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Background: Fibroblast growth factor (FGF23) is a protein that is produced by osteoblasts and osteocytes. Increased serum levels of FGF23 have been associated with increased risks of osteoporotic fractures and cardiovascular disease, particularly in participants with poor renal function. Serum iron (Fe) has been suggested as a regulator of FGF23 homeostasis. Objective: To determine whether Fe and iron status are determinants of the levels of intact FGF23 (iFGF23) in elderly men. Methods: The MrOS study is a population-based study of elderly men (N = 1010; mean age, 75.3 years; range, 69–81 years). The levels of Fe, transferrin saturation (TS), and ferritin were evaluated in relation to the serum concentrations of iFGF23 before and after adjustments for confounders. Results: TS < 15% was found in 3.5% (34/977) of the participants, who had a higher median level iFGF23 compared with the remaining subjects (47.4 μmol/L vs. 41.9 μmol/L, p = 0.008). The levels of iFGF23 correlated negatively (un-adjusted) with the levels of Fe (r = − 0.17, p < 0.001), TS (r = − 0.16, p < 0.001) and serum ferritin (r = − 0.07, p = 0.022). In addition, in participants with estimated glomerular filtration rate eGFRCystatin C > 60 mL/min, the levels of iFGF23 correlated (age-adjusted) negatively with the levels of Fe (r = − 0.15, p < 0.001) and TS (r = − 0.17, p < 0.001). The level of iFGF23 correlated positively (un-adjusted) with lumbar spine bone mineral density (BMD) (r = 0.14, p < 0.001), total body BMD (r = 0.11, p = 0.001), and total hip BMD (r = 0.09, p = 0.004). The corresponding correlations, when adjusted for age, weight, and height were: r = 0.08, p = 0.018; r = 0.05, p = 0.120; and r = 0.02, p = 0.624, respectively. No associations were found between BMD and the levels of Fe or TS. Multiple step-wise linear regression analyses [adjusting for age, body mass index (BMI), comorbidity index, cystatin C, C-reactive protein (hs-CRP), serum vitamin D 25-OH (25OHD), phosphate, calcium, parathyroid hormone (PTH), erythropoietin, hemoglobin, lumbar spine BMD, apolipoprotein B/A1 ratio] were performed in three separate models with Fe, TS or ferritin as potential explanatory variables. Fe and TS, but not ferritin, were independent predictors of iFGF23 level (standardized β-values: − 0.10, p < 0.001; − 0.10, p < 0.001; and − 0.05, p = 0.062, respectively). Conclusion: Low levels of Fe in elderly men are associated with high levels of iFGF23, independently of markers of inflammation and renal function, suggesting an iron-related pathway for FGF23 regulation.

Leukocyte telomere length (LTL) is related to the aging of somatic cells. We hypothesized that LTL is inversely associated with mortality in elderly men. LTL was measured in 2744 elderly men (mean age 75.5, range 69-81years) included in the prospective population-based MrOS-Sweden study. Mortality data were obtained from national health registers with no loss of follow-up. During the follow-up (mean 6.0years), 556 (20%) of the participants died. Using Cox proportional hazards regression, tertile of LTL did not associate with all-cause mortality [tertile 1 (shortest) or 2 (middle) vs. tertile 3 (longest); hazard ratio (HR)=1.05, 95% confidence interval (CI) 0.85-1.28 and HR=0.97, 95% CI 0.79-1.19, respectively]. Furthermore, LTL did not associate with cancer (197 events) or cardiovascular disease (CVD, 206 events) mortality (tertile 1 vs. tertile 3; HR=0.94, 95% CI 0.67-1.34 and HR=0.94, 95% CI 0.68-1.30, respectively). The lack of association between LTL and mortality remained also after adjustment for multiple covariates. Our results demonstrate that LTL is not associated with all-cause mortality or mortality due to cancer or CVD in elderly men. Further studies are needed to determine whether LTL can predict the risk of mortality in elderly women.

Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to bone disorders. Long-term treatment with glucocorticoids results in osteoporosis and increased risk of fractures. Much remains unclear regarding the effects of these compounds in bone cells. In the current study, human osteosarcoma Saos-2 cells and primary human osteoblasts were found to express mRNA for the vitamin D receptor as well as activating and deactivating enzymes in vitamin D3 metabolism. These bone cells exhibited CYP24A1-mediated 24-hydroxylation which is essential for deactivation of the active vitamin form. However, bioactivating vitamin D3 hydroxylase activities could not be detected in either of these cells. Several glucocorticoids, including prednisolone, down regulated CYP24A1 mRNA and CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblasts. Also, prednisolone significantly suppressed a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells co-transfected with the glucocorticoid receptor. Thus, the results of the present study show suppression by glucocorticoids on CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of this study we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid with glucocorticoid activity, which can bind the glucocorticoid receptor. Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblasts suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may interfere with regulation of active vitamin D levels.

Fracture healing is a complex interplay between endochondral and intramembranous bone formation processes. The canonical Wnt/β-catenin pathway enhances new bone formation and may play a role in fracture healing. Glycogen synthase kinase 3β (GSK3β) is a key regulator of β-catenin degradation. In this study, we investigate the effects of AZD2858, an orally bioactive GSK3 inhibitor, on fracture healing. Femoral fractures were produced in rats after the insertion of a femoral nail. The rats were treated with oral administration of AZD2858 at a dose of 30 μmol/kg (20mg/kg) daily for up to 3 weeks, while control animals were administered vehicle. At 4days, and at 1, 2 and 3 weeks, histological analysis was performed, and at the 2 and 3 week time points, we performed peripheral quantitative computed tomography (pQCT), X-rays, and four-point bending tests. Peripheral QCT showed an increase in both mineral density (of 28% at 2 weeks and 38% at 3weeks) and mineral content (of 81% at 2 weeks and 93% at 3 weeks) in the calluses from AZD2858 treated animals as compared to vehicle treated animals. Histological analysis demonstrated that rats treated with GSK3 inhibitor healed their fractures rapidly, but without the pre-formation of cartilage tissue. Furthermore, four-point bending tests of fractured femora from animals treated for 2 and 3 weeks showed an increase in strength in treated animals compared to their vehicle-treated controls. In conclusion, AZD2858, a potent GSK3 inhibitor, has a substantial impact on fracture healing. The fractures healed with a bony callus without an obvious endochondral component, suggesting that AZD2858 drives mesenchymal cells into the osteoblastic pathway. This leads to direct bone repair in an unstable fracture milieu.

Glycogen synthase kinase 3β (GSK-3β) actions are central in the canonical Wnt pathway, important in many biological processes and a potential drug target for treating several diseases. It is appreciated that a balanced Wnt canonical signaling is crucial for the maintenance of normal bone mass. In this study we investigated the effects of a potent orally active GSK-3 inhibitor, AZD2858, on bone mass in rats. Treatment (1 μM) of human osteoblast cells with AZD2858 in vitro increased β-catenin levels after a short period of time. In rats, oral AZD2858 treatment caused a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20 mg/kg once daily (total BMC: 172% of control; p

During a median 5-year follow-up, 485 CV events occurred. Both total testosterone and SHBG levels were inversely associated with the risk of CV events (trend over quartiles: p 0.009 and p 0.012, respectively). Men in the highest quartile of testosterone (550 ng/dl) had a lower risk of CV events compared with men in the 3 lower quartiles (hazard ratio: 0.70, 95% confidence interval: 0.56 to 0.88). This association remained after adjustment for traditional CV risk factors and was not materially changed in analyses excluding men with known CV disease at baseline (hazard ratio: 0.71, 95% confidence interval: 0.53 to 0.95). In models that included both testosterone and SHBG, testosterone but not SHBG predicted CV risk. Conclusions High serum testosterone predicted a reduced 5-year risk of CV events in elderly men. (J Am Coll Cardiol 2011

Genetic variation plays an important role in osteoporosis and a prime candidate gene is Collagen alpha2(I) (COL1A2). A coding polymorphism (rs42524) in COL1A2 has previously been associated with intracranial aneurysms. Here the effects of this polymorphism have been studied in relation to bone mineral density (BMD) and prevalences of stroke and myocardial infarction (MI). rs42524 was genotyped in elderly men (n = 2004) from the Swedish MrOS cohort. Genotypes were analysed for association to BMD and certain health parameters. Significant associations (overall P < 0.05), were observed between rs42524 genotype and BMD at several skeletal sites. Surprisingly, the heterozygote genotype class exhibited lower BMD than either homozygote group. When subjects were classified as heterozygotes or homozygotes, the heterozygous genotype was found to confer a lower BMD at total hip, femoral neck and trochanter Furthermore, the heterozygote genotype had an increased risk of stroke and MI, with population Attributable Risks being 0.12 and 0.08, respectively.

The WHO criteria for osteoporosis are based on bone mineral density (BMD) values in comparison to a reference population of healthy young adults. The aim of this study was to create BMD references for ethnic Swedish women, and to investigate whether the use of these T-score measurements influence the amount of Swedish postmenopausal patients that are diagnosed as having osteoporosis. A bone density reference was created by measuring a population-based sample of 335 randomly selected Swedish women aged 20-39yr. BMD was measured by dual-energy X-ray absorptiometry (DXA) at the lumbar spine, proximal femur, and total body. These locally derived T-score values were subsequently used to diagnose a sample of 300 consecutive postmenopausal Swedish patients referred to the Uppsala Osteoporosis Unit. There was a slight age-dependent decrease in femoral neck BMD, whereas no age effect was seen at other sites such as total hip, lumbar spine, or total body. This suggests that the cohort represents the steady state BMD at the ages of expected peak bone mass in Swedish women. The correlation between BMD measures at different sites differed from r=0.55 (lumbar spine BMD vs femoral neck BMD [FNBMD]) to r=0.92 (total hip BMD vs FNBMD). Central DXA-generated T-scores were calculated from this cohort, and these were significantly higher (0.3-0.5 SD) as compared with manufacturers and NHANESIII reference populations. This indicates that young Swedish women have a higher peak bone mass than the subjects included in the reference populations currently used for clinical measurements. The T-score in total hip derived from the investigated cohort was subsequently used to diagnose 300 clinical patients (mean age 63yr) referred for a DXA scan by their physicians. The use of this locally established and ethnic representative, T-score reference increased the prevalence of osteoporosis in femoral neck and total hip with 53-106%. A Swedish female BMD reference representing peak bone mass has been established and the normative data are presented. Notably, this cohort has considerably higher BMD as compared to the NHANESIII and manufacturer's reference populations. The use of the present T-score reference therefore causes approximately a 2-fold increase in the amount of Swedish postmenopausal women that fulfill the WHO criteria for osteoporosis. This demonstrates the problems with using T-score as diagnostic threshold for osteoporosis and is an argument for future strategies to obtain standardized densitometric cut-offs, for example, mg/cm(2).

Objective To report on a novel strategy for tumor localization in a 62-year-old man with hypophosphatemic tumor-induced osteomalacia (TIO). Methods Repeated computed tomographic and magnetic resonance imaging scans failed to localize any tumor in a patient with adult-onset hypophosphatemic osteomalacia. Therefore, venous sampling for fibroblast growth factor-23 (FGF23)—a circulating hormone that has been identified as a causative factor for TIO—in major veins was conducted. Serum FGF23 was measured from collected samples by an intact FGF23 enzyme-linked immunosorbent assay. Results Venous sampling suggested a local increase in serum FGF23 in the left femoral vein; this finding prompted performance of octreotide scintigraphy restricted to the left leg. A tumor was located at the lateral condyle of the left femur, which was also confirmed by magnetic resonance imaging. Surgical resection of the tumor normalized the serum phosphorus and 1,25-dihydroxyvitamin D3 levels within 5 to 10 days, and FGF23 declined to normal levels within 24 hours. Histologic analysis supported the diagnosis of a soft-tissue giant cell tumor. Conclusion Our study case demonstrates the diagnostic complexity and difficulties in localizing a small tumor in a patient with TIO. Venous sampling for FGF23 may be helpful in tumor localization in sporadic cases of hypophosphatemic osteomalacia, especially when noninvasive diagnostic techniques prove insufficient. (Endocr Pract. 2008;14:362-367)

Sex steroids are important for growth and maintenance of the skeleton. Catechol-O-methyltransferase (COMT) is an estrogen degrading enzyme. The COMT val158met polymorphism results in a 60-75% difference in enzyme activity between the val (high activity=H) and met (low activity=L) variants. We have previously reported that this polymorphism is associated with bone mineral density (BMD) in young men. The aim of this study was to investigate associations between COMT val158met, BMD and fractures in elderly men.Population-based study of Swedish men 75.4, SD 3.2, years of age. Fractures were reported using standardized questionnaires. Fracture and genotype data were available from 2,822 individuals.Total number of individuals with self-reported fracture was 989 (35.0%). Prevalence ofor=1 fracture was 37.2% in COMT(LL), 35.7% in COMT(HL) and 30.4% in COMT(HH) (p0.05). Early fractures (or=50 years of age) were less common in COMT(HH) than in the combined COMT(LL+HL) genotype, OR 0.78 (95% CI 0.63-0.97). No associations were found for late fractures (50 years of age). The OR for fracture of the non-weight bearing skeleton in COMT(HH) compared with COMT(LL+HL) was 0.74 (95% CI 0.59-0.92). No associations between COMT val158met and BMD were found in this cohort of elderly men.The COMT val158met polymorphism is associated with life time fracture prevalence in elderly Swedish men. This association is mainly driven by early fractures (or=50 years of age).

OBJECTIVES: This study sought to determine whether serum levels of testosterone and estradiol associate with lower extremity peripheral arterial disease (PAD) in a large population-based cohort of elderly men. BACKGROUND: Few studies have explored the relationship between serum sex steroids and lower extremity PAD in men. METHODS: The Swedish arm of the MrOS (Osteoporotic Fractures in Men) study (n = 3,014; average age 75.4 years) assessed ankle-brachial index (ABI) and defined lower extremity PAD as ABI

Postmenopausal hormone replacement therapy (HRT) has positive effects on fracture incidence before any effects on bone mineral density can be demonstrated. This has been attributed to increased muscle strength by HRT. This study was designed to evaluate the effect of 6 months of HRT on muscle strength in postmenopausal women.Forty postmenopausal women, aged 60-78 were included in the study. They were randomly divided in two groups with 20 women in each group. One group received Menorest 50 microg/24 h (estradiol 4.3 mg) and Gestapuran 2.5 mg (medroxyprogesteron) daily and the other group received placebo treatment. The study was conducted as a double blinded, prospective and placebo controlled trial. Hand grip strength, isokinetic knee flexion and extention, and physical activity were measured before treatment, after 3 and 6 months. Physical activity was estimated using a classification system of physical activity. A JAMAR hydraulic hand dynamometer and a Cybex II dynamometer were used to evaluate muscle strength.Hand grip strength in the right hand, increased significantly in both groups (HRT P0.001 and placebo P0.01) and in the left hand in the HRT group (P0.01). However, there were no differences in muscle strength between the two groups. There was no significant change in isokinetic knee flexion or extension after 6 months in either of the groups. The estimated physical activity increased slightly in the placebo group, but there was no significant difference compared to the treatment group.Our data suggest that 6 months of HRT does not influence muscle strength in postmenopausal women.

Osteoprotegerin (OPG), a member of the tumor necrosis receptor family, is produced by various tissues and inhibits osteoclast differentiation and activity. Since the metastasis of prostate cancer to bone often induces osteosclerosis, the possibility that these tumor cells secrete OPG is of interest. We have investigated whether the prostate cancer cell lines LNCaP, PC-3, and DU-145 produce and secrete OPG in vitro and if the production might be regulated by cytokines involved in remodeling of bone. OPG transcripts were detected by RT-PCR in all cell lines. OPG in culture media was analyzed by ELISA. In all three lineages, treatment with tumor necrosis factor-alpha and interleukin-1 beta dose dependently (5-5000 pM) stimulated the OPG secretion. Treatment with tumor necrosis factor-beta in increasing concentrations (1-1000 pM) stimulated OPG secretion in PC-3 but had no effect on the DU-145 and LNCaP cells. Dexamethasone (100 pM) had a small, but not significant, inhibitory effect on OPG secretion from DU-145 and LNCaP. In human non-malignant prostate cells, used as controls, there was no effect of IL-1 or TNFs on the secretion rate of OPG.

Risk Of Major Osteoporotic Fracture (Hip, Vertebral, Radius, Humerus [Mof]) After First, Second And Third Fragility Fracture In A Swedish General Population Cohort

Interleukin-13 (IL-13) inhibits cell proliferation and stimulates interleukin-6 (IL-6) formation in isolated human osteoblasts (hOBs). Because the related cytokine, interleukin-4 (IL-4), is known to exert effects similar to IL-13 in other tissues, and because IL-4 has been implicated as a regulator of bone metabolism, we compared the effects of IL-13 and IL-4 on cell proliferation, IL-6 synthesis, the expression of osteoblastic phenotypic markers in hOB cultures. Also, the receptor proteins mediating these effects in hOBs have been partly characterized. IL-4 and IL-13 dose-dependently inhibited [(3)H]-thymidine incorporation into the DNA of human osteoblasts and stimulated secretion of IL-6 into culture supernatants. IL-13 and IL-4 also increased the mRNA levels of IL-6, as measured by RNAse protection assay. Furthermore, IL-13 and IL-4 dose-dependently enhanced alkaline phosphatase (ALP) activity, but did not affect osteocalcin or collagen type I synthesis. IL-4 was tenfold more potent than IL-13 in inducing both ALP activity and IL-6 secretion, whereas the cytokines were equipotent as inhibitors of cell proliferation. The expression of mRNA for receptor subunits previously implicated in IL-4 and IL-13 signaling was investigated by reverse transcriptase-polymerase chain reaction. IL-13R, IL-13Ralpha, and IL-4Ralpha mRNA were repeatedly detected in hOBs, whereas mRNA for IL-2Rgamma(C) was not detected. Receptor-blocking antibodies to IL-4Ralpha inhibited the induction of IL-6 formation by both IL-4 and IL-13, indicating that both cytokines utilize this receptor subunit in signaling. However, the antibodies did not affect the IL-4/-13-induced inhibition of [(3)H]-thymidine incorporation or the stimulation of alkaline phosphatase (ALP), suggesting that IL-4Ralpha does not mediate these effects of IL-4/-13 in hOBs. We conclude that the cytokines IL-13 and IL-4, through sharing of receptor components, induce similar effects on hOBs, causing inhibition of cell proliferation, stimulation of IL-6, and enhanced ALP activity.

Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis. OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG. In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells. An ELISA was developed for measuring the concentration of OPG in culture medium. OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion. Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells. The cells were also stimulated with inflammatory mediators and glucocorticoids. Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40%. In conclusion, an ELISA measuring OPG in cell culture media was developed. Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone.

The recently cloned osteoclastogenesis inhibitory factor, or osteoprotegerin (OPG), has been shown to be a potent inhibitor of osteoclast formation. The inhibition is believed to be mediated through specific binding of OPG to a cell surface ligand on osteoblastic stromal cells. In this report we have studied the effect of the bone resorbing agent prostaglandin E2 (PGE2) on OPG mRNA levels in primary cultures of human bone marrow stroma cells (hBMSC). PGE2 dose- and time-dependently down-regulated the mRNA levels of OPG, as measured by RNAse protection assay. After 4 hours of stimulation with 1 microM PGE2, OPG mRNA levels were significantly decreased. The inhibitory effect was seen at and above 1 nM of PGE2. To elucidate whether the OPG mRNA levels are regulated via the proteinkinase A and/or the proteinkinase C pathways we stimulated cells with either forskolin (FSK) or phorbolic ester (PDbu) respectively. FSK (10 microM) decreased OPG mRNA levels to 50 % of control, whereas PE (10 nM) upregulated the mRNA levels to 250 % of control. These data show that PGE2 down-regulates the expression of OPG mRNA in hBMSC, probably via an increase in cAMP. This mechanism might be involved in PGE2-induced bone resorption.

Parathyroid hormone acts on the osteoblast to induce osteoclastic bone resorption. Parathyroid hormone utilises cyclic AMP as a second messenger in osteoblasts, but may also cause an increase in cytoplasmatic free calcium ions ([Ca2+]i) in the same cell. To investigate the role of osteoblastic [Ca2+]i in the induction of bone resorption, we have compared the effects of parathyroid hormone and the Ca2+-ionophore, A23187, as well as the adenylate cyclase stimulating agent, forskolin, and the phorbol ester, phorbole 12,13 dibutyrate (PDB), on bone resorption in neonatal mouse calvarial bones. Parathyroid hormone (0.1 and 1 nM) dose dependently stimulated the release of prelabelled 45Ca2+ in 72 h culture. Parathyroid hormone-induced bone resorption was not affected by the addition of 1 microM indomethacin to the incubation media, and was therefore, not mediated by local prostaglandin formation. A23187 stimulated the release of 45Ca2+ at 1-10 nM. Above 100 nM, A23187 inhibited bone resorption. The A23187 (3 and 10 nM)-induced bone resorption was abolished by the cyclooxygenase inhibitor, indomethacin (1 microM), indicating that the stimulatory effect was mediated via prostaglandin formation. The adenylate cyclase stimulating agent, forskolin, dose dependently stimulated bone resorption at and above 1 microM. There was no additive or synergistic effect of forskolin and A23187 on 45Ca2+ release. Forskolin-induced bone resorption was, as with parathyroid hormone but in contrast to ionophore-induced bone resorption, not abolished by indomethacin (1 microM). The protein kinase C activator, PDB, at 10 and 1000 nM stimulated the release of prelabelled 45Ca2+. The stimulatory effect of the protein kinase C stimulating phorbol ester, PDB, on bone resorption was abolished by the addition of indomethacin. In summary, bone resorption induced by a Ca2+-ionophore is abolished by indomethacin. This indicates that bone resorbing agents known to increase [Ca2+]i subsequently enhance local prostaglandin formation. Bone resorption induced by the protein kinase C activator, PDB, was also abolished by indomethacin, whereas, forskolin and parathyroid hormone-induced bone resorption was unaffected. These data indicate that cyclic AMP, but not [Ca2+]i, is involved as a second messenger in parathyroid-induced bone resorption.

Polymorphisms in the Klotho (Kl) gene, which is central for vitamin D regulation by fibroblast growth factor 23 (FGF23), have been associated with longevity, coronary disease and stroke. The CC genotype of the single nucleotide polymorphism (SNP) rs577912 in the Kl-gene is associated with decreased Kl expression, as well as increased mortality in end stage renal disease. We examined if SNP in the Kl-gene was associated with mortality in the community derived cohort of 70 to 80 year old males of MrOS Sweden (N = 3014).High throughput genotyping of the KLOTHO SNPs was achieved by use of SequenomR MassEXTEND/Mass/ARRAY technology. 2738 subjects had a valid result for rs577912: CC 73.1% and CA + AA 26.9%. There were no differences in the serum levels of FGF23, phosphate, parathyroid hormone or renal function between genotypes CC and CA + AA. During a follow-up of a median of 4.5 years there were 337 deaths, 253 (12.6%) in the CC group and 84 (11.4%) in the CA + AA group. With log rank analysis there were no differences in mortality between the genotypes for all cause mortality (P = 0.39) or cardiovascular mortality (P = 0.60). None of the other SNPs in the Kl gene was associated with mortality in this cohort either.Conclusion:There is no association between the SNP rs577912 in the Kl-gene and mortality among elderly men.

Objective: Osteogenesis imperfecta (OI) is a rare heterogeneous disease of connective tissue leading to varying degrees of bone fragility. The worst form (type II) is peri-natally lethal whereas the mildest form (type I) is compatible with a normal life span. Over 1000 mutations causing OI have been described in the genes encoding collagen type I. As COL1A1 and COL1A2 are large genes, there are still many codon positions where no mutations have been reported and only a fraction of theoretically possible glycine substitutions have been described. In this study the spectrum of mutations causing OI in Sweden will be investigated and genotype–phenotype correlations as well as pharmacogenetics will be studied. Method: All patients with OI cared for at the Uppsala Osteoporosis Unit (Uppsala University Hospital) or Astrid Lindgren's Paediatric Hospital (Karolinska Institutet, Stockholm) were offered to enter the study. Patients from 140 unrelated families with OI accepted participation; 77 type I, 34 type IV, 20 type III, 5 without previous diagnosis and 4 with unclear OI type. Extensive clinical data is currently being collected on enrolled patients. Exons and flanking intron sequences of COL1A1 and COL1A2 are being sequenced in these families. Results: So far 133/140 families have been completely analyzed and in 27 no mutation was found. A total of 120 mutations have been detected, of which 104 are of a typical OI-type. In COL1A1 73 mutations were found and in COL1A2 31 mutations were noted. In 7 families 2 mutations were present, but only one of these was a typical OI-causing mutation. To date 16 amino acid changing mutations that were not of a typical OI-causing type have been noted and the majority of these have an unclear significance. Calculations of delta BMD Z-score response to bisphosphonate treatment did not show a difference in treatment response between groups with different types of OI or between patients with OI type I due to a qualitative vs. a quantitative collagen type I defect. Conclusion: The spectrum of mutations causing OI described in this Swedish cohort is of the expected type, with the exception of the amino acid changing mutations. It is notable that in seven families two separate mutations were identified. Calculations do not support a mutation dependent response to bisphosphonate treatment.

The effect of forskolin on parathyroid hormone (PTH) stimulated bone resorption, as assessed in vitro by the release of 45Ca from prelabelled neonatal mouse calvarial bones, and cyclic AMP formation in mouse calvarial bones and osteoblast-like cells was investigated. Much higher concentrations (100–300-times) of PTH were required to stimulate cyclic AMP accumulation than to stimulate mineral mobilization in murine calvarial bones. PTH, in the absence of phosphodiesterase inhibitor, stimulated cyclic AMP formation in mouse calvarial bones at and above concentrations of 3–10 nmol/l with EC50 at 10–15 nmol/l. In the presence of forskolin ( 1 or 10 μmol/l) the minimal concentration required to obtain a cyclic AMP response to PTH was decreased by a factor of 30–100 and the EC50 value was decreased to 1–2 nmol/l. Similar results were seen in osteoblast-cnriched cells. In addition, the magnitude of the PTH-induced cyclic AMP response was substantially potentiated by forskolin, both in calvarial bones and in isolated osteoblasts. Forskolin, in the absence of PTH, stimulated cyclic AMP levels in mouse calvaria at and above 1 μmol/l. In the presence of PTH, the response to forskolin was potentiated over the whole dose-response curve with apparent EC50 value at 1–2 μmol/l of forskolin. Forskolin (1 μmol/l) did not affect the magnitude of the 45Ca release response to PTH in 24 or 48 h cultures. In 96 h cultures, forskolin, in an additive manner, potentiated the effect of PTH on calcium mobilization. These results show that forskolin, in mouse calvarial bones and in isolated osteoblasts, in addition to directly stimulating cyclic AMP, can enhance receptor-mediated activation of adenylate cyclase. The finding that forskolin did not synergistically potentiate PTH-induced bone resorption suggests that there is no simple relationship between PTH-induccd cyclic AMP formation and stimulation of bone resorption.