The clinician's guide to prevention and treatment of osteoporosis.

Osteoporosis is the most common metabolic bone disease in the USA and the world. It is a subclinical condition until complicated by fracture(s). These fractures place an enormous medical and personal burden on individuals who suffer from them and take a significant economic toll. Any new fracture in an adult aged 50 years or older signifies imminent elevated risk for subsequent fractures, particularly in the year following the initial fracture. What a patient perceives as an unfortunate accident may be seen as a sentinel event indicative of bone fragility and increased future fracture risk even when the result of considerable trauma. Clinical or subclinical vertebral fractures, the most common type of osteoporotic fractures, are associated with a 5-fold increased risk for additional vertebral fractures and a 2- to 3-fold increased risk for fractures at other sites. Untreated osteoporosis can lead to a vicious cycle of recurrent fracture(s), often resulting in disability and premature death. In appropriate patients, treatment with effective antifracture medication prevents fractures and improves outcomes. Primary care providers and medical specialists are critical gatekeepers who can identify fractures and initiate proven osteoporosis interventions. Osteoporosis detection, diagnosis, and treatment should be routine practice in all adult healthcare settings. The Bone Health and Osteoporosis Foundation (BHOF) - formerly the National Osteoporosis Foundation - first published the Clinician's Guide in 1999 to provide accurate information on osteoporosis prevention and treatment. Since that time, significant improvements have been made in diagnostic technologies and treatments for osteoporosis. Despite these advances, a disturbing gap persists in patient care. At-risk patients are often not screened to establish fracture probability and not educated about fracture prevention. Most concerning, the majority of highest risk women and men who have a fracture(s) are not diagnosed and do not receive effective, FDA-approved therapies. Even those prescribed appropriate therapy are unlikely to take the medication as prescribed. The Clinician's Guide offers concise recommendations regarding prevention, risk assessment, diagnosis, and treatment of osteoporosis in postmenopausal women and men aged 50 years and older. It includes indications for bone densitometry as well as fracture risk thresholds for pharmacologic intervention. Current medications build bone and/or decrease bone breakdown and dramatically reduce incident fractures. All antifracture therapeutics treat but do not cure the disease. Skeletal deterioration resumes sooner or later when a medication is discontinued-sooner for nonbisphosphonates and later for bisphosphonates. Even if normal BMD is achieved, osteoporosis and elevated risk for fracture are still present. The diagnosis of osteoporosis persists even if subsequent DXA T-scores are above - 2.5. Ongoing monitoring and strategic interventions will be necessary if fractures are to be avoided. In addition to pharmacotherapy, adequate intake of calcium and vitamin D, avoidance of smoking and excessive alcohol intake, weight-bearing and resistance-training exercise, and fall prevention are included in the fracture prevention armamentarium. Where possible, recommendations in this guide are based on evidence from RCTs; however, relevant published data and guidance from expert clinical experience provides the basis for recommendations in those areas where RCT evidence is currently deficient or not applicable to the many osteoporosis patients not considered for RCT participation due to age and morbidity.

Impact of gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) on glucose and lipid homeostasis.

LRP5 loss-of-function mutations have been shown to cause profound osteoporosis and have been associated with impaired insulin sensitivity and dysregulated lipid metabolism. We hypothesized that gain-of-function mutations in LRP5 would also affect these parameters. We therefore studied individuals with LRP5 gain-of-function mutations exhibiting high bone mass (HBM) phenotypes and found that while there was no detected change in insulin sensitivity, there was a significant reduction in serum LDL. INTRODUCTION: Wnt signaling through LRP5 represents a newly appreciated metabolic pathway, which potentially represents a target for drug discovery in type 2 diabetes and hyperlipidemia. Studies in animal models suggest a physiologic link between LRP5 and glucose and lipid homeostasis; however, whether it plays a similar role in humans is unclear. As current literature links loss-of-function LRP5 to impaired glucose and lipid metabolism, we hypothesized that individuals with an HBM-causing mutation in LRP5 would exhibit improved glucose and lipid homeostasis. Since studies in animal models have suggested that Wnt signaling augments insulin secretion, we also examined the effect of Wnt signaling on glucose-stimulated insulin secretion on human pancreatic islets. METHODS: This was a matched case-control study. We used several methods to assess glucose and lipid metabolism in 11 individuals with HBM-causing mutations in LRP5. Affected study participants were recruited from previously identified kindreds with HBM-causing LRP5 mutations and included 9 males and 2 females. Two subjects that were being treated with insulin for type 2 diabetes were excluded from our analysis, as this would have obscured our ability to determine the impact of gain-of-function LRP5 mutations on glucose metabolism. The mean age of the evaluated study subjects was 55 ± 7 with a mean BMI of 27.2 ± 2.0. Control subjects were matched and recruited from the general community at an equivalent ratio, with 18 males and 4 females (mean age 56 ± 4; mean BMI 27.2 ± 1.0). Study testing was conducted at an academic medical center. RESULTS: There were no statistically significant differences between affected and matched control populations for HbA1c (p = 0.06), eAG (p = 0.06), insulin (p = 0.82), HOMA-B (p = 0.34), or HOMA-IR (p = 0.66). The mean Insulin Sensitivity Index (ISI) was also similar between control and affected individuals. Total cholesterol (p = 0.43), triglycerides (TG) (p = 0.56), and HDL (p = 0.32) were not different between the same two groups. In a small subset of studied subjects, intramyocellular and hepatic lipid content were similar in the affected individuals and controls when quantified by proton magnetic resonance spectroscopy (MRS). However, the mean value for serum LDL was significantly lower (p = 0.04) in affected individuals. In primary human islets, there were no differences between control and Wnt treatment groups for insulin secretion measured as area under the curve (AUC) for first phase (p = 0.17) or second phase (p = 0.33) insulin secretion. CONCLUSIONS: Although our sample size was small, our data do not support the hypothesis that HBM-causing LRP5 mutations, associated with increased Wnt signaling, improve glucose metabolism in humans. However, it does appear that LRP5 variants may affect LDL metabolism, a major risk factor for coronary artery disease. The molecular mechanisms underpinning this effect warrant further study.

Increased bone resorption during tenofovir plus lopinavir/ritonavir therapy in Chinese individuals with HIV.

UNLABELLED: We sought to evaluate the effects of antiretroviral therapy on skeletal metabolism in Chinese individuals with human immunodeficiency virus. Patients switched to tenofovir/lamivudine + lopinavir/ritonavir after treatment failure had an increase in bone resorption marker levels by nearly 60%, which is greater than the magnitude previously described in non-Chinese populations. INTRODUCTION: Few studies have evaluated the effects of antiretroviral therapy on skeletal metabolism in Asian populations infected with human immunodeficiency virus (HIV). METHODS: We performed a secondary analysis of bone turnover markers (BTM) at baseline and 2 years in stored plasma samples collected from 2/2009 to 1/2013 as part of a multi-center trial. Two groups were compared: (1) treatment-naïve patients initiated on zidovudine (AZT)/lamivudine (3TC) plus nevirapine (NVP) and (2) patients who failed first-line therapy and were switched to tenofovir (TDF)/3TC plus lopinavir/ritonavir (LPVr). Tests included the bone resorption marker, C-terminal cross-linking telopeptide of type-1 collagen (CTX), and the bone formation marker procollagen type 1 N-terminal propeptide (P1NP). RESULTS: In the TDF/3TC + LPVr group, samples were available from 59 patients at baseline and 56 patients at 2 years. Of these, 36 patients had samples available from both time points. In the AZT/3TC + NVP group, plasma samples were analyzed from 82 participants at baseline and of those, 61 had samples at 2 years. Median change over 2 years was greater in the TDF/3TC + LPVr group for both CTX (+0.24 ng/mL, interquartile ranges (IQR) 0.10-0.43 vs. +0.09 ng/mL, IQR -0.03 to 0.18, p = 0.001) and P1NP (+25.5 ng/mL, IQR 2.4-51.3 vs. +7.11 ng/mL, IQR -4.3 to 21.6, p = 0.012). Differences remained after adjusting for potential confounders in the multivariable analysis. CONCLUSIONS: Switching to TDF/3TC + LPVr after treatment failure resulted in greater increases in BTMs than initiation with AZT/3TC + NVP in Chinese patients with HIV. Following this change, bone resorption marker levels increased by nearly 60 %, which is greater than the 25-35% increase from baseline described previously in non-Chinese populations. Further studies are warranted to elucidate these findings.

Serum levels of sclerostin, Dickkopf-1, and secreted frizzled-related protein-4 are not changed in individuals with high bone mass causing mutations in LRP5.

SUMMARY: We compared circulating levels of Wnt inhibitors among patients with high bone mass mutations in LRP5, unaffected kindred, and unrelated normal controls. Inhibitors were unchanged in affected and unaffected kindred. We saw no meaningful differences between controls and affected individuals. LRP5 signaling may not influence circulating levels of these inhibitors. INTRODUCTION: It is thought that gain-of-function mutations in LRP5 result in high bone mass syndromes because these allelic variants confer resistance to the actions of endogenous inhibitors of Wnt signaling. We therefore attempted to determine if circulating levels of Wnt inhibitors are altered in patients with gain-of-function mutations in LRP5. METHODS: This is a cross-sectional study in a university research center. Serum was collected from consented volunteers known to have either the G171V or N198S gain-of-function mutations in LRP5, kindred members affected with either mutation, unrelated kindred, and unrelated normal age-matched controls. BMD was provided or measured on site. RESULTS: There were no significant differences found in the serum levels of sclerostin (SOST), Dickkopf-1 (Dkk-1), or secreted frizzled-related protein-4 (SFRP-4) in affected vs. unaffected individuals from different kindreds or when compared to age-matched unrelated normal individuals. Mean serum SOST values in affected and unaffected kindred members and unrelated normal controls were 52.7 ± 6.1, 36.5 ± 9.6, and 54.8 ± 5.4, respectively. For Dkk-1, the values were 25.9 ± 3.4, 25.7 ± 3.0, and 17.3 ± 2.3 and for SFRP-4, 38.1 ± 2.3, 39.8 ± 3.6, and 28.5 ± 1.7. Serum levels of RANKL and osteoprotegerin (OPG) were not different in the three groups. CONCLUSIONS: Circulating levels of endogenous Wnt inhibitors do not change in patients with gain-of-function mutations in LRP5 including Dkk1, which is suppressed by Wnt signaling. It may be that circulating levels of Wnt inhibitors do not reflect changes in target tissues. It is also possible that other mechanisms besides or in addition to resistance in Wnt inhibitors explains the skeletal effects of these mutations.

Adequate dietary protein is associated with better physical performance among post-menopausal women 60-90 years.

OBJECTIVES: Sarcopenia, the involuntary loss of skeletal muscle with age, affects up to one-quarter of older adults. Evidence indicates a positive association between dietary protein intake and lean muscle mass and strength among older persons, but information on dietary protein's effect on physical performance in older adults has received less attention. DESIGN: Cross-sectional observational analysis of the relationship of dietary protein on body composition and physical performance. SETTING: Clinical research center. PARTICIPANTS: 387 healthy women aged 60 - 90 years (mean 72.7 ± 7.0 y). MEASUREMENTS: Measures included body composition (fat-free mass, appendicular skeletal mass and fat mass) via dual x-ray absorptiometry (DXA), physical performance (Physical Performance Test [PPT] and Short Physical Performance Battery [SPPB]), handgrip strength, Physical Activity Scale in the Elderly (PASE), quality of life measure (SF-8), falls, fractures, nutrient and macromolecule intake (four-day food record). Independent samples t-tests determined mean differences between the above or below RDA protein groups. STATISTICAL ANALYSIS: Analysis of covariance was used to control for body mass index (BMI) between groups when assessing physical performance, physical activity and health-related quality of life. RESULTS: The subjects consumed an average of 72.2 g protein/day representing 1.1 g protein/kg body weight/day. Subjects were categorized as below the recommended daily allowance (RDA) for protein (defined as less than 0.8 g protein/kg) or at or above the RDA (equal to or higher than 0.8 g protein/kg). Ninety-seven subjects (25%) were in the low protein group, and 290 (75%) were in the higher protein group. Women in the higher protein group had lower body mass, including fat and lean mass, and fat-to-lean ratio than those in the lower-protein group (p <0.001). Composite scores of upper and lower extremity strength were impaired in the group with low protein intake; SPPB score was 9.9±1.9 compared to 10.6±1.6 in those with higher protein intake and PPT was 19.8± 2.9 compared to 20.9± 2.1 in the low and higher protein groups, respectively. The results were attenuated by correction for BMI, but remained significant. The physical component of the SF-8 was also lower in the low protein group but did not remain significant when controlling for BMI. No significant differences were found in hand grip strength or reported physical activity. CONCLUSION: Healthy, older postmenopausal women consumed, on average, 1.1 g/kg/d protein, although 25% consumed less than the RDA. Those in the low protein group had higher body fat and fat-to-lean ratio than those who consumed the higher protein diet. Upper and lower extremity function was impaired in those who consumed a low protein diet compared to those with a higher protein intake. Protein intake should be considered when evaluating the multi-factorial loss of physical function in older women.

An investigation of the association between omega 3 FA and bone mineral density among older adults: results from the National Health and Nutrition Examination Survey years 2005­2008.

SUMMARY: The relation of omega 3 fatty acids (n-3 FA) with bone mineral density (BMD) was assessed among adults >60 years; NHANES data (2005-2008). The association of dietary n-3 FA with measures of hip BMD was equivocal, but n-3 FA supplement use was significantly associated with higher spine BMD - a finding that deserves further study. INTRODUCTION: Associations between polyunsaturated fatty acids and bone mineral density are not well understood. PURPOSE: To evaluate the cross-sectional relation between dietary omega 3 fatty acid intake (specifically docosahexaenoic acid, eicosapentaenoic acid, and octadecatetraenoic) and BMD at the hip and spine among older adults. METHODS: Omega 3 FA intake (g/day) was assessed from two 24-h recalls using the National Health and Nutrition Examination Survey (NHANES, in 2005-2008); and omega 3 FA supplement use (yes/no) via questionnaire. Multivariable regression models were developed to explain variance in femoral neck, total femur, and lumbar spine BMD among 2,125 men and women over 60 years. RESULTS: Mean age was 70 years. In adjusted models, dietary omega 3 FA were marginally associated with greater femoral neck BMD (p = 0.0505), but not with total femur BMD (p = 0.95) or lumbar spine BMD (p = 0.74). Omega 3 supplement use was significantly positively associated with lumbar spine BMD (p = 0.005) but not with femoral neck or total femur BMD. CONCLUSIONS: Dietary intakes of omega 3 FA were marginally associated with femoral neck BMD; however, omega 3 supplement use was significantly associated with higher lumbar spine BMD in older adults. These results emphasize the need for assessment of total omega 3 intakes (diet and supplements) to provide a greater range of intake and a more accurate picture of the relation between omega 3 FA and BMD.

CSF-1 induces fos gene transcription and activates the transcription factor Elk-1 in mature osteoclasts.

Mice with targeted deletion of the fos gene fail to develop mature osteoclasts, reflecting an absolute requirement for the c-Fos proto-oncogene in osteoclast precursors. C-Fos is also expressed in mature osteoclasts; however, the regulation of fos in these cells has not been studied. By using cultured murine osteoclast-like cells (OCLs) we found that treatment with colony-stimulation factor 1 (CSF-1) induced a 3.9-fold increase in c-Fos rnRNA at 30 minutes and a 2.6-fold increase at 60 minutes. With use of mature osteoclasts isolated from transgenic mice expressing the bacterial Lac-Z gene under the control of the murine fos promoter, we were able to directly demonstrate transcriptional activation of fos by CSF-1 in these cells. Transcriptional activation was 2.6-fold greater at 5 minutes and 2.8-fold greater at 15 minutes in CSF-1-treated cells than in vehicle-treated cells. CSF-1 induced nuclear protein binding to the fos serum response element that was significantly attenuated by antibodies to the transcription factor Elk-1 but not by Sap-1a. Treatment of mature osteoclasts with CSF-1 for 2 hours resulted in a significant increase in the levels of nuclear c-Fos protein. These data demonstrate that CSF-1 upregulates c-fos expression in mature osteoclasts at least in part via transcriptional activation of fos. CSF-1 induced binding of Elk-1 to the fos gene serum response element appears to be part of the molecular mechanism by which this occurs.

A role for cell-surface CSF-1 in osteoblast-mediated osteoclastogenesis.

CSF-1 is required for osteoblast-mediated osteoclast formation. Osteoblasts produce soluble (sCSF-1) and cell-surface forms of CSF-1 (also known as membrane-bound CSF-1, mCSF-1) but their individual contributions to osteoclastogenesis remain unclear. Using glutaraldehyde-fixed primary murine osteoblasts as a source of mCSF-1, osteoblasts from op/op mice as a source for other osteoblast-derived osteoclastogenic factors and murine bone marrow as a source of osteoclast progenitors, osteoclast-like cells (OCL) formation was observed after 7-9 days of co-culture. In contrast, no OCL formation occurred when mCSF-1 expressed by primary murine osteoblasts was blocked by CSF-1 antibody pretreatment or when op/op osteoblasts were substituted for primary murine osteoblasts in the co-culture system. Osteoclast formation was also significantly inhibited when murine primary osteoblasts were pretreated with an antisense phosphorothioate oligonucleotide against mCSF-1. Finally, mCSF-1 and sCSF-1 were synergistic in stimulating OCL formation. These data support the conclusion that mCSF-1 plays an important role in osteoblast-mediated osteoclastogenesis within the bone microenvironment.

IL-6 negatively regulates IL-11 production in vitro and in vivo.

IL-6 and IL-11 are two cytokines that increase osteoclast formation and augment bone resorption. PTH stimulates the production of both cytokines by human osteoblast-like cells. Circulating levels of IL-6 are elevated in patients with states of PTH excess and correlate strongly to markers of bone resorption. In contrast, serum levels of IL-11 were significantly reduced in patients with primary hyperparathyroidism compared with values in euparathyroid controls. Further, after successful parathyroid adenomectomy, circulating levels of IL-6 fell, whereas IL-11 levels increased. Five-day infusions of human PTH-(1--84) in rodents resulted in a significant decline in mean circulating levels of IL-11, whereas IL-6 levels significantly increased. Pretreatment of cells and mice with neutralizing serum to IL-6 enhanced PTH-induced IL-11 production compared with the effect of pretreatment with nonimmune sera. These data indicate that IL-6 negatively regulates IL-11 production in vivo and in vitro. Analysis of steady state mRNA levels in SaOS-2 cells indicated that this effect is posttranscriptional. As both IL-6 and IL-11 stimulate osteoclast formation, down-regulation of IL-11 by IL-6 may help modulate the resorptive response to PTH.

Parameters of high bone-turnover predict bone loss in renal transplant patients: a longitudinal study.

BACKGROUND: Osteoporosis is a serious complication of kidney transplantation. Various factors have been postulated to contribute to posttransplant bone loss, among them treatment with corticosteroids, the use of cyclosporine and cyclosporine-like agents, and persistent hyperparathyroidism. In a previous cross-sectional study of long-term renal transplant recipients, we observed that osteoporosis or osteopenia was present in 88% of patients. Because biochemical markers of bone formation (serum osteocalcin) and bone resorption (urine pyridinoline, PYD, and deoxypyridinoline, DPD) were elevated in the majority of study subjects, we hypothesized that elevated rates of bone-turnover contribute to posttransplant bone loss in long-term renal transplant patients. This study was performed to examine this hypothesis. METHODS: The study population was composed of 62 patients who were more than 1-year postrenal transplantation and who had preserved renal function. They were followed prospectively for 1 year. Biochemical markers of bone-turnover were measured at study entry, and patients were classified as having high bone-turnover based on elevated urinary levels of at least one marker of bone resorption (i.e., PYD or DPD) and/or serum osteocalcin (group 1). If none of these were present, they were classified as having normal bone-turnover (group 2). Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry (DEXA) at time of entry into the study and again after 1 year of follow-up. The changes in BMD at the lumbar spine, hip, and wrist over the period of the study were compared between the high and normal bone-turnover groups. RESULTS: Forty-three patients (69%) were classified as having high bone-turnover (Group 1), and 19 patients (31%) were classified as having normal bone-turnover (Group 2). There was a statistically significant difference in change in BMD between the two groups at the lumbar spine (-1.11+/-0.42%, high bone-turnover, vs. 0.64+/-0.54%, normal bone-turnover; P=0.02) and the hip (-0.69+/-0.38%, high bone-turnover, vs. 1.36+/-0.66%, normal bone-turnover; P=0.006). Whereas group 2 had stable bone mass, group 1 exhibited bone loss at these skeletal sites. CONCLUSIONS: Our results indicate that bone loss is greater in renal transplant recipients with elevated biochemical markers of bone-turnover, suggesting that these markers may be useful in identifying patients at risk for continued bone loss. These data support the hypothesis that continued bone loss in long-term renal transplant recipients is associated with high bone-turnover. If accelerated bone resorption does play a role in posttransplant bone loss, this would provide a strong rationale for use of antiresorptive therapy for the prevention and treatment of this complication.

Parathyroid hormone induces hepatic production of bioactive interleukin-6 and its soluble receptor.

Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.

Nuclear factor-kappaB p50 is required for tumor necrosis factor-alpha-induced colony-stimulating factor-1 gene expression in osteoblasts.

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-alpha, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-kappaB site in the CSF-1 promoter is required for TNF-alpha-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-kappaB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IkappaB alpha, IkappaB gamma, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-alpha-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-alpha-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-kappaB p50 knock-out mice. Our findings support the conclusion that the NF-kappaB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

Evidence for a functional association between phosphatidylinositol 3-kinase and c-src in the spreading response of osteoclasts to colony-stimulating factor-1.

Osteoclasts are bone-resorbing cells whose normal function depends in part upon their ability to migrate over the bone surface to initiate new sites of bone resorption. The growth factor/cytokine, colony-stimulating factor-1 (CSF-1), potently stimulates osteoclast motility, in a c-src-dependent fashion. The intracellular signaling molecules that participate with c-src in CSF-1-induced remodeling of the osteoclast cytoskeleton have not been identified. Here we demonstrate, using the inhibitors wortmannin and LY294002, that activation of phosphatidylinositol 3-kinase (PI3-K) is required for CSF-1-induced spreading in osteoclasts. After CSF-1 treatment of osteoclast-like cells, PI3-K activity associated with the CSF-1 receptor c-fms, is increased, and the 85-kDa regulatory subunit of PI3-K and c-src coimmunoprecipitate. CSF-1 induces redistribution of PI3-K to the periphery of the cell. The association between p85 and c-src is due in part to a direct interaction between the proline-rich sequences of p85 and the SH3 domain of c-src. In vitro, the c-src SH3 domain stimulates PI3-K activity. Taken together, the current data suggest that c-src, via its SH3 domain, may participate in CSF-1-induced activation of PI3-K and that PI3-K and c-src are in the signaling pathway that subserves CSF-1-induced cytoskeletal changes in osteoclasts.

A threshold for low-protein-diet-induced elevations in parathyroid hormone.

BACKGROUND: We reported previously that lowering dietary protein intake in young healthy women to 0.7 g/kg depressed intestinal calcium absorption and was accompanied by elevations in parathyroid hormone (PTH). Moderate amounts of dietary protein (1.0 g/kg) did not appear to perturb calcium homeostasis. OBJECTIVE: The purpose of this study was to evaluate the effect of graded intakes of dietary protein (0.7, 0.8, 0.9, and 1.0 g/kg) on calcium homeostasis. DESIGN: The experiment consisted of 2 wk of a well-balanced diet containing moderate amounts of calcium, sodium, and protein followed by 4 d of an experimental diet containing 1 of 4 amounts of protein. Eight young healthy women received the 4 amounts of protein in random order. The average age of the subjects was 23.1 +/- 2.3 y, their weight was 64 +/- 3 kg, and their body mass index (in kg/m(2)) was 24.3 +/- 0.9. RESULTS: Elevations in PTH developed by day 4 of the diets containing 0.7 and 0.8 g protein/kg but not during the diets containing 0.9 or 1.0 g protein/kg. By day 4 of the 0.7- and 0. 8-g/kg diets, midmolecule PTH, calcitriol, and nephrogenous cyclic adenosine monophosphate were 1.5-3.5-fold higher than on day 0. Calcitropic hormones on day 4 of the diets containing 0.8 and 0.9 g protein/kg were within the normal range and 23-57% lower than values observed with the 0.7- and 0.8-g/kg diets (P < 0.005). Mean 24-h urinary calcium was 3.29 +/- 0.35 mmol with the diet containing 0.7 g protein/kg and 3.54 +/- 0.46 mmol with the diet containing 1.0 g protein/kg. CONCLUSIONS: Our data suggest that in young healthy women consuming a well-balanced diet, the current recommended dietary allowance for protein (0.8 g/kg) results in short-term perturbations in calcium homeostasis.

Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro.

Interleukin (IL)-6 promotes osteoclastogenesis and is thought to play a role in the bone loss that follows estrogen withdrawal. In vitro studies have demonstrated that IL-6 is produced in response to PTH by cells in the osteoblast lineage and that PTH-induced bone resorption is inhibited by a neutralizing antibody to the IL-6 receptor. In addition, we have recently reported that IL-6 plays a role in PTH-induced bone resorption in humans with chronic PTH excess and in experimental animals during the short-term infusion of PTH. In the current study, we examined whether estrogen withdrawal augments PTH-induced IL-6 production. When cultured in the absence of estrogen, human osteosarcoma cells (Saos-2) treated with PTH demonstrated significantly greater release of IL-6 than cells grown under estrogen-replete conditions, 30-fold vs. 15-fold (P = 0.005). A similar effect but of lesser magnitude was seen with primary human osteoblasts. In vivo, PTH induced IL-6 production was also increased in the estrogen-deficient state (ovx) such that at the end of a 5-day PTH infusion, the mean circulating level of IL-6 was significantly higher in ovx vs. sham/ovx mice (60.1 vs. 16.9 pg/ml; P < 0.0001). The greater increase in circulating levels of IL-6 in PTH-treated ovx mice was paralleled by a greater rise in bone resorption markers with the mean level of urine collagen cross-links in the PTH-treated ovx group being more than 2.5-fold higher than in the PTH-treated sham/ovx animals (236 vs. 88.5 microg/mmol creatinine, P < 0.0001). Mean serum collagen cross-link values were 17.4 microg/liter in PTH-treated ovx vs. 7.4 microg/liter in PTH-treated sham/ovx animals (P < 0.0001). Treatment of animals with estrogen prevented the exaggerated response to PTH infusion such that the increase in both circulating levels of IL-6 and bone turnover markers in estrogen-treated animals were similar to those observed in sham/ovx animals and significantly lower than those in PTH-treated ovx animals. These findings may help to explain the increased skeletal sensitivity to the resorbing effects of PTH seen in the estrogen-deficient state.

Parathyroid hormone-related protein induces spontaneous osteoclast formation via a paracrine cascade.

Experiments in vivo have established that tooth eruption fails in the absence of parathyroid hormone (PTH)-related protein (PTHrP) action in the microenvironment of the tooth because of the failure of osteoclastic bone resorption on the coronal tooth surface to form an eruption pathway. To elucidate the effects of PTHrP on osteoclast regulation in this environment, we established primary cultures of epithelial stellate reticulum cells and mesenchymal dental follicle (DF) cells surrounding the teeth. When cocultured, these cells are fully capable of supporting the formation of functional osteoclasts in the absence of added splenic osteoclast precursors, osteoblasts, or vitamin D/PTH/PTHrP. Neutralizing the effects of PTHrP resulted in a decrease in the number of osteoclasts formed, suggesting that stellate reticulum-derived PTHrP drives osteoclast formation. DF cells were found to express functional PTH/PTHrP type I receptors, and conditioned media collected from PTHrP-treated DF cells were able to induce bone resorption in the fetal-rat long-bone assay. PTHrP treatment also induced an increase in osteoclast differentiation factor expression and a concomitant decrease in osteoclastogenesis inhibitory factor expression in DF cells. The addition of osteoclastogenesis inhibitory factor resulted in a decrease in the number of osteoclasts formed in the cocultures, suggesting that osteoclast formation is mediated by osteoclast differentiation factor. Thus, PTHrP seems to regulate osteoclast formation via mediation of the DF, in a manner analogous to the osteoblast-mediated process in the peripheral skeleton. The primary coculture system of dental crypt cells also offers a system for the study of osteoclast formation and regulation.

Posttransplant bone disease: evidence for a high bone resorption state.

Loss of bone is a significant problem after renal transplant. Although bone loss in the first post transplant year has been well documented, conflicting data exist concerning bone loss after this time. It is equally unclear whether bone loss in long-term renal transplant recipients correlates with bone turnover as it does in postmenapausal osteoporosis. To examine these issues, we conducted a cross-sectional study to define the prevalence of osteoporosis in long-term (> 1 year) renal transplant recipients with preserved renal function (mean creatinine clearance 73 +/- 23 ml/min). Bone mineral density (BMD) was measured at the hip, spine and wrist by DEXA in 69 patients. Markers for bone formation (serum osteocalcin) and bone resorption [urinary levels of pyridinoline (PYD) and deoxypyridinoline (DPD)] were also measured as well as parameters of calcium metabolism. Correlations were made between these parameters and BMD at the various sites. The mean age of the patients was 45 +/- 11 years. Eighty eight percent of patients were on cyclosporine (12% on tacrolimus) and all but 2 were on prednisone [mean dose 9 +/- 2 mg/day)]. Osteoporosis (BMD more than 2.5 SD below peak adult BMD) at the spine or hip was diagnosed in 44% of patients and osteopenia was present in an additional 44%. Elevated levels of intact parathyroid hormone (i PTH) were observed in 81% of patients. Elevated urinary levels of PYD or DPD were present in 73% of patients and 38% had elevated serum levels of osteocalcin. Levels of calcium, and of 25(OH) and 1,25(OH)2 vitamin D were normal. In a stepwise multiple regression model that included osteocalcin, PYD, DPD, intact PTH, age, years posttransplant, duration of dialysis, cumulative prednisone dose, smoking, and diabetes: urinary PYD was the strongest predictor of bone mass. These results demonstrate that osteoporosis is common in long-term renal transplant recipients. The data also suggest that elevated rates of bone resorption contribute importantly to this process.