA-B*58:01/57:01. RESULTS: The newly developed assay has the sensitivity of 100% (95% CI: 79.4-100.0%), the specificity of 98.8% (95% CI: 93.6-99.9%), the positive predictive value of 94.1% (95% CI: 71.3-99.9%) and the negative predictive value of 100.0% (95% CI: 95.7-100.0%). The lowest limit of detection is 0.04 ng/µl of DNA. CONCLUSION: The present method is a rapid and robust assay that is appropriate for screening of HLA-B*58:01/*57:01 alleles.

Association Between Circulating Osteogenic Progenitor Cells and Disability and Frailty in Older Persons: The Nepean Osteoporosis and Frailty Study.

Circulating osteogenic progenitor (COP) cells are considered as surrogates of the mesenchymal repository in the body. In this study, we hypothesized that COP cells decrease with age and that lower levels of COP cells are associated with greater frailty and disability in older persons. Using well-established clinical criteria, we quantified physical performance and disability and stratified frailty in a random sample of community-dwelling individuals enrolled in the Nepean Osteoporosis and Frailty (NOF) Study (mean age 82.8; N = 77; 70% female; 27 nonfrail, 23 prefrail, and 27 frail). Percentage of COP cells was quantified by flow cytometry. Logistic regression models estimated the relationship between the percentage of COP cells and prevalent disability, poor physical performance, and frailty. We found that aging is associated with a significant decrease in COP cells (p < .001). Lower percentages of COP cells were associated with disability and poor physical performance (p < .001). Older adults with COP cells in the lower quartile were more likely to be frail (odds ratio 2.65, 95% confidence interval 2.72-3.15, p < .001). In conclusion, COP cells in the circulation decrease with age. Lower percentages of COP cells in late life are associated with prevalent frailty and disability. Further longitudinal studies are needed to understand COP cells as a risk stratifier, biomarker, or therapeutic target and to predict disability in frail older persons.

Validation of a novel real-time PCR assay for detection of HLA-B*15:02 allele for prevention of carbamazepine - Induced Stevens-Johnson syndrome/Toxic Epidermal Necrolysis in individuals of Asian ancestry.

OBJECTIVES: Screening for the HLA-B*15:02 allele has been recommended to prevent carbamazepine (CBZ) - induced Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) in individuals with Asian ancestry. We aimed, therefore, to develop and validate a robust and inexpensive method for detection of the HLA-B*15:02 allele. METHODS: Real-time PCR using TaqMan® probes followed by SYBR® Green was used to detect the HLA-B*15:02 allele prior to treatment with CBZ therapy. RESULTS: A total of 121 samples were tested. The assay has a sensitivity of 100% (95% CI: 76.84-100.0%), a specificity of 100% (95% CI: 96.61-100%), a positive predictive value of 100% (95% CI: 76.84-100%) and a negative predictive value of 100.0% (95% CI: 96.61-100.0%), respectively. There was 100% agreement between our results and genotyping using Luminex SSO/SBT/SSP. The lowest limit of detection of the TaqMan® probe is 0.05ng/µl and the SYBR® Green is 0.5ng/µl of DNA. The unit cost of using the TaqMan® probe followed by SYBR® Green is only $4.7 USD. CONCLUSION: We developed a novel assay for the detection of the HLA-B*15:02 allele, which is robust, inexpensive and suitable for screening individuals of Asian ancestry in the prevention of CBZ-induced SJS/TEN.

Mechanisms of palmitate-induced lipotoxicity in human osteoblasts.

The interest in the relationship between fat and bone has increased steadily during recent years. Fat could have a lipotoxic effect on bone cells through the secretion of fatty acids. Palmitate is the most prevalent fatty acid secreted by adipocytes in vitro. Considering that palmitate has shown a high lipotoxic effect in other tissues, here we characterized the lipotoxic effect of palmitate on human osteoblasts (Obs). Initially we tested for changes in palmitoylation in this model. Subsequently we compared the capacity of Obs to differentiate and form bone nodules in the presence of palmitate. From a mechanistic approach, we assessed changes in nuclear activity of ß-catenin and runt-related transcription factor 2 (Runx2)/phosphorylated mothers against decapentaplegic (Smad) complexes using Western blotting and confocal microscopy. Quantitative real-time PCR showed negative changes in gene expression of palmitoyltransferase genes. Furthermore, palmitate negatively affected differentiation and bone nodule formation and mineralization by Obs. Although the expression of ß-catenin in palmitate-treated cells was not affected, there was a significant reduction in the transcriptional activities of both ß-catenin and Runx2. Confocal microscopy showed that whereas Runx2 and Smad-4 and -5 complex formation was increased in bone morphogenetic protein-2-treated cells, palmitate had a negative effect on protein expression and colocalization of these factors. In summary, in this study we identified potential mechanisms of palmitate-induced lipotoxicity, which include changes in palmitoylation, defective mineralization, and significant alterations in the ß-catenin and Runx2/Smad signaling pathways. Our evidence facilitates the understanding of the relationship between fat and bone and could allow the development of new potential therapies for osteoporosis in older persons.

Biochemical changes induced by strontium ranelate in differentiating adipocytes.

Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1-2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu.

Mechanisms of palmitate-induced cell death in human osteoblasts.

Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis.

Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice.

Infiltration of bone marrow with fat is a prevalent feature in people with age-related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator-activated receptor gamma (PPARγ). Inhibition of PPARγ thus represents a potential therapeutic approach for age-related bone loss. In this study, we examined the effect of PPARγ inhibition on bone in skeletally mature C57BL/6 male mice. Nine-month-old mice were treated with a PPARγ antagonist, bisphenol-A-diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH](2) D(3) ) for 6 weeks. Micro-computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE + 1,25(OH)(2) D(3) had significantly increased bone volume and improved bone quality compared with vehicle-treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE + 1,25(OH)(2) D(3) -treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca(+) remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARγ may represent an effective anabolic therapy for osteoporosis in the near future.