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.

Alzheimer's disease pathogenesis: Is there a role for folate?

Epigenetic modifications, including changes in DNA methylation, have been implicated in a wide range of diseases including neurological diseases such as Alzheimer's. The role of dietary folate in providing methyl groups required for maintenance and modulation of DNA methylation makes it a nutrient of interest in Alzheimer's. Late onset Alzheimer's disease is the most common form of dementia and at present its aetiology is largely undetermined. From epidemiological studies, the interactions between folate, B-vitamins and homocysteine as well as the long latency period has led to difficulties in interpretation of the data, thus current evidence exploring the role of dietary folate in Alzheimer's is contradictory and unresolved. Therefore, examining the effects at a molecular level and exploring potential epigenetic mechanisms could increase our understanding of the disease and aetiology. The aim of this review is to examine the role that folate could play in Alzheimer's disease neuropathology and will focus on the effects of folate on DNA methylation which link to disease pathology, initiation and progression.

Methyl Donor Status Influences DNMT Expression and Global DNA Methylation in Cervical Cancer Cells.

BACKGROUND: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyldonor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. MATERIALS AND METHODS: The human cervical cancer cell line, C4 II, was grown in complete medium and medium depleted of folate (FM+) and folate and methionine (FM). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. RESULTS: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant downregulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. CONCLUSIONS: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

Physiology of Bone.

Bone serves three main physiological functions: its mechanical nature provides support for locomotion and offers protection to vulnerable internal organs, it forms a reservoir for the storage of calcium and phosphate in the body, and it provides an environment for bone marrow production and haematopoietic cell development. The traditional view of bone as a passive tissue that responds to hormonal and dietary influences has changed over the past half century to one of bone as a dynamic adaptive tissue that responds to mechanical demands. This chapter gathers together some recent advances in bone physiology and molecular cell biology and discusses the potential application of the functional adaptation of bone to loading to enhance bone strength during childhood and adolescence.

Lack of prolidase causes a bone phenotype both in human and in mouse.

The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and µCT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest.

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.

Physiology of bone.

Bone serves three main physiological functions. Its mechanical nature provides support for locomotion and offers protection to vulnerable internal organs, it forms a reservoir for storage of calcium and phosphate in the body, and it provides an environment for bone marrow and for the development of haematopoietic cells. The traditional view of a passive tissue responding to hormonal and dietary influences has changed over the past half century to one of a dynamic adaptive tissue responding to mechanical demands. This chapter gathers together some recent advances in bone physiology and molecular cell biology and discusses the potential application of the bone's functional adaptation to loading in enhancing bone strength during childhood and adolescence.

The peroxisome proliferator activator receptor alpha/delta agonists linoleic acid and bezafibrate upregulate osteoblast differentiation and induce periosteal bone formation in vivo.

We showed previously that some actions of prostaglandin E(2) (PGE(2)) on bone are caused by its degradation product, PGA(2), which mediates its effects via a class of nuclear receptors known as the peroxisome proliferator activator receptors (PPARs), suggesting that the PPARs may be involved in the regulation of bone formation. The aims of this study were to determine the effects of PPARalpha/delta agonists on bone in vitro and in vivo. PPAR agonists were examined in vitro using the fibroblastic colony-forming unit (CFU-f) assay. The PPARalpha/delta agonists linoleic acid (LA) and bezafibrate (Bez) were then administered to intact male rats by daily s.c. injection for 12 weeks with either vehicle (10% dimethyl sulfoxide), LA (0.3 mg/kg), or Bez (1 mg/kg). CFU-f assays were performed on stromal cells ex vivo. Bone mineral density (BMD) and serum markers of formation and resorption were measured. Bone histomorphometry was performed at cancellous and cortical bone sites. PPARalpha/delta agonists increased significantly the number of osteoblastic colonies as demonstrated by increased alkaline phosphatase activity, collagen production, and calcification. This increase was typically equal to or greater than that achieved with the known bone anabolic agent PGE(2). In intact male rats, LA and Bez increased metaphyseal BMD by 7% and 11%, respectively. Increased BMD was associated with an increase in total bone area, although no changes were observed in bone formation rate within the trabecular compartment. Serum osteocalcin and osteoprogenitor numbers were increased, whereas there was no change in either tartrate-resistant acid phosphatase 5b or osteoclast number. Both LA and Bez increased cortical bone area by approximately 38%, periosteal perimeter by 15%, and periosteal bone formation by 221% and 140%, respectively. There was no effect on medullary cavity area or endocortical perimeter. These data suggest that PPARalpha/delta may have roles in bone anabolism, specifically in the regulation of periosteal bone formation. They are potential therapeutic targets for osteoporosis therapy.

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.

Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL.

Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor-KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.