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

De la délégation en cascade

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

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

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

Comparison of the RNAseq datasets to gene with known involvement in bone biology from genome-wide association studies (GWAS).

<p>The nuclear ERE-dependent, nuclear ERE-independent, and extra-nuclear datasets were intersected with the 72 genes identified by GWAS studies. The overlapped genes for each mode of ERα action are listed and color-coded for clarity. The directionality of the estrogen-dependent regulation is denoted with an arrow following the gene symbol.</p

Estrogen receptor expression in hFOB cells.

<p>A) hFOB cells were infected with Ad-ERα, Ad-NERKI and Ad-NOER and cultured for 24 h. Protein extracts were prepared and a western blot was performed using the anti-FLAG and anti-β-actin antibodies. B) Densitometry was performed and the data are expressed as ER expression relative to the β-actin control.</p