87 research outputs found

    Circulating levels of dickkopf-1, osteoprotegerin and sclerostin are higher in old compared with young men and women and positively associated with whole-body bone mineral density in older adults

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    Summary: Bone mineral density declines with increasing older age. We examined the levels of circulating factors known to regulate bone metabolism in healthy young and older adults. The circulating levels of dickkopf-1, osteocalcin, osteoprotegerin and sclerostin were positively associated with WBMD in older adults, despite the average WBMD being lower and circulating dickkopf-1, osteoprotegerin and sclerostin being higher in old than young. Purpose: To investigate the relationship between whole-body bone mineral density (WBMD) and levels of circulating factors with known roles in bone remodelling during 'healthy' ageing. Methods: WBMD and fasting plasma concentrations of dickkopf-1, fibroblast growth factor-23, osteocalcin, osteoprotegerin, osteopontin and sclerostin were measured in 272 older subjects (69 to 81 years; 52% female) and 171 younger subjects (18-30 years; 53% female). Results: WBMD was lower in old than young. Circulating osteocalcin was lower in old compared with young, while dickkopf-1, osteoprotegerin and sclerostin were higher in old compared with young. These circulating factors were each positively associated with WBMD in the older adults and the relationships remained after adjustment for covariates (r-values ranging from 0.174 to 0.254, all p<0.01). In multivariate regression, the body mass index, circulating sclerostin and whole-body lean mass together accounted for 13.8% of the variation with WBMD in the older adults. In young adults, dickkopf-1 and body mass index together accounted for 7.7% of variation in WBMD. Conclusion: Circulating levels of dickkopf-1, osteocalcin, osteoprotegerin and sclerostin are positively associated with WBMD in community-dwelling older adults, despite the average WBMD being lower and circulating dickkopf-1, osteoprotegerin and sclerostin being higher in old than young

    Small molecules and targeted therapies in distant metastatic disease

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    Chemotherapy, biological agents or combinations of both have had little impact on survival of patients with metastatic melanoma. Advances in understanding the genetic changes associated with the development of melanoma resulted in availability of promising new agents that inhibit specific proteins up-regulated in signal cell pathways or inhibit anti-apoptotic proteins. Sorafenib, a multikinase inhibitor of the RAF/RAS/MEK pathway, elesclomol (STA-4783) and oblimersen (G3139), an antisense oligonucleotide targeting anti-apoptotic BCl-2, are in phase III clinical studies in combination with chemotherapy. Agents targeting mutant B-Raf (RAF265 and PLX4032), MEK (PD0325901, AZD6244), heat-shock protein 90 (tanespimycin), mTOR (everolimus, deforolimus, temsirolimus) and VEGFR (axitinib) showed some promise in earlier stages of clinical development. Receptor tyrosine-kinase inhibitors (imatinib, dasatinib, sunitinib) may have a role in treatment of patients with melanoma harbouring c-Kit mutations. Although often studied as single agents with disappointing results, new targeted drugs should be more thoroughly evaluated in combination therapies. The future of rational use of new targeted agents also depends on successful application of analytical techniques enabling molecular profiling of patients and leading to selection of likely therapy responders

    Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

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    There is currently no data regarding the expression of specific genes or pathways in purified human osteoblasts that have not been subjected to extensive in vitro culture. Since the latter is likely to alter the RNA expression profile of these cells, we have developed methods to obtain progressively enriched human osteoblast populations within 2-4 hr of obtaining a small needle biopsy (1-2 mm x 1 cm) and coupled this to high-throughput RNA sequencing (RNAseq) and focused QPCR analyses. Needle biopsies were obtained from the posterior iliac crest of 8 human male subjects and subjected to serial collagenase digests. Because the 1st30 minute digest(~100 million cells) contained surface hematopoietic and loosely adherent cells, we used the 2nd60 minute digest (~10 million cells), which contained mineralizing cells and was highly enriched for osteoblast marker genes (col1a1, 5-fold; osteocalcin [ocn],8-fold;bsp, 11-fold; osteopontin [opn], 10-fold). The 2ndfraction was then stained with an AP antibody and the AP+ cells (~1 million cells) were rapidly isolated (within ~2 hr from biopsy) using magnetic activated cell sorting. Relative to AP- cells, the AP+ cells contained all of the mineralizing cells and were further enriched for osteoblast marker genes (AP, 10-fold;col1a1, 1000-fold;ocn, 300-fold;bsp, 300-fold;opn, 50-fold). We then further purified the AP+ cells by depletion of cells expressing CD45, CD34, orCD31 by FACS to obtain AP+/CD45/34/31- (AP+/—) cells (within ~4 hr from biopsy), which represented a highly enriched human osteoblast preparation devoid of hematopoietic/endothelial cells. In addition to in vitro mineralization, AP+/— cells expressed very low to undetectable levels of osteocyte marker genes, including E11,dmp-1, phex, fgf23,andsost.Finally, we used high-throughput RNAseq analysis to compare the transcriptome of the AP+/— cells (n = 6) to human fibroblasts (n = 3). By Ingenuity PathwayAnalysis, we identified a set of unique pathways reflecting genes expressed only inhuman osteoblasts (AP+/— cells)in vivo but not in fibroblasts, including (all P,0.05)Osteoblast Related, VDR/RXR Activation, LXR/RXR Activation, NF-kB Signaling,CXCR4 Signaling, and GPCR Signaling.In summary, we describe a novel approach to isolate and interrogate highly enriched human osteoblast populations without in vitro culture which should bebroadly applicable to studying the pathogenesis of osteoporosis and other metabolic bone diseases

    De la délégation en cascade

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    <p>Selected Pathways of Interest with Known Function in Bone in the “Nuclear ERE-independent” Dataset.</p

    Effects of age on molecular pathways regulating bone formation in humans: A key role for notch and Rorβ signaling

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    Despite extensive studies in mice, there is currently little information on the molecular pathways contributing to age-related bone loss in humans. In part, this stems from the difficulty of obtaining trephine bone biopsies (~5-7 mm diameter) in normal volunteers. Thus, we used the standard clinical approach hematologists employ for bone marrow aspirates and biopsies to obtain needle biopsies (1-2 mm diameter) from the posterior iliac crest in 20 young (30¡5 yr) and 20 old (73¡7 yr)women. We coupled this to customized, in-house QPCR analyses of 288 genes related to bone metabolism, including genes reflecting 17 pre-specified clusters/pathways (e.g.,Wnt targets) and 71 genes linked to SNPs from GWAS studies (Nat Genet 44:491,2012). Genes in pre-specified pathways were analyzed using a cluster analysis (O’Brien Umbrella Test) which tests for concordant changes in multiple genes in the pathway. One of the most highly upregulated pathways in the old women was Notch (P =0.003), which is known to modulate age-related bone loss in mice (Nat Med 14:306,2008). Individual significant (P,0.05) gene changes in this pathway werehes1(1.6x),hey1(1.8x),heyL(1.5x), andJag1(1.2x). In addition, recent studies have identified retinoic acid receptor-related orphan receptorβ (Rorβ) as an important regulator of osteogenesis that is markedly upregulated in bone marrow mesenchymal cells from aged versus young mice (JBMR 27:891, 2012).Rorβ itself (1.6x) as well as multiple Rorβ target genes (P = 0.001 for the pathway) were also upregulated in the biopsies from the old women. Both Notch and Rorβ signaling inhibit runx2 activity, there by potentially blocking osteoblast differentiation. Interestingly, a panel of stem cell markers was significantly upregulated with aging (P = 0.022), including nestin (2.0x),CD146(1.4x), and nanog (1.3x), suggesting that activation of Notch and Rorβ signaling may result in a block in osteoblast differentiation with resultant expansion of the stem cell pool within bone. Of the 71 GWAS genes, 11 were significantly altered with aging, most notablymmp7(4.0x). Other individual gene changes of interest with aging included rankl (1.6x) and fgf23(2.0x).In summary, we describe a novel approach coupling needle biopsies of bone to customized QPCR analyses to study genes/pathways regulating bone metabolism in humans. Our work validates, in humans, several pathways associated with age-related bone loss in mice, including Notch and Rorβ signaling

    Estrogen reduces bone sost mRNA and circulating sclerostin levels in postmenopausal women

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    Studies in postmenopausal women have shown that estrogen (E) reduces circulating sclerostin levels (JBMR 26:27, 2011). However, recent studies in mice(JBMR 28:618, 2013) found no significant effects of ovariectomy on serum sclerostin levels and lack of a relationship between circulating sclerostin and sost mRNA levels in various bones. To resolve this issue in humans, we measured serum sclerostin and bone sost mRNA levels in needle biopsies (1-2 mm diameter) from 20 postmenopausal women (71¡5 yr) treated with transdermal estradiol (0.05mg/d) for 3 weeks and 20untreated control women (73¡7 yr). Serum sclerostin levels were 29% lower (P =0.008) in the E-treated compared to the control women. Concomitantly, bone sost mRNA levels were reduced by 48% (P = 0.03) in the E-treated women. Interestingly ,bone sost mRNA levels were significantly correlated with serum sclerostin levels in the E-treated (r = 0.57, P = 0.008), but not in the control women (r = -0.25, P = 0.280). In addition, mRNA levels of the sclerostin domain-containing protein 1 (sostdc1), a sclerostin-related protein that is another Wnt/BMP inhibitor, were also reduced in the bones of the E-treated compared to the control women (by 54%, P = 0.01).We further extended these studies using customized, in-house QPCR analyses to examine the mRNA expression of genes in other pathways related to bone metabolism, as well as the expression of 71 genes linked to SNPs from GWAS studies (Nat Genet 44:491, 2012). Consistent with studies in mice showing that ovariectomy upregulated components of NFkB signaling, leading to impaired osteoblastic bone formation (Nat Med 15:682, 2009), we found significantly reduced mRNA levels of bothNFkB2andrelB, along with an overall trend (P = 0.028 by cluster analysis) for lower mRNA levels of multiple inflammatory markers in the bone biopsies of the E-treated compared to the control women. Of the 71 GWAS-related genes examined, 14 were modulated in vivo by E treatment. In summary, our studies demonstrate that, in humans, E reduces both bone sost mRNA and circulating sclerostin levels. Further, since bone loss following E deficiency is associated with impaired bone formation relative to bone resorption, our findings point to increases in two key inhibitors of Wnt/BMP signaling, sclerostin andsostdc1, along with increased NFkB signaling, as mediating this relative deficit in bone formation in E-deficient postmenopausal women
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