Biomedical subjects
Publications and source records attributed to Roberto Civitelli.
Recent microarray studies of mouse and human osteoblast differentiation in vitro have identified novel transcription factors that may be important in the establishment and maintenance of differentiation. These findings help unravel the pattern of gene-expression changes that underly the complex process of bone formation.
Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.
The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.
BACKGROUND: We conducted a 3-year, double-blind, randomized, placebo-controlled study to determine whether the positive effects of hormone/estrogen replacement therapy (H/ERT) on postcranial bone density are accompanied by similar positive effects on oral bone mass. METHODS: A total of 135 postmenopausal women (aged 41-70 years) with no evidence of moderate or severe periodontal disease were randomized to receive daily oral conjugated estrogen (Premarin; 0.625 mg) alone or in combination with medroxyprogesterone acetate (Prempro; 0.625 and 2.5 mg, respectively) or placebo. All subjects received calcium carbonate (1000 mg/d) and cholecalciferol (400 [corrected] IU/d) supplements. The primary efficacy end points were the changes in alveolar crest height and alveolar bone density. Alveolar crest height was measured on bite-wing radiographs, and changes in alveolar bone mass were assessed by means of digital-subtraction radiography. Postcranial bone density was measured in the lumbar spine and left proximal femur by means of dual-energy x-ray absorptiometry. RESULTS: Hormone/estrogen replacement therapy significantly increased alveolar bone mass compared with placebo (+1.84% vs +0.95% [P =.04]), and tended to improve alveolar crest height (+4.83% vs +3.46% [P =.34]). Bone mineral density of the proximal femur significantly increased in the H/ERT compared with the placebo group (total proximal femur, +3.59% vs +0.22% [P =.001]; neck, +2.05% vs -0.34% [P =.02]; trochanter, +3.49% vs +0.08% [P<.001]), but not the lumbar spine (+1.01% vs +0.17% [P =.39]). Changes in alveolar bone mass correlated with bone density changes in the total femur (r = 0.28 [P =.02]) and femoral trochanter (r = 0.25 [P =.04]) in the H/ERT but not in the placebo group. CONCLUSIONS: Postcranial and oral bone mass were increased in postmenopausal women receiving H/ERT. Improvement in oral bone health constitutes an additional benefit of H/ERT.
BACKGROUND: The loss of ovarian function at menopause is associated with loss of postcranial and oral bone. Hormone/estrogen replacement therapy (HRT/ERT) has a positive effect on both postcranial and oral bone. The objective of the study was to determine if the positive effect of HRT/ERT on alveolar crest height (ACH) is generalized or site specific. METHODS: The sample consisted of 49 women who completed a 3-year, HRT/ERT prospective study. Cemento-enamel junction distances (ACH) were measured on digitized images of bitewing radiographs. Lumbar spine and proximal femur bone mineral densities (BMDs) were determined with dual-energy x-ray absorptiometric scans. Measurements were made at baseline and at the end of year 3. For the 3-year study period, mean change in ACH was determined for each patient. In addition, the sites with the greatest, second and third greatest ACH changes were determined for each patient. Correlations between changes in ACH (as determined by the various methods) and postcranial BMD were determined. RESULTS: Mean ACH changes had an average correlation (r) of -0.24 with femoral and lumbar spine BMDs. Although the largest site-specific change in ACH resulted in a mean correlation of -0.21, the correlations for the second and third largest changes in ACH dropped to -0.15 and -0.12. Overall, the correlations for site-specific changes were substantively smaller than those for generalized change. CONCLUSIONS: The data of this study indicate that ACH change attributable to HRT/ERT is generalized rather than site specific. Studies of the effect of HRT/ERT on ACH should employ multiple measurements to minimize measurement errors associated with site-specific measurements.
BACKGROUND: There are physiological reasons to expect an association between bone mineral density of the spine and hip and attachment loss. To this point, however, most studies have found no correlation. METHODS: The 135 patients in this report were part of a randomized controlled trial of estrogen replacement. All patients were in good oral health at entry and received annual oral prophylaxis as part of the study. Standard probing measurements were made with a pressure sensitive probe at 6 sites on each tooth. Bone mineral density was measured with dual-energy x-ray absorbtiometry at the lumbar spine (anterior-posterior and lateral) and proximal femur (neck, trochanter, intertrochanter, Ward's triangle, and total area). These procedures were performed at baseline and at annual intervals for 3 years. RESULTS: Correlations between cross-sectional measurements of clinical attachment level and bone mineral density were very weak, and did not approach statistical significance (-0.06 < or =r < or =0.10, 0.15 < or =P < or =0.75). A few somewhat stronger correlations were found between longitudinal changes in bone mineral density and attachment (-0.20 < or = r < or =-0.02, 0.02 < or = P < or =0.81). Although the correlations in the longitudinal changes were weak, they were consistently in the direction of greater bone mineral density being associated with less attachment loss. CONCLUSIONS: There is no clear association between clinical attachment level and bone mineral density of the lumbar spine and proximal femur, whether examined on a cross-sectional or longitudinal basis. Patterns in the data suggest there may be a weak association in the longitudinal changes.
Signaling between osteoblasts and osteoclasts is important in bone homeostasis. We previously showed that human osteoblasts propagate intercellular calcium signals via two mechanisms: autocrine activation of P2Y receptors, and gap junctional communication. In the current work we identified mechanically induced intercellular calcium signaling between osteoblasts and osteoclasts and among osteoclasts. Intercellular calcium responses in osteoclasts required P2 receptor activation but not gap junctional communication. Pharmacological studies and reverse transcriptase-PCR amplification demonstrated that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling to osteoclasts. Thus these studies show that calcium signaling between osteoblasts and osteoclasts occurs via activation of P2 receptors, but that different families of P2 receptors are required for calcium signaling in these two cell types. Intercellular calcium signaling among bone cells is therefore amenable to pharmacological manipulation that will specifically affect only bone-forming or bone-resorbing cells. P2 receptors may be important drug targets for the modulation of bone turnover.
BACKGROUND: Antiresorptive agents for the treatment of osteoporosis suppress bone remodeling and reestablish bone turnover at a lower rate to reduce bone loss. Recombinant teriparatide (human parathyroid hormone 1-34) stimulates bone formation, increases bone mass, and improves bone microarchitecture. We contrasted the effects of once-daily doses of 20 mug of teriparatide and 10 mg of alendronate sodium on bone mineral density (BMD) and markers of bone turnover. METHODS: Markers of bone turnover and areal BMD were assessed in 203 postmenopausal women with osteoporosis in an 18-month randomized parallel double-blind study; volumetric BMD was measured in a subset of women. RESULTS: Teriparatide significantly increased markers of bone turnover that peaked at 6 months (serum procollagen type I N-terminal propeptide, 218%, and urinary N-telopeptide corrected for creatinine, 58%; P<.001); alendronate significantly decreased the markers at 6 months (-67% and -72%, respectively; P<.001). At 18 months, areal and volumetric spine BMDs were significantly higher with teriparatide than with alendronate (10.3% vs 5.5% [P<.001] and 19.0% vs 3.8% [P<.01], respectively). Areal femoral neck BMD was significantly higher than baseline in the teriparatide and alendronate groups (3.9% and 3.5%, respectively). There were no significant differences in trabecular femoral neck BMD between the teriparatide and alendronate groups (4.9% and 2.2%, respectively). Cortical volumetric femoral neck BMD was significantly different between the teriparatide and alendronate groups (-1.2% and 7.7%, respectively; P = .05). CONCLUSION: Two distinct options for the management of osteoporosis lead to increases in BMD by opposite mechanisms of action on bone remodeling.
Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a "floxed" Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colalpha1(I) promoter. Homozygous Cx43(fl/fl) mice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43(+/-); Colalpha1(I)-Cre or Cx43(+/-); OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43(fl/-); Colalpha1(I)-Cre or Cx43(fl/-); OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.
As human beings venture into space in the 21st century, they will be confronted with a "hypodynamic" and thus hostile environment for the bone homeostasis, that could potentially compromise their mobility in general and skeletal strength in particular after landing. From this point of view, space flight studies are especially interesting and intriguing models for scientists. Space studies, however, must not only overcome enormous technical problems but are also limited in size and frequency. Therefore, ground-based models have also been developed to evaluate the effects of skeletal unloading. The most popular model for human studies is prolonged bed rest with normal volunteers, although studies with paraplegics have also been undertaken. In animals, the hindlimb elevation (tail suspension) model simulates space flight models and is well tolerated by the animals with minimal evidence of stress. Although negative calcium balance and bone loss have been observed in all the aforementioned models of skeletal unloading, the exact mechanism(s) by which this occurs are still unknown and mainly speculative.