Carolina Sañudo

Genome‐wide profiling of bone reveals differentially methylated regions in osteoporosis and osteoarthritis

OBJECTIVE To determine genome-wide methylation profiles of bone from patients with hip osteoarthritis (OA) and those with osteoporotic (OP) hip fractures. METHODS Trabecular bone pieces were obtained from the central part of the femoral head of 27 patients with hip fractures and 26 patients with hip OA. DNA was isolated, and methylation was explored with Illumina methylation arrays. RNA was extracted, pooled, and deep-sequenced to obtain the whole transcriptome. Differentially methylated regions were identified, and connections between genes with differentially methylated regions were explored by pathway and text-mining analyses. RESULTS After quality control, methylation of 23,367 CpG sites (13,463 genes) was analyzed. There was a genome-wide inverse relationship between methylation and gene expression in both patient groups. Comparison of OP and OA bones revealed 241 CpG sites, located in 228 genes, with significant differences in methylation (false discovery rate<0.05). Of them, 217 were less methylated in OP than in OA. The absolute methylation differences were >5% in 128 CpG sites and >10% in 45 CpG sites. The differentially methylated genes were enriched for association with bone traits in the genome-wide association study catalog. Pathway analysis and text-mining analysis with Gene Relationships Across Implicated Loci software revealed enrichment in genes participating in glycoprotein metabolism or cell differentiation, and particularly in the homeobox superfamily of transcription factors. CONCLUSION Genome-wide methylation profiling of bone samples revealed differentially methylated regions in OP and OA. These regions were enriched in genes associated with cell differentiation and skeletal embryogenesis, such as those in the homeobox superfamily, suggesting the existence of a developmental component in the predisposition to these disorders.

DNA methylation contributes to the regulation of sclerostin expression in human osteocytes

Sclerostin, encoded by the SOST gene, is a potent inhibitor of bone formation, produced by osteocytes, not by osteoblasts, but little is known about the molecular mechanisms controlling its expression. We aimed to test the hypothesis that epigenetic mechanisms, specifically DNA methylation, modulate SOST expression. We found two CpG‐rich regions in SOST: region 1, located in the proximal promoter, and region 2, around exon 1. qMSP and pyrosequencing analysis of DNA methylation showed that region 2 was largely methylated in all samples analyzed. In contrast, marked differences were observed in region 1. Whereas the CpG‐rich region 1 was hypermethylated in osteoblasts, this region was largely hypomethylated in microdissected human osteocytes. Bone lining cells showed a methylation profile between primary osteoblasts and osteocytes. Whereas SOST expression was detected at very low level or not at all by RT‐qPCR in several human osteoblastic and nonosteoblastic cell lines, and human primary osteoblasts under basal conditions, it was dramatically upregulated (up to 1300‐fold) by the demethylating agent AzadC. Experiments using reporter vectors demonstrated the functional importance of the region −581/+30 of the SOST gene, which contains the CpG‐rich region 1. In vitro methylation of this CpG‐island impaired nuclear protein binding and led to a 75 ± 12% inhibition of promoter activity. In addition, BMP‐2‐induced expression of SOST was markedly enhanced in cells demethylated by AzadC. Overall, these results strongly suggest that DNA methylation is involved in the regulation of SOST expression during osteoblast–osteocyte transition, presumably by preventing the binding of transcription factors to the proximal promoter. To our knowledge, our data provide first ever evidence of the involvement of DNA methylation in the regulation of SOST expression and may help to establish convenient experimental models for further studies of human sclerostin.

A new pentaplex system to study short tandem repeat markers of forensic interest on X chromosome

A new method has been optimised to amplify five X chromosome short tandem repeat (STR) markers of interest in forensic medicine: human phosphoribosyl transferase (HPRTB), DXS101, androgen receptor (ARA), DXS7423 and DXS8377. Markers were conveniently amplified in a single PCR reaction with fluorochrome-labelled primers, which allowed the analysis of fragment sizes after injection into a capillary electrophoresis system. The most common alleles of each locus were sequenced and used in a control ladder to type unknown samples.

Role of DNA methylation in the regulation of the RANKL-OPG system in human bone

Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.

Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage

Epigenetic mechanisms play an important role in the tissue-specific regulation of gene expression. This study analyzed the relationship between tissue non-specific alkaline phosphatase (ALPL) gene expression and the methylation of a CpG island located in its proximal region. Gene expression was analyzed by real time RT-qPCR in primary human osteoblasts (hOBs), the osteoblastic cell line MG-63, the mammary cell line MCF-7, and bone tissue. DNA methylation was analyzed by qMSP in those cells and also in lining osteoblasts and in osteocytes obtained from human bone samples by laser-assisted capture. hOBs expressed much more ALPL mRNA than MG-63 cells (7.3±3.2 vs. 0.2±0.1 arbitrary units, respectively). hOBs showed a very weak DNA methylation (<10%), whereas MG-63 had a higher degree of methylation (58±6%). Likewise, MCF-7 cells, which scarcely expressed ALPL, had a hypermethylated CpG island. Thus, the degree of methylation in the CpG island was inversely associated with the transcriptional levels of ALPL in the studied cells. Furthermore, treatment with the DNA demethylating agent AzadC induced a 30-fold increase in ALPL expression, in MG-63 cells, accompanied by a parallel increase in alkaline phosphatase activity. However, AzadC did not affect ALPL levels in the already hypomethylated hOBs. In addition, in microdissected osteocytes, which do not express alkaline phosphatase, the CpG island was highly methylated (>90%), whereas lining osteoblasts showed an intermediate degree of methylation (58±13%). These results suggest an important role of DNA methylation in the regulation of ALPL expression through the osteoblast-osteocyte transition.

Association of the Aromatase Gene Alleles With BMD:Epidemiological and Functional Evidence†‡

BMD has a strong heritable component. Estrogen activity depends on the aromatization of androgenic precursors in nongonadal tissues both in postmenopausal women and men. Therefore, aromatase is an appealing candidate gene to explain, in part, the genetic component of BMD. In fact, an association between aromatase polymorphisms and BMD has been previously reported in some relatively small groups. In this study, we explored the relationship between several SNPs in the aromatase region and hip BMD in 1163 postmenopausal women. We found significant differences across genotypes, particularly in older women. The BMD differences between homozygous women with opposing genotypes were 4.2% in the whole group and 7.3% in women >67 yr of age. Body weight was significantly associated with BMD also, but there was no evidence for a statistically significant interaction between body weight and aromatase polymorphisms. Electrophoretic mobility shift assays suggested the binding of the CEBPβ transcription factor to the C/G rs1062033 locus, located ∼12 kb upstream of the translation start site. Experiments of transient transfection of osteoblastic cells with luciferase reporters showed differences in the transcriptional activity of alleles C and G at this locus, with different responses to the co‐transfection of a CEBPβ expression vector. Furthermore, evidence for differential allelic expression was found in bone tissue samples. In conclusion, polymorphisms in a 12‐kb region of the aromatase gene are associated with BMD in postmenopausal women, particularly during the late postmenopausal period. In vitro functional studies point to rs1062033 as a true regulatory polymorphism.

Interaction between CYP19 Aromatase and Butyrylcholinesterase Genes Increases Alzheimer’s Disease Risk

Biological evidence supports a role of aromatase and butyrilcholinesterase (BCHE) enzymes in the disruption of the cholinergic neurotransmission observed in Alzheimer’s disease (AD). Estrogens may reduce the risk of AD through enhancing or preserving cholinergic neurotransmission, and aromatase, the product of the CYP19 gene, is a critical enzyme in the peripheral synthesis of estrogens. BCHE is a hydrolytic enzyme associated with acetylcholine synaptic degradation, and the BCHE K genetic variant confers some protective effect for AD by reducing the activity of the enzyme. We investigated whether a 5′-UTR CYP19 polymorphism and the BCHE K variant might be responsible for susceptibility to AD by studying a clinically well-defined group of 187 sporadic AD patients and 172 control subjects from a Spanish population. We have shown that the CYP19 C/C genotype is overrepresented in AD patients who carry the BCHE non-K allele when compared with controls (OR = 1.85, p = 0.03). Our findings suggest that the CYP19 and BCHE polymorphisms may interact in determining the risk of AD.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs). Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST. These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Differential analysis of genome-wide methylation and gene expression in mesenchymal stem cells of patients with fractures and osteoarthritis

ABSTRACT Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation.

Osterix and RUNX2 are Transcriptional Regulators of Sclerostin in Human Bone

Sclerostin, encoded by the SOST gene, works as an inhibitor of the Wnt pathway and therefore is an important regulator of bone homeostasis. Due to its potent action as an inhibitor of bone formation, blocking sclerostin activity is the purpose of recently developed anti-osteoporotic treatments. Two bone-specific transcription factors, RUNX2 and OSX, have been shown to interact and co-ordinately regulate the expression of bone-specific genes. Although it has been recently shown that sclerostin is targeted by OSX in mice, there is currently no information of whether this is also the case in human cells. We have identified SP-protein family and AML1 consensus binding sequences at the human SOST promoter and have shown that OSX, together with RUNX2, binds to a specific region close to the transcription start site. Furthermore, we show that OSX and RUNX2 activate SOST expression in a co-ordinated manner in vitro and that SOST expression levels show a significant positive correlation with OSX/RUNX2 expression levels in human bone. We also confirmed previous results showing an association of several SOST/RUNX2 polymorphisms with bone mineral density.