ALS-derived fibroblasts exhibit reduced proliferation rate, cytoplasmic TDP-43 aggregation and a higher susceptibility to DNA damage.

BACKGROUND: Dermic fibroblasts have been proposed as a potential genetic-ALS cellular model. This study aimed to explore whether dermic fibroblasts from patients with sporadic-ALS (sALS) recapitulate alterations typical of ALS motor neurons and exhibit abnormal DNA-damage response. METHODS: Dermic fibroblasts were obtained from eight sALS patients and four control subjects. Cellular characterization included proliferation rate analysis, cytoarchitecture studies and confocal immunofluorescence assessment for TDP-43. Additionally, basal and irradiation-induced DNA damage was evaluated by confocal immunofluorescence and biochemical techniques. RESULTS: sALS-fibroblasts showed decreased proliferation rates compared to controls. Additionally, whereas control fibroblasts exhibited the expected normal spindle-shaped morphology, ALS fibroblasts were thinner, with reduced cell size and enlarged nucleoli, with frequent cytoplasmic TDP-43aggregates. Also, baseline signs of DNA damage were evidenced more frequently in ALS-derived fibroblasts (11 versus 4% in control-fibroblasts). Assays for evaluating the irradiation-induced DNA damage demonstrated that DNA repair was defective in ALS-fibroblasts, accumulating more than double of γH2AX-positive DNA damage foci than controls. Very intriguingly, the proportion of fibroblasts particularly vulnerable to irradiation (with more than 15 DNA damage foci per nucleus) was seven times higher in ALS-derived fibroblasts than in controls. CONCLUSIONS: Dermic-derived ALS fibroblasts recapitulate relevant cellular features of sALS and show a higher susceptibility to DNA damage and defective DNA repair responses. Altogether, these results support that dermic fibroblasts may represent a convenient and accessible ALS cellular model to study pathogenetic mechanisms, particularly those related to DNA damage response, as well as the eventual response to disease-modifying therapies.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

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 2 = 0.970) and with the results obtained by the amplification of 38 Indels (r 2 = 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.

A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders.

We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing ß-catenin levels and explored potential genetic and epigenetic mechanisms involved. ß-Catenin gene expression and nuclear levels were analyzed by real time PCR and confocal immunofluorescence. Increased nuclear ß-catenin was found in osteoblasts isolated from patients with osteoarthritis (99 ± 4 units vs. 76 ± 12, p=0.01, n=10), without differences in gene transcription, which is consistent with a post-translational down-regulation of ß-catenin and decreased Wnt pathway activity. Twenty four single nucleotide polymorphisms (SNPs) of genes showing differential expression between fractures and osteoarthritis (WNT4, WNT10A, WNT16 and SFRP1) were analyzed in DNA isolated from blood of 853 patients. The genotypic frequencies were similar in both groups of patients, with no significant differences. Methylation of Wnt pathway genes was analyzed in bone tissue samples (15 with fractures and 15 with osteoarthritis) by interrogating a CpG-based methylation array. Six genes showed significant methylation differences between both groups of patients: FZD10, TBL1X, CSNK1E, WNT8A, CSNK1A1L and SFRP4. The DNA demethylating agent 5-deoxycytidine up-regulated 8 genes, including FZD10, in an osteoblast-like cell line, whereas it down-regulated other 16 genes. In conclusion, Wnt activity is reduced in patients with hip fractures, in comparison with those with osteoarthritis. It does not appear to be related to differences in the allele frequencies of the Wnt genes studied. On the other hand, methylation differences between both groups could contribute to explain the differences in Wnt activity.

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.

Chimerism analysis in transplant patients: a hypothesis-free approach in the absence of reference genotypes.

INTRODUCTION: During routine analysis of chimerism in bone marrow transplant patients pre-transplant genotype of the recipient or the donor might lack. We aimed to develop a new method to analyze DNA results suitable when reference genotypes are not available. METHODS: The method was based on the balance between heterozygotes. It was implemented in a standard computer spreadsheet, and considered the hypothetical donor-recipient genotype combinations. Hypotheses with peak height ratios and allele sharing tendency above a critical threshold were accepted. The results were compared with those obtained with prior knowledge of reference genotypes. RESULTS: The algorithm predicted correctly the proportion of donor/recipient chimerism, even in the absence of reference genotypes. In fact, the predicted values were closely correlated (r(2)>0.98) and free of systematic bias (slope 0.98-1.04), in comparison with the reference values obtained with prior knowledge of the donor and recipient genetic profiles. CONCLUSIONS: This study constitutes a proof-of-concept of the application of the heterozygote balance for the quantitative study of chimerism. The algorithm computes post-transplant chimerism in an easy and time-efficient way, even when the donor and recipient reference genotypes are unavailable. Therefore, it can be a useful tool for laboratories involved in chimerism analysis.

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.

Osteocyte deficiency in hip fractures.

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 sclerostin-positive 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.

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.

Polymorphisms of the WNT10B gene, bone mineral density, and fractures in postmenopausal women.

Wnt ligands are important regulators of skeletal homeostasis. Wnt10B tends to stimulate the differentiation of common mesenchymal precursors toward the osteoblastic lineage, while inhibiting adipocytic differentiation. Hence, we decided to explore the association of WNT10B allelic variants with bone mineral density and osteoporotic fractures. A set of tag SNPs capturing most common variations of the WNT10B gene was genotyped in 1438 Caucasian postmenopausal women, including 146 with vertebral fractures and 432 with hip fractures. We found no association between single SNPs and spine or hip bone mineral density (BMD). In the multilocus analysis, some haplotypes showed a slight association with spine BMD (P = 0.03), but it was not significant after multiple-test correction. There was no association between genotype and vertebral or hip fractures. Transcripts of WNT10B and other Wnt ligands were detected in human bone samples by real-time PCR. However, there was no relationship between genotype and RNA abundance. Thus, WNT10B is expressed in the bone microenvironment and may be an important regulator of osteoblastogenesis, but we have not found evidence for a robust association of common WNT10B gene allelic variants with either BMD or fractures in postmenopausal women.

Aromatase expression in osteoarthritic and osteoporotic bone.

OBJECTIVE: Estrogen deprivation is a central mechanism in the development of osteoporosis with aging. Results from recent studies also suggest the involvement of estrogens in the pathophysiology of osteoarthritis (OA). Aromatization of androgenic precursors in peripheral tissue is the main source of estrogens in postmenopausal women and in men. However, the importance of aromatase expression in bone is a subject of controversy. This study was undertaken to determine aromatase expression in bone samples from patients with hip fracture and patients with OA. METHODS: We studied 104 patients with hip fracture (n = 60) or primary hip OA (n = 44). Aromatase expression was determined in trabecular bone samples from the femoral neck and in osteoblast cultures grown by the primary explant technique (n = 62), using real-time reverse transcriptase-polymerase chain reaction. RESULTS: Aromatase RNA was detected in bone samples at levels similar to those found in adipose tissue. Transcript levels were significantly lower in bone tissue samples obtained from patients with OA than in those obtained from patients with fracture (P = 0.00001). Likewise, primary cultures of osteoblast cells from OA patients revealed lower aromatase expression than those of cells from fracture patients (P = 0.012). Results were independent of age or sex differences. CONCLUSION: Our findings indicate that the aromatase gene is expressed in bone tissue in high amounts, similar to those found in adipose tissue, but transcript levels are lower in tissue samples and osteoblast cultures from patients with OA than in those from patients with hip fracture. Since estrogens may help to prevent local cartilage degradation, it can be speculated that such a reduced expression of aromatase could facilitate the development of OA.