Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.

BACKGROUND AND AIM: The Receptor Activity Modifying Proteins (RAMPs) are a group of accessory proteins, of which there are three in humans, that interact with a number of G-protein coupled receptors (GPCR) and play various roles in regulation of endocrine signaling. Studies in RAMP3 knockout (KO) mice reveal an age related phenotype with altered metabolic regulation and high bone mass. To translate these findings into a clinically relevant perspective, we investigated the association between RAMP3 gene variants, body composition and bone phenotypes in two population-based cohorts of Swedish women. METHODS: Five single nucleotide polymorphisms (SNP) in the vicinity of the RAMP3 gene were genotyped in the PEAK-25 cohort (n = 1061; 25 years) and OPRA (n = 1044; 75 years). Bone mineral density (BMD), fat mass and lean mass (total body; regional) were measured by DXA at baseline, 5 and 10 year follow-up. RESULTS: BMD did not differ with RAMP3 genotype in either cohort, although fracture risk was increased in the elderly women (OR 2.695 [95% CI 1.514-4.801]). Fat mass tended to be higher with RAMP3 SNPs; although only in elderly women. In the young women, changes in BMI and fat mass between ages 25-35 differed by genotype (p = 0.001; p < 0.001). CONCLUSION: Variation in RAMP3 may contribute to age-related changes in body composition and risk of fracture.

Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.

BACKGROUND AND AIM: The Receptor Activity Modifying Proteins (RAMPs) are a group of accessory proteins, of which there are three in humans, that interact with a number of G-protein coupled receptors (GPCR) and play various roles in regulation of endocrine signaling. Studies in RAMP3 knockout (KO) mice reveal an age related phenotype with altered metabolic regulation and high bone mass. To translate these findings into a clinically relevant perspective, we investigated the association between RAMP3 gene variants, body composition and bone phenotypes in two population-based cohorts of Swedish women. METHODS: Five single nucleotide polymorphisms (SNP) in the vicinity of the RAMP3 gene were genotyped in the PEAK-25 cohort (n = 1061; 25 years) and OPRA (n = 1044; 75 years). Bone mineral density (BMD), fat mass and lean mass (total body; regional) were measured by DXA at baseline, 5 and 10 year follow-up. RESULTS: BMD did not differ with RAMP3 genotype in either cohort, although fracture risk was increased in the elderly women (OR 2.695 [95% CI 1.514-4.801]). Fat mass tended to be higher with RAMP3 SNPs; although only in elderly women. In the young women, changes in BMI and fat mass between ages 25-35 differed by genotype (p = 0.001; p < 0.001). CONCLUSION: Variation in RAMP3 may contribute to age-related changes in body composition and risk of fracture.

The transient receptor potential ion channel TRPV6 is expressed at low levels in osteoblasts and has little role in osteoblast calcium uptake.

BACKGROUND: TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6-/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. RESULTS: Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P=0.77) or TE-85 (P=0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9×10(-5) relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0×10(-2) and 2.38±0.28×10(-4) the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24×10(-5) relative to GAPDH, in contrast with 4.3±1.5×10(-2) relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. CONCLUSIONS: TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake.

Failure to repair the c.338C>T mutation in carnitine palmitoyl transferase 2 deficient skin fibroblasts using chimeraplasty.

Chimeraplasty, using oligonucleotides to target gene repair, was heralded as an efficient alternative approach to conventional gene therapy. We designed oligonucleotides to target a common mutation in the carnitine palmitoyl transferase 2 gene and developed a specific and sensitive assay to detect gene repair in human skin fibroblasts homozygous for the mutation. We failed to repair the gene under a variety of conditions and believe this approach is of little value until cellular DNA repair mechanisms are much better understood.

CMT3 alters mitochondrial function in murine osteoclast lineage cells.

Chemically modified tetracyclines (CMTs 1-10) were developed as non-antibiotic inhibitors of matrix metalloproteinases (MMPs). We previously demonstrated that MMP inhibition alone is insufficient to explain the pro-apoptotic action of CMTs in osteoclast lineage cells and we have explored additional mechanisms of action. We compared the characteristics of apoptosis in RAW264.7 murine monocyte and osteoclast cultures treated with pharmacologically relevant concentrations of CMT3 or the bisphosphonate alendronate, which induces osteoclast apoptosis through inhibition of farnesyl diphosphate synthase. CMT3 induced apoptosis rapidly (2-3h), whereas alendronate-induced apoptosis was delayed (>12h). CMT3-treated cells did not accumulate unprenylated Rap1A in contrast to cells treated with alendronate. Importantly, CMT3 induced a rapid loss of mitochondrial stability in RAW264.7 cells measured by loss of Mitotracker((R)) Red fluorescence, while bongkrekic acid protected polykaryons from CMT3-induced apoptosis. Modulation of mitochondrial function is therefore a significant early action of CMT3 that promotes apoptosis in osteoclast lineage cells.