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Delgado-Calle J.,University of Cantabria | Sanudo C.,University of Cantabria | Bolado A.,University of Cantabria | Fernandez A.F.,University of Oviedo | And 7 more authors.
Journal of Bone and Mineral Research | Year: 2012

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. Copyright © 2012 American Society for Bone and Mineral Research.


Delgado-Calle J.,University of Cantabria | Sanudo C.,University of Cantabria | Sanchez-Verde L.,Histology and Immunohistochemistry Unit | Garcia-Renedo R.J.,Hospital Um Valdecilla | And 2 more authors.
Bone | Year: 2011

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. © 2011 Elsevier Inc.


Delgado-Calle J.,University of Cantabria | Arozamena J.,University of Cantabria | Garcia-Renedo R.,Hospital Um Valdecilla | Garcia-Ibarbia C.,University of Cantabria | And 3 more authors.
Calcified Tissue International | Year: 2011

Osteocytes play a central role in the regulation of bone remodeling. The aim of this study was to explore osteocyte function, and particularly the expression of SOST, a Wnt inhibitor, in patients with hip fractures. Serum sclerostin levels were measured by ELISA. The expression of several osteocytic genes was studied by quantitative PCR in trabecular samples of the femoral head of patients with hip fractures, hip osteoarthritis and control subjects. The presence of sclerostin protein and activated caspase 3 was revealed by immunostaining. There were no significant differences in serum sclerostin between the three groups. Patients with fractures have fewer lacunae occupied by osteocytes (60 ± 5% vs. 64 ± 6% in control subjects, P = 0.014) and higher numbers of osteocytes expressing activated caspase 3, a marker of apoptosis. The proportion of sclerostin-positive lacunae was lower in patients with fractures than in control subjects (34 ± 11% vs. 69 ± 10%, P = 2 × 10 -8). The proportion of sclerostinpositive osteocytes was also lower in patients. RNA transcripts of SOST, FGF23 and PHEX were also less abundant in fractures than in control bones (P = 0.002, 5 × 10 -6, and 0.04, respectively). On the contrary, in patients with osteoarthritis, there was a decreased expression of SOST and FGF23, without differences in PHEX transcripts or osteocyte numbers. Osteocyte activity is altered in patients with hip fractures, with increased osteocyte apoptosis and reduced osteocyte numbers, as well as decreased transcription of osteocytic genes. Therefore, these results suggest that an osteocyte deficiency may play a role in the propensity to hip fractures. © Springer Science+Business Media, LLC 2010.


Santurtun A.,University of Cantabria | Riancho J.A.,University of Cantabria | Yanez L.,Hospital Um Valdecilla | Santurtun M.,University of Cantabria | Zarrabeitia M.T.,University of Cantabria
Bone Marrow Transplantation | Year: 2014

In order to detect chimerism after allogeneic hematopoietic SCT (HSCT), several methods have been developed. In this study we describe the use of a set of insertion/deletion (Indel) polymorphic loci to determine the level of donor cell engraftment. We analyzed 50 DNA samples from patients who had undergone HSCT, and also several artificial chimeric samples created by mixing different DNA specimens from non-transplanted donors in various proportions. A specific set of 38 autosomic Indel polymorphisms were analyzed. For comparison purposes, a set of 15 short tandem repeats (STRs) were analyzed using the Identifiler Plus Amplification Kit. Our results suggest that Indel-based and STR-based procedures behave similarly in most cases. However, Indel analysis may provide additional information in some cases with a small minor chimeric component or when the presence of stutter bands complicates chimerism estimation. © 2014 Macmillan Publishers Limited.


Santurtun A.,University of Cantabria | Riancho J.A.,University of Cantabria | Arozamena J.,University of Cantabria | Lopez-Duarte M.,Hospital Um Valdecilla | Zarrabeitia M.T.,University of Cantabria
International Journal of Legal Medicine | Year: 2016

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r2 = 0.970) and with the results obtained by the amplification of 38 Indels (r2 = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough. © 2016 Springer-Verlag Berlin Heidelberg


Delgado-Calle J.,University of Cantabria | Arozamena J.,University of Cantabria | Perez-Lopez J.,University of Cantabria | Bolado-Carrancio A.,University of Cantabria | And 6 more authors.
Molecular and Cellular Endocrinology | Year: 2013

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. © 2013 Elsevier Ireland Ltd.


Delgado-Calle J.,University of Cantabria | Sanudo C.,University of Cantabria | Fernandez A.F.,Instituto Universitario Of Oncologia Del Principado Of Asturias Iuopa | Garcia-Renedo R.,Hospital Um Valdecilla | And 2 more authors.
Epigenetics | Year: 2012

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 NFκB 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. © 2012 Landes Bioscience.


Garcia-Ibarbia C.,University of Cantabria | Perez-Nunez M.I.,Hospital Um Valdecilla | Olmos J.M.,University of Cantabria | Valero C.,University of Cantabria | And 5 more authors.
Osteoporosis International | Year: 2013

Summary: Two missense polymorphisms of WNT16 were associated with hip bone mineral density (BMD), the buckling ratio of the femoral neck, calcaneal ultrasound and hip fractures in individuals under 80 years of age. These results confirm the association of the WNT16 gene with bone mass and osteoporotic fractures. Introduction: Osteoporosis has a strong genetic component. Wnt ligands stimulate the differentiation of osteoblast precursors and play a major role in skeletal homeostasis. Therefore, the aim of this study was to explore the association of allelic variants of the WNT16 gene with BMD, other structural parameters of bone and osteoporotic hip fractures. Methods: Six single nucleotide polymorphisms were analysed in 1,083 Caucasian individuals over 49 years of age. Results: Two missense polymorphisms (rs2908004 and rs2707466) were associated with femoral neck BMD, with average differences across genotypes of 35 mg/cm2 (p = 0.00037 and 0.0015, respectively). Likewise, the polymorphisms were associated with calcaneal quantitative ultrasound parameters (p = 0.00004 and 0.0014, respectively) and the buckling ratio, an index of cortical instability of the femoral neck (p = 0.0007 and 0.0029, respectively). Although there were no significant differences in the genotype frequency distributions between 294 patients with hip fractures and 670 controls, among the subgroup under 80 years of age, TT genotypes were underrepresented in patients with fractures (odds ratio 0.50; CI 0.27-0.94). Conclusion: Common missense polymorphisms of the WNT16 gene are associated with BMD at the hip, calcaneal ultrasound and the buckling ratio of the femoral neck, as well as with hip fractures in individuals under 80 years of age. Overall, these results confirm the association of the WNT16 locus with BMD identified in genome-wide association studies and support its role in determining the risk of osteoporotic fractures. © 2013 International Osteoporosis Foundation and National Osteoporosis Foundation.


PubMed | University of Cantabria and Hospital Um Valdecilla
Type: Comparative Study | Journal: Bone marrow transplantation | Year: 2014

In order to detect chimerism after allogeneic hematopoietic SCT (HSCT), several methods have been developed. In this study we describe the use of a set of insertion/deletion (Indel) polymorphic loci to determine the level of donor cell engraftment. We analyzed 50 DNA samples from patients who had undergone HSCT, and also several artificial chimeric samples created by mixing different DNA specimens from non-transplanted donors in various proportions. A specific set of 38 autosomic Indel polymorphisms were analyzed. For comparison purposes, a set of 15 short tandem repeats (STRs) were analyzed using the Identifiler Plus Amplification Kit. Our results suggest that Indel-based and STR-based procedures behave similarly in most cases. However, Indel analysis may provide additional information in some cases with a small minor chimeric component or when the presence of stutter bands complicates chimerism estimation.


PubMed | University of Cantabria and Hospital Um Valdecilla
Type: Journal Article | Journal: International journal of legal medicine | Year: 2016

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r

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