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

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

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

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

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

De la délégation en cascade

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

Effects of age on bone mRNA levels of sclerostin and other genes relevant to bone metabolism in humans

Although aging is associated with a decline in bone formation in humans, the molecular pathways contributing to this decline remain unclear. Several previous clinical studies have shown that circulating sclerostin levels increase with age, raising the possibility that increased production of sclerostin by osteocytes leads to the age-related impairment in bone formation. Thus, in the present study, we examined circulating sclerostin levels as well as bone mRNA levels of sclerostin using quantitative polymerase chain reaction (QPCR) analyses in needle bone biopsies from young (mean age, 30.0years) versus old (mean age, 72.9years) women. In addition, we analyzed the expression of genes in a number of pathways known to be altered with skeletal aging, based largely on studies in mice. While serum sclerostin levels were 46% higher (p\u3c0.01) in the old as compared to the young women, bone sclerostin mRNA levels were no different between the two groups (p=0.845). However, genes related to notch signaling were significantly upregulated (p=0.003 when analyzed as a group) in the biopsies from the old women. In an additional analysis of 118 genes including those from genome-wide association studies related to bone density and/or fracture, BMP/TGFβ family genes, selected growth factors and nuclear receptors, and Wnt/Wnt-related genes, we found that mRNA levels of the Wnt inhibitor, SFRP1, were significantly increased (by 1.6-fold, p=0.0004, false discovery rate [q]=0.04) in the biopsies from the old as compared to the young women. Our findings thus indicate that despite increases in circulating sclerostin levels, bone sclerostin mRNA levels do not increase in elderly women. However, aging is associated with alterations in several key pathways and genes in humans that may contribute to the observed impairment in bone formation. These include notch signaling, which represents a potential therapeutic target for increasing bone formation in humans. Our studies further identified mRNA levels of SFRP1 as being increased in aging bone in humans, suggesting that this may also represent a viable target for the development of anabolic therapies for age-related bone loss and osteoporosis

Notion d'expéditeur : Cass., com., 28 octobre 2008, pourvoi n°07-20.786, SARL Transports Page contre SA Charal

<p>Most Highly Regulated Genes in the Nuclear ERE-dependent Dataset.</p

Dissection of Estrogen Receptor Alpha Signaling Pathways in Osteoblasts Using RNA-Sequencing

<div><p>The effects of 17-β-estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER)α and ERβ. ERs function through three general modes of action: DNA-binding dependent through estrogen response elements (EREs; designated nuclear ERE signaling); nuclear signaling via protein-protein interactions to other transcription factors (nuclear non-ERE signaling); and extra-nuclear signaling (membrane-bound functions of ERs). Identification of the specific transcriptional signatures regulated by each of these modes of action should contribute to an enhanced understanding of estrogen signaling in osteoblasts. To achieve this goal, we utilized specific mutations of ERα that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ERα knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ERα (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ERα (WT) pattern provides mode-specific data concerning transcriptional regulation by ERα. We expressed these constructs in the ER-negative osteoblastic cell line hFOB (−/+ estrogen) and performed global RNA-sequencing. Using a series of pair-wise comparisons, we generated three lists of genes that were regulated either by the nuclear ERE-dependent, nuclear ERE-independent, or extra-nuclear actions of ERα. Pathway and gene ontology analyses revealed that genes regulated through the nuclear ERE and nuclear non-ERE pathways were largely involved in transcriptional regulation, whereas genes regulated through extra-nuclear mechanisms are involved in cytoplasmic signaling transduction pathways. We also intersected our data with genes linked to bone density and fractures from a recent genome-wide association study and found 25 of 72 genes (35%) regulated by estrogen. These data provide a comprehensive list of genes and pathways targeted by these specific modes of ERα action and suggest that “mode-specific” ligands could be developed to modulate specific ERα functionality in bone.</p></div

Effects of estrogen on bone mRNA levels of sclerostin and other genes relevant to bone metabolism in postmenopausal women

Context: Studies in postmenopausal women have shown that estrogen reduces circulating sclerostin levels, but effects of estrogen on skeletal sclerostin mRNA levels are unknown.Objective: The objective of the study was to evaluate the effects of short-term estrogen treatment on bone mRNA levels of sclerostin and other genes relevant to bone metabolism.Design, setting, and patients: Needle bone biopsies were obtained from 20 postmenopausal women treated with transdermal estrogen for 3 weeks and 20 untreated controls. Quantitative PCR analyses were used to examine the expression of sclerostin and other genes related to bone metabolism, including 71 additional genes linked to bone density/fracture from genome-wide association studies.Results: Estrogen treatment was associated with lower bone sclerostin mRNA levels (by 48%, P\u3c.05) and with lower (by 54%, P\u3c.01) mRNA levels of the sclerostin-related protein, sclerostin domain-containing protein 1 (SOSTDC1), which is also a Wnt/bone morphogenetic protein inhibitor. Consistent with studies in mice showing that ovariectomy increased nuclear factor-κB (NF-κB) activation, we found that estrogen treatment was associated with a significant reduction in inflammatory genes as a group (P=.028), with bone mRNA levels of NFKB2 and RELB (both encoding proteins in the NF-κB transcription factor complex) being significantly reduced individual genes. Eight of the 71 genome-wide association study-related genes examined were modulated by estrogen (P\u3c.05, false discovery rate\u3c0.10).Conclusion: In humans, estrogen-induced decreases in two key inhibitors of Wnt/bone morphogenetic protein signaling, sclerostin and SOSTDC1, along with reductions in NF-κB signaling, may be responsible for at least part of the protective effects of estrogen on bone