Anna Enjuanes

Two new single-nucleotide polymorphisms in the COL1A1 upstream regulatory region and their relationship to bone mineral density.

Single‐nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of a gene product in response to external signals. Under this hypothesis, we scanned an 800‐base pair (bp) region within the COL1A1 promoter, known to harbor cis elements important for in vivo expression, and we found two new polymorphisms: −1663indelT and −1997 G/T. The G to T transversion at −1997 was associated with lumbar spine BMD (p = 0.015) when tested in a cohort of 256 postmenopausal women after adjusting by age, body weight, and years since menopause; a lower degree of association was detected also for femoral neck BMD in a subgroup of 146 women in univariate analysis and after adjusting by age (p = 0.044). The polymorphism −1663indelT, which corresponds to a deletion of a T in a tract of eight T residues (−1670 to −1663), did not show significant association with BMD. Interestingly, −1663indelT is in strong linkage disequilibrium (LD) with the previously described Sp1 polymorphism of intron 1, which in this study did not show association with BMD either. Significant interaction between −1997 G/T and −1663indelT (p = 0.019), and between −1997 G/T and Sp1 (p = 0.045) was observed also. Individuals heterozygous for the three polymorphisms showed the highest mean BMD value. Gel retardation assays showed that oligonucleotides containing either the −1663 or the −1997 polymorphic sites specifically bind primary osteoblast nuclear proteins. We named these binding sites as PCOL1 and PCOL2, respectively. In summary, this study describes two new SNPs in the COL1A1 promoter, which may affect bone mass determination.

Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG‐63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein‐2 (BMP2) gene expression in primary human osteoblast (hOB) and MG‐63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10−9 M, 10−8 M, 10−7 M, 10−6 M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide α1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real‐time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG‐63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone‐forming effect of statins, probably through the BMP2 pathway. J. Cell. Biochem. 101: 1430–1438, 2007.

Effects of bilirubin and sera from jaundiced patients on osteoblasts: Contribution to the development of osteoporosis in liver diseases†

Low bone formation is considered to be the main feature in osteoporosis associated with cholestatic and end‐stage liver diseases, although the consequences of retained substances in chronic cholestasis on bone cells have scarcely been studied. Therefore, we analyzed the effects of bilirubin and serum from jaundiced patients on viability, differentiation, mineralization, and gene expression in the cells involved in bone formation. The experiments were performed in human primary osteoblasts and SAOS‐2 human osteosarcoma cells. Unconjugated bilirubin or serum from jaundiced patients resulted in a dose‐dependent decrease in osteoblast viability. Concentrations of bilirubin or jaundiced serum without effects on cell survival significantly diminished osteoblast differentiation. Mineralization was significantly reduced by exposure to 50 μM bilirubin at all time points (from −32% to −55%) and jaundiced sera resulted in a significant decrease on cell mineralization as well. Furthermore, bilirubin down‐regulated RUNX2 (runt‐related transcription factor 2) gene expression, a basic osteogenic factor involved in osteoblast differentiation, and serum from jaundiced patients significantly up‐regulated the RANKL/OPG (receptor activator of nuclear factor‐κB ligand/osteoprotegerin) gene expression ratio, a system closely involved in osteoblast‐induced osteoclastogenesis. Conclusion: Besides decreased cell viability, unconjugated bilirubin and serum from jaundiced patients led to defective consequences on osteoblasts. Moreover, jaundiced serum up‐regulates the system involved in osteoblast‐induced osteoclastogenesis. These results support the deleterious consequences of increased bilirubin in advanced chronic cholestasis and in end‐stage liver diseases, resulting in disturbed bone formation related to osteoblast dysfunction. (HEPATOLOGY 2011)

Leptin Receptor (OB‐R) Gene Expression in Human Primary Osteoblasts: Confirmation

In their recent paper, Reseland et al. studied the leptin transcription, translation, and secretion in primary cultures of human osteoblasts (HO). Leptin, the obese gene (OB) product, which is synthesized and secreted mainly by adipocytes, is becoming a relevant factor for bone-formation regulation, in addition to its role as a hormonal regulator of body weight. Although leptin action on osteoblasts has been described via hypothalamus, we have started a study on a possible direct action of the hormone on HO in primary culture. The first step in our work has been to analyze if human osteoblasts in primary culture express OB-R, mainly OB-RL (isoform receptor with intracellular signal-transducing capabilities). In concordance with the results reported by Reseland et al. we detected, by reverse-transcription polymerase chain reaction (RT-PCR), transcripts corresponding to the isoform OB-RL of the leptin receptor in HO, obtained from two men and two women. However, Ducy et al. could not detect OB-RL in human osteoblasts. To ensure that this finding is reliable, in our analysis we included samples obtained from other tissues such as kidney and skeletal muscle as negative controls. No signal was found in either of the tissues, verifying the true positivity of the OB-RL in the osteoblasts and reinforcing the validity of the findings obtained both by Reseland et al. and by ourselves. This is important because other authors using nonquantitative PCR detected these transcripts in a large number of different tissues. OB-RS isoform was detected in all samples. In summary, although our results strongly suggest OB-RL expression by osteoblasts, we believe that further functional studies showing the signal transduction by this receptor in HO are necessary.

Efectos del tabaquismo sobre los niveles plasmáticos de osteoprotegerina en adultos jóvenes sanos

El tabaco es un conocido factor de riesgo de osteoporosis, pero los mecanismos por los que se produce esta asociacion siguen siendo objeto de debate. Estudios previos han evaluado las variaciones de osteoprotegerina plasmatica, una citoquina que regula la osteoclastogenesis y la resorcion osea, en fumadores, con resultados dispares. En este estudio se analizaron los niveles de osteoprotegerina plasmatica en un grupo de 49 voluntarios jovenes sanos, pero no existieron diferencias entre fumadores y no fumadores. Tampoco se encontraron diferencias tras ajustar por las diferencias basales existentes en estrona en varones y en edad e ingesta de calcio en mujeres. En conclusion, fumar no parece influir en los niveles plasmaticos de osteoprotegerina en voluntarios jovenes sanos. PALABRAS CLAVE: osteoprotegerina, tabaco, jovenes. While tobacco smoke is a known risk factor for osteoporosis, the mechanisms that produce this association must still elucidated. Several previous studies have evaluated the variations in serum levels of osteoprotegerin, a cytokine that regulates osteoclastogenesis and bone resorption in smokers, however, the results have been inconclusive. This study, which makes an analysis of serum levels of osteoprotegerin in a group of 49 healthy young volunteers, did not find any influence of smoking on this parameter. There were also no differences following adjustment for baseline differences in estrone in men and for age and calcium intake in women. In conclusion, tobacco smoking does not seem to influence osteoprotegerin serum levels in healthy young volunteers.