Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation.

Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate(214), we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1(D214N)) on skeletal homeostasis. ARTD1(D214N), unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1(D214N) altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1(D214N)-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation.

Serotonin-norepinephrine reuptake inhibitor therapy in late-life depression is associated with increased marker of bone resorption.

UNLABELLED: Antidepressants are associated with bone loss and fractures in older adults. We treated depressed older adults with an antidepressant and examined its effects on bone turnover by comparing blood samples before and after treatment. Bone resorption increased after antidepressant treatment, which may increase fracture risk. INTRODUCTION: Antidepressants have been associated with increased bone loss and fractures in older adults in observational studies, but the mechanism is unclear. We examined the effects of a serotonin-norepinephrine reuptake inhibitor, venlafaxine, on biomarkers of bone turnover in a prospective treatment study of late-life depression. METHODS: Seventy-six individuals aged 60 years and older with current major depressive disorder received a 12-week course of venlafaxine XR 150-300 mg daily. We measured serum C-terminal cross-linking telopeptide of type I collagen (ß-CTX) and N-terminal propeptide of type I procollagen (P1NP), measures of bone resorption and formation, respectively, before and after treatment. We then analyzed the change in ß-CTX and P1NP within each participant. Venlafaxine levels were measured at the end of the study. We assessed depression severity at baseline and remission status after treatment. RESULTS: After 12 weeks of venlafaxine, ß-CTX increased significantly, whereas P1NP did not significantly change. The increase in ß-CTX was significant only in participants whose depression did not remit (increase by 10 % in non-remitters vs. 4 % in remitters). Change in ß-CTX was not correlated with serum levels of venlafaxine or norvenlafaxine. CONCLUSION: Our findings suggest that the primary effect of serotonergic antidepressants is to increase bone resorption. However, such an increase in bone resorption seemed to depend on whether or not participants' depression remitted. Our results are in agreement with prior observational studies reporting increased bone loss in older adults taking serotonergic antidepressants. These negative effects on bone homeostasis could potentially contribute to increased fracture risk in older adults.

Calcium and phosphate homeostasis: concerted interplay of new regulators.

Calcium and phosphate are essential to many vital physiological processes, making the maintenance of their homeostasis crucial for survival. A tightly controlled balance of calcium and phosphorus is maintained by hormonal control of transport in the intestine, bone, and kidney. The kidneys participate by modulating calcium and phosphate reabsorption from the glomerular filtrate according to the body needs. This process is mediated by ion transporters. Besides the classical endocrine factors (i.e., PTH and vitamin D metabolites), new factors have been identified that are involved in maintaining calcium and primarily phosphate balance. Fibroblast growth factor-23 (FGF23) regulates urinary phosphate excretion by interacting with FGF receptors. Klotho, a transmembrane protein, facilitates this interaction, with the result of reducing phosphate reabsorption by the kidney leading to hypophosphatemia. More recently, dental matrix protein-1, an osteocyte product, has been shown to participate in FGF23-mediated regulation of phosphorus homeostasis. Transgenic mouse models have been of great value in the elucidation of Klotho and FGF23 function. This review highlights current knowledge into calcium and phosphate homeostasis in health and disease.

Alveolar bone measurement precision for phosphor-plate images.

OBJECTIVES: The aim was to demonstrate methods for determining measurement precision and to determine the precision of alveolar bone measurements made with a vacuum-coupled positioning device and phosphor plate images. STUDY DESIGN: Subjects were rigidly attached to the x-ray tube by means of a vacuum coupling device and custom cross-arch bite plates. Original and repeat radiographs (taken within minutes of each other) were obtained of the mandibular posterior teeth of 51 subjects, and cementoenamel junction-alveolar crest (CEJ-AC) distances were measured on both sets of images. In addition, x-ray transmission (radiodensity) and AC height differences were determined by subtracting one image from the other. Image subtractions and measurements were performed twice. Based on duplicate measurements, the root mean square standard deviation (precision) and least significant change (LSC) were calculated. LSC is the magnitude of change in a measurement needed to indicate that a true biologic change has occurred. RESULTS: The LSCs were 4% for x-ray transmission, 0.49 mm for CEJ-AC distance, and 0.06 mm for crest height. CONCLUSION: The LSCs for our CEJ-AC and x-ray transmission measurements were similar to what has been previously reported. The LSC for AC height (determined with image subtraction) was <0.1 mm. Compared with findings from earlier studies, this represents a highly precise measurement of AC height. The methods demonstrated for calculating LSC can be used by investigators to determine how large changes in radiographic measurements need to be before the changes can be considered to be (with 95% confidence) true biologic changes and not noise (i.e., equipment/observer error).

Calcium and vitamin D use among adults in periodontal disease maintenance programmes.

OBJECTIVES: To determine the level of calcium and vitamin D oral supplementation in patients in periodontal disease maintenance programmes. DESIGN: Convenience survey. SETTING: St. Louis Metropolitan region. SUBJECTS AND METHODS: Patients (n = 228) in two university-based, periodontal disease maintenance programmes. MAIN OUTCOME MEASURES: Reported amounts of oral calcium and vitamin D supplementation were tested for differences based on gender and race. RESULTS: The last published recommended daily intakes from the United States (US) Food and Nutrition Board (FNB) for adults >50 years of age are 1,200 mg calcium and 400 IU vitamin D (or 600 IU if over 70). The mean age of the 228 patients (125 females and 103 males) was 63.6 +/- 11.0 years (standard deviation). Of the 228 patients surveyed: (1) 204 (89%) were >50 years of age and of these, only 15 (7%) met the US FNB's recommended intakes of calcium and vitamin D from supplementation; (2) 138 (66%) reported that they took no oral supplementation, with significantly more males (n = 82) than females (n = 56) not taking supplementation (p = 0.03); (3) 88 (39%) took calcium supplementation, with females (947 +/- 511 mg/day) taking significantly (p <0.001) more than males (632 +/- 907 mg/day); and (4) 66 (29%) took vitamin D supplementation, with females(420 +/- 227 IU/day) taking approximately the same amount as males (443 +/- 317 IU/day, p >0.05). The amounts of oral supplementation did not vary with race (p >0.05). CONCLUSION: The use of calcium and vitamin D supplementation has been promoted for years, yet the numbers of adults taking supplements remains low and the level of supplementation varies greatly. Knowledge of the benefits of supplementation needs to be better disseminated and research needs to be conducted to determine optimal levels of calcium and vitamin D supplementation.

Bone turnover markers: understanding their value in clinical trials and clinical practice.

While bone mineral density (BMD) by dual-energy X-ray absorptiometry is the primary method of determining fracture risk, assessing bone turnover may add valuable information for the management of patients with low bone mass. Bone turnover markers (BTMs) are used in clinical trials where they can provide essential information on the biological efficacy of osteoporosis treatments. In such population-based studies, BTMs can predict fracture risk independent of BMD. When combined with BMD, they improve the fracture risk estimate above and beyond BMD alone in postmenopausal osteoporotic women. Since changes in bone turnover after the initiation of therapy with bone resorption inhibitors occur much more rapidly than changes in BMD, treatment efficacy could, in theory, be determined within weeks of using BTMs. However, such predictive value is limited by the large biological variability of these biochemical markers, even though newer automated methods have reduced their analytical variability. Consequently, widespread adoption as a means of predicting treatment efficacy in fracture prevention for individual patients cannot yet be recommended. BTMs may be useful for monitoring adherence to antiresorptive therapy and may aid in identifying patients for whom antiresorptive therapy is most appropriate. Thus, although BTMs are currently confined to clinical research applications, further improvement in assay precision may extend their diagnostic value in clinical settings.

Epidemiology of glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis is the leading cause of medication-induced osteoporosis. The incidence of new fractures after one year of glucocorticoid therapy can be as high as 17%, and observational studies suggest that fractures, which are often asymptomatic, occur in 30-50% of chronic glucocorticoid-treated patients. Fractures can occur within 3 months of initiation of steroid therapy and with daily doses as low as 2.5 mg of prednisone, indicating that there is no "safe dose" of glucocorticoid therapy in terms of skeletal safety. Even inhaled steroids can lead to bone loss, if used for prolonged periods of time. Importantly in glucocorticoid- treated patients, fractures tend to occur at bone mineral density levels that usually carry lower risk in women with post-menopausal osteoporosis, thus implying effects independent of bone mass. Glucocorticoids seem to affect skeletal sites that are mostly composed of trabecular bone, although fractures can occur at cortical sites as well. The combination of high dose, long duration of treatment, and a continuous pattern of administration significantly increase the relative risk of fractures. The rapid and profound bone loss that occurs after initiation of glucocorticoid therapy has some very practical implications with regards to the site and the timing of bone mineral density measurements and fracture prevention strategies.

Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts.

In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.

Risedronate rapidly reduces the risk for nonvertebral fractures in women with postmenopausal osteoporosis.

Prevention of nonvertebral fractures, which account for a substantial proportion of osteoporotic fractures, is an important goal of osteoporosis treatment. Risedronate, a pyridinyl bisphosphonate, significantly reduces clinical vertebral fracture incidence within 6 months. To determine the effect of risedronate on osteoporosis-related nonvertebral fractures, data from four large, randomized, double-blind, placebo-controlled, Phase III studies were pooled and analyzed. The population analyzed consisted of postmenopausal women, with and without vertebral fractures, who had low bone mineral density (lumbar spine T-score <-2.5). Patients received placebo (N = 608) or risedronate 5 mg daily (N = 564) for 1 to 3 years. At baseline, 58% had at least one prevalent vertebral fracture, and the mean lumbar spine T-score was -3.4. Among placebo-treated patients, the presence of prevalent vertebral fractures did not increase the risk of incident nonvertebral fractures overall, although fractures of the humerus and hip and pelvis were more common in patients who had prevalent vertebral fractures than in those who did not. Risedronate 5 mg significantly reduced the incidence of nonvertebral fractures within 6 months compared with control. After 1 year, nonvertebral fracture incidence was reduced by 74% compared with control ( P = 0.001), and after 3 years, the incidence was reduced by 59% ( P = 0.002). The results indicate that risedronate significantly reduces the incidence of osteoporosis-related nonvertebral fractures within 6 months.

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells.

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.

Sequence and structure of the mouse connexin45 gene.

The connexin45 (Cx45) gene was cloned from a mouse genomic Bacterial Artificial Chromosome library. Approximately 8.4 kb of the genomic DNA was sequenced, and the structure of the Cx45 gene was determined. The mouse Cx45 gene is composed of 3 exons, with the entire coding sequence contained within exon III (EMBL Accession Number AJ300716). This structure is unique for the Cx45 gene, since all other members of the connexin family have only two exons. In addition, computer analysis reveals a potential TATA box and two putative AP-1 binding sites in the 5' region of the gene. Sequence alignment with connexin43 indicates substantial homology in the intronic sequences upstream of the 3' exons of the two genes, suggesting that the Cx45 gene is inherently similar to the rest of the connexin family, and that it probably evolved from an ancestor common to the other connexins.

Connexin45 interacts with zonula occludens-1 in osteoblastic cells.

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

Proliferation, differentiation and apoptosis in connexin43-null osteoblasts.

Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

A dominant negative cadherin inhibits osteoblast differentiation.

We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCaddeltaC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCaddeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally, 45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCaddeltaC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and beta-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCaddeltaC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.

The oxidative metabolism of estradiol conditions postmenopausal bone density and bone loss.

Because lifelong exposure to estrogen is a strong determinant of bone mass, we asked whether metabolic conversion of estrogen to either inactive or active metabolites would reflect postmenopausal bone mineral density (BMD) and rate of bone loss. Biochemical markers of inactive estrogen metabolites, urinary 2-hydroxyestrogen (2OHE1) and 2-methoxyestrogen (2MeOE1), and active metabolites, urinary 16alpha-hydroxyestrone (16alphaOHE1), estradiol (E2), and estriol (E3), were determined in 71 untreated, healthy postmenopausal women (age, 47-59 years) followed prospectively for 1 year. Urinary 2MeOE1 was correlated negatively with baseline vertebral (anteroposterior [AP] projection, r = -0.23 andp < 0.05; lateral view, r = -0.27 and p < 0.05) and proximal femur bone density measured by dual-energy X-ray absorptiometry (DXA; total, r = -0.38 and p < 0.01; neck, r = -0.28 and p = 0.02; trochanter, r = -0.44 and p < 0.01). BMDs of women in the lowest quartile of urinary 2MeOE1 (< 15 ng/g) were significantly higher than those in the highest quartile at all skeletal sites (p < 0.05). Likewise, women in the lowest quartile of urinary 2OHE1/16alphaOHE1 ratio (< 1.6) did not experience bone loss after 1 year, in contrast to women in the higher quartiles. We propose that the rate of inactivation of estrogens through 2-hydroxylation may contribute to postmenopausal osteoporosis.

Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.

Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987-995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249-2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.

Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways.

Cadherins, a family of cell-cell adhesion molecules, provide recognition signals that are important for cell sorting and aggregation during tissue development. This study was performed to determine whether distinct cadherin repertoires define tissue-specific lineages during differentiation of immature C3H10T1/2 and C2C12 mesenchymal cells. Both cell lines expressed mRNA for N-cadherin (N-cad), cadherin-11 (C11), and R-cadherin (R-cad). After induction of osteogenesis by recombinant human BMP-2 (rhBMP-2) treatment, steady state N-cad mRNA slightly increased in C3H10T1/2 cells. Likewise, the abundance of C11 mRNA increased in both cell lines, although the changes were more remarkable in C2C12 cells. By contrast, R-cad expression was almost shut off by rhBMP-2. The immature but committed osteoblastic MC3T3-E1 cells exhibited only minor changes in N-cad and C11 mRNA abundance after rhBMP-2 treatment. Whereas adipogenic differentiation was associated with a net decrease of N-cad and C11 expression in C3H10T1/2 cells, induction of myogenesis in C2C12 cells resulted in up-regulation of N-cad, while R-cad mRNA became undetectable in either case. Similarly, the adipocytic 3T3-L1 cells expressed very low levels of all cadherins when fully differentiated. Therefore, the repertoire of cadherins present in undifferentiated mesenchymal cells undergoes distinct changes during transition to mature cell phenotypes. Although neither N-cad nor C11 represent strict tissue-specific markers, the relative abundance of these mesenchymal cadherins defines lineage-specific signatures, perhaps providing recognition signals for aggregation and differentiation of committed precursors.

Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms.

Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction-mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.

Alveolar bone height and postcranial bone mineral density: negative effects of cigarette smoking and parity.

BACKGROUND: Our objective was to test the association between cemento-enamel junction, alveolar-crest distance (CEJ-AC, as measured on digitized vertical bite-wing radiographs) and postcranial bone mineral density (BMD) relative to clinical, dietary, and demographic variables. METHODS: Data were collected in a cross-sectional study of 134 postmenopausal women. CEJ-AC distances were determined from digitized vertical bite-wing radiographs. Lumbar spine and proximal femur BMDs were determined from dual-energy x-ray absorptiometric scans. Correlation analysis and Student t tests were used to identify those variables most associated with CEJ-AC distance. The selected variables were modeled with a backward stepwise regression analysis, with CEJ-AC distance as the dependent variable. RESULTS: Parity (number of pregnancies to term), cigarette smoking, and the interaction of lateral spine BMD with cigarette smoking were independent predictors of CEJ-AC distance (P < or =0.05). Statistical models containing these variables accounted for 19% of the variation in CEJ-AC distances. CONCLUSIONS: CEJ-AC distance in postmenopausal women is the result of a complicated interaction of many effects, including but not limited to, parity, cigarette smoking, and skeletal BMD.

Differential regulation of cadherins by dexamethasone in human osteoblastic cells.

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.