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.

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.

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.

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43.

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.

Stimulation of inositol trisphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone.

Parathyroid hormone (PTH) produced a dose-dependent immediate stimulation of inositol triphosphate and diacylglycerol production in the opossum kidney cell line, primary culture proximal tubular cells, and basolateral membranes from canine proximal tubular segments. The increase in inositol triphosphate production was accompanied by a minor increase in inositol phosphate and no significant increase in inositol bisphosphate production. Associated with the changes in inositol triphosphate and diacylglycerol, there was an immediate hydrolysis of phosphatidylinositol 4'5-bisphosphate. The effect on phospholipid hydrolysis was followed by stimulation of phosphorylation of phosphatidylinositol 4' monophosphate and phosphatidylinositol. PTH produced a sudden increase in cytoplasmic Ca2+ in opossum kidney cells that persisted for approximately 1 min. Inositol triphosphate transiently increased cytoplasmic Ca2+ in saponin-treated opossum kidney and primary culture proximal tubule cells. The effects of PTH were not mimicked by cyclic nucleotides. In fact, cyclic AMP appeared to diminish inositol triphosphate production. These results demonstrate that PTH may activate renal tubular epithelial cells by the production of inositol triphosphate and diacylglycerol.

Estrogen status and heredity are major determinants of premenopausal bone mass.

To analyze their relative effects on premenopausal bone mass, we have studied the impact of lifelong estrogen exposure, assessed by an estrogen score (ES; computed on age at menarche, average length of menstrual cycles since menarche, and use of birth control pills), heredity, and some environmental factors on vertebral bone density (VBD), of 63 premenopausal women (age, 19-40 yr). Compared with women with normal bone density (Z score > -1), subjects with low VBD (Z score < -1) had significantly lower ES (15.1 +/- 3.9 vs. 18.7 +/- 2.4, P = 0.001), higher age at menarche (13.8 +/- 1.7 vs. 12.6 +/- 1.4 yr, P = 0.005), and lower serum estradiol (46.9 +/- 37 vs. 86.6 +/- 57 pg/ml, P = 0.023) and estrone levels (107.4 +/- 60 vs. 178.8 +/- 9.0 pg/ml, P = 0.05). Likewise, women in the lowest quartile for VBD had significantly lower ES (15.3 +/- 4.5 vs. 18.1 +/- 2.7, P = 0.006) and higher age at menarche (13.9 +/- 1.9 vs. 12.8 +/- .4, P = 0.02) than those in the upper three quartiles. A higher proportion of subjects with irregular menses (52 vs. 23%, P = 0.03) and a positive family history of osteoporosis (86 vs. 61%, P = 0.04) was found in the low VBD group compared with subjects with normal VBD. VBD correlated positively with ES (r = 0.44, P = < 0.001) and negatively with age at menarche (r = -0.30, P = 0.03) by simple linear regression, whereas no correlation was found between VBD and age, body mass index, parity, lactation, physical activity, sunlight exposure, and dietary calcium and vitamin D intakes. The correlation between VBD and ES improved after correcting for the effect of all the other variables by partial correlation analysis (Pearson partial r = 0.57, P = < 0.01), which also disclosed a significant contribution of dietary calcium to VBD. However, ES was the only significant independent determinant of VBD, by stepwise multiple regression analysis (R2 = 0.24). Therefore, premenopausal estrogen exposure, and possibly genetic predisposition, rather than environmental factors, are the major determinants for the development of peak bone mass before menopause.

Bone turnover in postmenopausal osteoporosis. Effect of calcitonin treatment.

UNLABELLED: To investigate the effectiveness of calcitonin treatment of postmenopausal osteoporosis in relation to bone turnover, we examined 53 postmenopausal osteoporotic women before and after one year of therapy with salmon calcitonin (sCT), at the dose of 50 IU every other day. Baseline evaluation revealed that 17 (32%) patients had high turnover (HTOP), and 36 (68%) normal turnover osteoporosis (NTOP) as assessed by measurement of whole body retention (WBR) of 99mTc-methylene diphosphonate. The two groups did not differ in terms of bone mineral content (BMC) measured by dual photon absorptiometry at both lumbar spine and femoral diaphysis. However, HTOP patients had higher levels of serum osteocalcin (OC) and urinary hydroxyproline excretion (HOP/Cr). Multivariate regression analysis showed no correlation between parameters of bone turnover (WBR, OC, HOP/Cr) and both femoral and vertebral bone density; the latter being negatively correlated only with the years elapsed since menopause (R2 = 0.406). Treatment with sCT resulted in a significant increase of vertebral BMC in the 53 patients taken as a whole group (+/- 7%, P less than 0.001). When the results obtained in HTOP and NTOP were analyzed separately, only those with HTOP showed a marked increment of spinal BMC (+22%, P less than 0.001), NTOP subjects neither gained nor lost bone mineral during the study. Femoral BMC decreased in the whole group after sCT therapy (-3%, P less than 0.003). However, HTOP patients maintained initial BMC values, whereas those with NTOP lost a significant amount of bone during the study period (-5%, P less than 0.001). The increase of vertebral bone mass was associated with a marked depression of bone turnover detectable in both subsets of patients and in the whole group. IN CONCLUSION: (a) assessment of bone turnover cannot help predict the severity of bone loss in postmenopausal osteoporosis; (b) calcitonin therapy appears to be particularly indicated for patients with high-turnover osteoporosis, resulting in a net gain of bone mineral in the axial skeleton and a slowing of bone loss in the appendicular bones.

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

Gap junctional communication modulates gene expression in osteoblastic cells.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

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.

Does interleukin-1 affect intracellular calcium in osteoblast-like cells (UMR-106)?

Interleukin-1 (IL-1) enhances bone resorption and formation in vitro, presumably through a primary action on osteoblasts, but the mechanism by which IL-1 activates bone cells is unknown. We investigated the possibility that the effect of IL-1 on osteoblasts is mediated through an increase in intracellular calcium [Ca++]i by studying the effects of purified human monocyte-derived IL-1 (hIL-1) and recombinant human IL-1 alpha (rhIL alpha) and beta (rhIL-1 beta) on [Ca++]i in the rat osteogenic sarcoma cell line UMR 106 using indo-1, a new-generation fluorescent Ca++-sensitive probe. hIL-1 (1 U/ml) resulted in an 85.5% rise in [Ca++]i over baseline that reached a peak after 30 seconds and returned to basal levels within 60 seconds. A similar transient rise in calcium was obtained upon exposure of the UMR cells to both the hIL-1 suspension buffer and to the concentration of fetal bovine serum present in the hIL-1 buffer. This effect was not abolished either by heat inactivation of both hIL-1 and serum or by pretreatment of hIL-1 with specific rabbit antihuman-IL-1 antibody. Moreover, exposure of the UMR cells to either rhIL-1 alpha or rhIL-1 beta or to a mixture of both at concentrations of 1 to 100 U/ml was not followed by any change in [Ca++]i. Our data do not support the idea that IL-1 can stimulate osteoblasts through a calcium-mediated pathway.

Central osteosclerosis with ectodermal dysplasia: clinical, laboratory, radiologic, and histopathologic characterization with review of the literature.

IBIDS is a syndrome characterized by ichthyosis, brittle hair, impaired intelligence, decreased fertility, and short stature, but unassociated with skeletal lesions. This condition is considered a form of trichothiodystrophy because hair from several cases has been found to have a low sulfur content. We describe a 9-year and 10-month-old white boy whose clinical features resemble the IBIDS syndrome (ichthyosis, brittle hair, cataracts, and short stature), but who also has marked axial osteosclerosis and peripheral osteopenia. No abnormalities of mineral homeostasis were noted. Histopathologic assessment of nondecalcified bone specimens excluded osteopetrosis, but suggested slow skeletal remodeling. When subjected to polarized light microscopy, his hair exhibited the band-like pattern of birefringence described in trichothiodystrophy. Literature review disclosed 8 patients, 2 of whom had been diagnosed as trichothiodystrophy, with like clinical features including osteosclerosis. These skeletal abnormalities together with clinical features of the IBIDS/trichiodystrophy syndrome, we believe, reflect the prototype of a disorder that seems best described as central osteosclerosis with ectodermal dysplasia.

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.

Cyclic stretch enhances gap junctional communication between osteoblastic cells.

Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the "parachute assay," which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well-coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106-01 cells. The stretch-induced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady-state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell-to-cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106-01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43-specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43.

Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells.

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.

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.

An intact N terminus is required for the anabolic action of parathyroid hormone on adult female rats.

Intermittent administration of parathyroid hormone (PTH) peptides increases bone density in animal and human models of osteoporosis. In vitro studies have demonstrated that PTH analogs lacking the first two amino acids can stimulate cell proliferation in certain cell systems, whereas fragments with an intact N terminus can be antimitogenic. We have tested whether the truncated PTH(3-38) fragment may be a better "anabolic analog" than PTH(1-38) by monitoring bone density and biomechanical properties of the femur in 6-month-old ovariectomized (OVX) rats. Either PTH fragment was administered subcutaneously (8 micrograms/100 g of body weight) 5 days/week, for 4 weeks, starting 1 week after surgery. During the entire study, untreated OVX rats lost 12.1 +/- 4.4% of their initial bone density. PTH(1-38) reversed the initial bone loss, leading to complete restoration of presurgery values after 4 weeks of treatment. Conversely, administration of PTH(3-38) resulted in 13.2 +/- 5.8% bone loss, while continuous estrogen infusion (10 micrograms/kg/day) prevented bone loss but did not reverse it. Sham-operated animals also experienced significant bone loss in the vehicle and PTH(3-38)-treated groups (-4.5 +/- 6.7%, and -7.6 +/- 2.8%, respectively), whereas a significant gain in bone density (+4.4 +/- 5.6%) was observed in the rats treated with PTH(1-38). A bone quality factor (index of strain energy loss) and the impact strength (resistance to fracture) were 25% and 44% lower in femurs explanted from OVX animals treated with either vehicle or PTH(3-38), compared with sham-operated animals. On the contrary, no difference was observed between OVX and control animals after treatment with PTH(1-38), indicating a preservation of the capacity to withstand mechanical stress. Thus, PTH(1-38) counteracts estrogen-dependent loss of mineral density and bone biomechanical properties and increases bone density in estrogen-replete animals. An intact N terminus sequence is necessary for this anabolic action of PTH.

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.