Cinacalcet reduces plasma intact parathyroid hormone, serum phosphate and calcium levels in patients with secondary hyperparathyroidism irrespective of its severity.

AIMS: To evaluate the relationship between the severity of secondary hyperparathyroidism (SHPT) - defined in terms of baseline plasma intact parathyroid hormone (iPTH) level - and the magnitude of response to cinacalcet. MATERIALS AND METHODS: In this post hoc analysis, data were pooled from three randomized, placebo-controlled trials in which dialysis patients with iPTH ≥ 300 pg/ml were dose-titrated with cinacalcet or placebo in addition to conventional treatment to achieve iPTH ≤ 250 pg/ml. In 953 patients analyzed (cinacalcet, 545; placebo, 408), baseline iPTH levels were categorized in 100 pg/ml intervals (300 - ≥ 1,000 pg/ml), and the impact of baseline iPTH on changes in iPTH, phosphate (P), calcium (Ca) and calcium- phosphate product (Ca × P) was evaluated. RESULTS: Cinacalcet reduced iPTH (47% reduction), P (9%), Ca (7%), and Ca × P (15%) across all subgroups. For patients receiving cinacalcet, the mean percentage reduction from baseline in iPTH varied from 35 to 55%, being consistently decreased across the severity subgroups. The mean absolute change in iPTH was more pronounced in patients with higher baseline iPTH levels, particularly in the ≥ 1,000 pg/ml subgroup vs. the other subgroups. However, as baseline iPTH levels increased, iPTH ≤ 250 pg/ml was achieved in fewer patients. A trend towards greater absolute change from baseline was observed for P in patients with more severe disease (iPTH ≥ 800 pg/ml) treated with cinacalcet compared with patients with less severe disease (iPTH 300 - < 800 pg/ml). CONCLUSIONS: Cinacalcet lowers plasma iPTH and serum P, Ca and Ca × P levels in dialysis patients with SHPT, regardless of disease severity. Patients with more severe disease experienced greater reductions in PTH and P, but fewer achieved iPTH ≤ 250 pg/ml by the efficacy assessment phase. Use of cinacalcet when baseline PTH is lower may result in more stable control of SHPT and help to control bone and mineral alterations.

Development and progression of secondary hyperparathyroidism in chronic kidney disease: lessons from molecular genetics.

The identification of the calcium-sensing receptor (CaSR) and the clarification of its role as the major regulator of parathyroid gland function have important implications for understanding the pathogenesis and evolution of secondary hyperthyroidism in chronic kidney disease (CKD). Signaling through the CaSR has direct effects on three discrete components of parathyroid gland function, which include parathyroid hormone (PTH) secretion, PTH synthesis, and parathyroid gland hyperplasia. Disturbances in calcium and vitamin D metabolism that arise owing to CKD diminish the level of activation of the CaSR, leading to increases in PTH secretion, PTH synthesis, and parathyroid gland hyperplasia. Each represents a physiological adaptive response by the parathyroid glands to maintain plasma calcium homeostasis. Studies of genetically modified mice indicate that signal transduction via the CaSR is a key determinant of parathyroid cell proliferation and parathyroid gland hyperplasia. Because enlargement of the parathyroid glands has important implications for disease progression and disease severity, it is possible that clinical management strategies that maintain adequate calcium-dependent signaling through the CaSR will ultimately prove useful in diminishing parathyroid gland hyperplasia and in modifying disease progression.

Induction of de novo bone formation in the beagle. A novel effect of aluminum.

To define the primary effects of aluminum on bone in the mammalian species, we examined the dose/time-dependent actions of aluminum in normal beagles. Administration of low dose aluminum (0.75 mg/kg) significantly elevated the serum aluminum (151.7 +/- 19.9 micrograms/liter) compared with that in controls (4.2 +/- 1.35 micrograms/liter) but did not alter the calcium, creatinine, or parathyroid hormone. After 8 wk of therapy, bone biopsies displayed reduced bone resorption (2.6 +/- 0.63 vs. 4.5 +/- 0.39%) and osteoblast covered bone surfaces (2.02 +/- 0.51 vs. 7.64 +/- 1.86%), which was indicative of low turnover. In contrast, prolonged treatment resulted in increased bone volume and trabecular number (38.9 +/- 1.35 vs. 25.2 +/- 2.56% and 3.56 +/- 0.23 vs. 2.88 +/- 0.11/mm) which was consistent with uncoupled bone formation. Administration of higher doses of aluminum (1.20 mg/kg) increased the serum aluminum further (1242.3 +/- 259.8 micrograms/liter) but did not affect calcium, creatinine, or parathyroid hormone. However, after 8 wk of treatment, bone biopsies displayed changes similar to those after long-term, low-dose therapy. In this regard, an increased trabecular number (3.41 +/- 0.18/mm) and bone volume (36.5 +/- 2.38%) again provided evidence of uncoupled bone formation. In contrast, in this instance poorly mineralized woven bone contributed to the enhanced bone volume. High-dose treatment for 16 wk further enhanced bone volume (50.4 +/- 4.61%) and trabecular number (3.90 +/- 0.5/mm). These observations illustrate that aluminum may stimulate uncoupled bone formation and induce a positive bone balance. This enhancement of bone histogenesis contrasts with the effects of pharmacologic agents that alter the function of existing bone remodeling units.

Aluminum-induced de novo bone formation in the beagle. A parathyroid hormone-dependent event.

To examine the influence of osteoblast function on aluminum-induced neo-osteogenesis in the mammalian species, we compared the effects of aluminum in sham-operated and thyroparathyroidectomized (TPTX) beagles. TPTX dogs received sufficient calcium carbonate and calcitriol to maintain normal plasma calcium and calcitriol levels, but developed evidence of decreased osteoblast recruitment and activity, including diminished osteoid-covered trabecular bone surface (3.22 +/- 0.21 vs. 10.95 +/- 1.30%) and a decreased osteoblast number (27.8 +/- 8.1 vs. 139.0 +/- 26.0/mm). Administration of aluminum (1.25 mg/kg i.v., three times/wk) increased the serum aluminum levels in both sham (1,087.0 +/- 276.0 vs. 2.7 +/- 0.8 micrograms/liter) and TPTX animals (2,786.0 +/- 569.0 vs. 3.6 +/- 0.8 micrograms/liter) above normal but did not alter the plasma calcium, creatinine, or PTH from control levels in either sham or TPTX dogs. After 8 wk of therapy, however, bone biopsies from sham-operated beagles displayed evidence of neo-osteogenesis including an increased bone volume (47.0 +/- 1.0 vs. 30.4 +/- 0.9%) and trabecular number (4.1 +/- 0.2 vs. 3.2 +/- 0.2/mm). Much of the enhanced volume resulted from deposition of poorly mineralized woven bone (9.9 +/- 2.7%). In contrast, biopsies from aluminum-treated TPTX animals exhibited significantly less evidence of ectopic bone formation. In this regard, bone (35.5 +/- 1.7%) and woven tissue volume (1.4 +/- 0.8%) as well as trabecular number (3.3 +/- 0.1/mm) were significantly less than those of the aluminum-treated controls. These observations illustrate that aluminum reproducibly stimulates neo-osteogenesis and induces a positive bone balance. However, this effect apparently depends on the availability of a functional osteoblast pool which, if depleted by TPTX, limits the expression of aluminum-induced new bone formation.

Aluminum deposition at the osteoid-bone interface. An epiphenomenon of the osteomalacic state in vitamin D-deficient dogs.

Although aluminum excess is an apparent pathogenetic factor underlying osteomalacia in dialysis-treated patients with chronic renal failure, the mechanism by which aluminum impairs bone mineralization is unclear. However, the observation that aluminum is present at osteoid-bone interfaces in bone biopsies of affected patients suggests that its presence at calcification fronts disturbs the cellular and/or physiochemical processes underlying normal mineralization. Alternatively, aluminum at osteoid-bone interfaces may reflect deposition in preexistent osteomalacic bone without direct effects on the mineralization process. We investigated whether aluminum accumulates preferentially in osteomalacic bone and, if so, whether deposition of aluminum occurs at calcification fronts and specifically inhibits mineralization. Aluminum chloride (1 mg/kg) was administered intravenously three times per week for 3 wk to five normal and five vitamin D-deficient osteomalacic dogs. Before administration of aluminum the vitamin D-deficient dogs had biochemical and bone biopsy evidence of osteomalacia. Bone aluminum content in the osteomalacic dogs (15.1 +/- 2.2 micrograms/g) and the plasma aluminum concentration (10.4 +/- 2.1 micrograms/liter) were no different than those of normal dogs (10.5 +/- 3.5 micrograms/g and 11.9 +/- 1.2 microgram/liter, respectively). After the 3 wk of aluminum administration the plasma phosphorus, parathyroid hormone, and 25-hydroxyvitamin D concentrations were unchanged in normal and vitamin D-deficient dogs. Similarly, no alteration in bone histology occurred in either group. In contrast, bone aluminum content increased to a greater extent in the vitamin D-deficient dogs (390.3 +/- 24.3 micrograms/g) than in the normal dogs (73.6 +/- 10.6 micrograms/g). Moreover, aluminum localized at the osteoid-bone interfaces of the osteomalacic bone in the vitamin D-deficient dogs, covering 42.9 +/- 9.2% of the osteoid-bone surface. Further, in spite of continued aluminum chloride administration (1 mg/kg two times per week), vitamin D repletion of the vitamin D-deficient dogs for 11 wk resulted in normalization of their biochemistries. In addition, while normal dogs maintained normal bone histology during the period of continued aluminum administration, vitamin D repletion of the vitamin D-deficient dogs induced healing of their bones. Indeed, the appearance of aluminum in the cement lines of the healed bones indicated that mineralization had occurred at sites of prior aluminum deposition. These observations illustrate that aluminum deposition in osteomalacic bone may be a secondary event that does not influence bone mineralization. Thus, although aluminum may cause osteomalacia in chronic renal failure, its presence at mineralization fronts may not be the mechanism underlying this derangement.

Cloning and sequencing of human PEX from a bone cDNA library: evidence for its developmental stage-specific regulation in osteoblasts.

Inactivating mutations of the neutral endopeptidase, PEX, have been identified as the cause of X-linked hypophosphatemia (XLH). Though the function of PEX is unknown, current information suggests that impaired renal phosphate conservation in XLH is due to the failure of PEX to either degrade an undefined phosphaturic factor or activate a novel phosphate-conserving hormone. The physiologically relevant target tissue for the XLH mutation has not been identified. An apparent intrinsic defect of osteoblast function in XLH implicates bone as a possible site of PEX expression. In the current investigation, we employed a polymerase chain reaction (PCR) strategy to amplify a PEX cDNA from a human bone cell cDNA library. We found that the human PEX cDNA encodes a 749 amino acid protein belonging to the type II integral membrane zinc-dependent endopeptidase family. The predicted PEX amino acid sequence shares 96.0% identify to the recently cloned mouse Pex cDNA and has 27-38% identity to other members of the metalloendopeptidase family. Using reverse transcriptase (RT)-PCR with PEX-specific primers, we detected PEX transcripts in both human osteosarcoma-derived MG-63 osteoblasts and in differentiated mouse MC3T3-E1 clonal osteoblasts but not in immature MC3T3-E1 preosteoblasts. The association of impaired mineralization of bone in XLH and the apparent developmental stage-specific expression of PEX in osteoblasts suggest that bone is a physiologically relevant site of PEX expression and that PEX may play an active role in osteoblast-mediated mineralization.

Equivalency of various methods for estimating osteoid seam width.

We compared the true osteoid seam width (TOSW) as measured by a modification of the orthogonal intercept lengths with various methods of estimating seam widths, including (1) the commonly used length measurements at four equidistant points (O.Wi/4PT), (2) osteoid area divided by the osteoid perimeter (O.Ar/O/Pm) or the bone/osteoid interface (O.Ar/B.Bd), and (3) a novel method for estimating seam width defined as the osteoid area divided by the major axis of the seam (O.Ar/Axis). All methods for approximating osteoid seam width significantly exaggerated the true osteoid seam dimension by an amount that ranged from 16 to 23%. However, the relative accuracy of all methods of estimating osteoid seam width are equivalent as evidenced by the similar mean difference from the TOSW (3.4, 4.1, 5.1, and 3.8) demonstrated by O.Wi/5PT, O.Ar/Axis, O.Ar/O.Pm, O.Ar/B.Bd, respectively. Regression analysis of the various estimates of seam width on TOSW also demonstrated the equivalency of these methods. Moreover, all estimates could be employed to discriminate seams of normal dimensions from abnormally wide seams in bone specimens derived from patients with osteomalacia. Differences between the methods, however, were observed that may have practical importance. In this regard, the direct procedure of determining distance demonstrated less variance than the indirect estimate of width. As a result, the direct measurement required fewer samples (n = 13) to detect a significant difference to normal and could discriminate smaller deviations in seam width (1.7 microns) at a given sample size compared with O.Ar/Axis (n = 28; 2.9 microns), O.Ar/O.Pm (n = 42; 3.4 microns), and O.Ar/B.Bd (n = 42; n = 3.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines.

Whether the known calcium-sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti-CasR antisera (4637) and low stringency reverse transcriptase-polymerase chain reaction (RT-PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti-CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT-PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a approximately 165 kDa and approximately 140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild-type HEK 293 cells or Saos2, MG-63, or U-2 OS osteoblast-like cell lines. Standard two-step RT-PCR using CasR-specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT-PCR procedure amplified nonspecific products in wild-type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT-PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.

Aluminum-induced neo-osteogenesis: a generalized process affecting trabecular networking in the axial skeleton.

To determine if aluminum-induced neo-osteogenesis occurs in the axial skeleton, we compared spinal bone density and vertebral histology of beagles treated with aluminum for 8 and 16 weeks to that of untreated normals. Administration of aluminum (1.25 mg/kg) did not alter serum calcium, phosphorus, or creatinine but did result in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 (286.7 +/- 12.4 mg/ml) and 16 (361.7 +/- 46.5 mg/ml) weeks of treatment compared with controls (212.2 +/- 4.5 mg/ml). In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis, including the presence of woven bone, both mineralized and unmineralized, within the marrow space. The genesis of such woven bone units resulted after 16 weeks in a significant increase in trabecular bone volume, woven and lamellar (51.2 +/- 4.4 versus 32.4 +/- 1.2%; p less than 0.05), woven bone volume (9.1 +/- 3.6 versus 0 +/- 0%; p less than 0.05), and trabecular number (4.5 +/- 0.3 versus 3.5 +/- 0.2 per mm; p less than 0.05). In addition, scanning electron microscopic evaluation of the bone biopsies confirmed the existence of new trabecular plates that provided interconnections between existent units. These observations illustrate that aluminum-induced neo-osteogenesis positively influences trabecular networking in the axial skeleton. Such enhancement of bone histogenesis contrasts with the effects of other pharmacologic agents that solely alter the thickness of existing trabecular plates or rods within the vertebral spongiosa.

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia.

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.

A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor.

The presence of a cation-sensing mechanism in osteoblasts is suggested by the ability of specific cations to stimulate osteoblastic proliferation in culture and to induce de novo bone formation in some experimental models. Our study examines whether extracellular cations stimulate osteoblasts through the recently identified G protein-coupled calcium receptor (CaR). We found that CaR agonists, calcium (Ca2+), gadolinium (Gd3+), aluminum (Al3+), and neomycin, stimulated DNA synthesis in murine-derived MC3T3-E1 preosteoblasts, whereas magnesium (Mg2+), nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+) had no effect. With the exception of Mg2+, the cation specificities and apparent affinities were similar to that reported for CaR. CaR agonists also stimulated DNA synthesis in C3HT10(1/2) fibroblasts, but not in mesangial PVG, CHO, hepatic HTC, COS-7 cells, or malignant transformed ROS17/2.8 and UMR-106 osteoblasts. In addition, similar to other growth factors, CaR agonists activated transcription of a serum response element luciferase reporter construct (SRE-Luc) stably transfected into MC3T3-E1 osteoblasts, but had no effect on SRE-Luc transfected into CHO and COS-7 cells. We were unable to detect CaR expression by Northern analysis using a mouse CaR-specific probe or to amplify CaR mRNA by reverse transcribed polymerase chain reaction in MC3T3-E1 osteoblasts. These findings suggest that an extra-cellular cation-sensing mechanism is present in murine-derived osteoblasts that is functionally similar to but molecularly distinct from CaR.

Construction of a full-length murine pro alpha 2(I) collagen cDNA by the polymerase chain reaction.

Construction of large collagen cDNA has been hindered by the relatively large size and high G-C content of processed mRNA. We describe here the development of a rapid and efficient method for obtaining large full-length collagen cDNA. A full-length (4.3 kb) murine pro alpha 2(I) collagen cDNA was constructed by synthesis of a first-strand cDNA library with use of poly-A RNA (MC3T3-E1) and the oligo-dT17-adapter primer described by Frohman et al (Proc Natl Acad Sci USA 85:8998, 1988). Pro alpha 2(I) collagen cDNA were specifically amplified by the polymerase chain reaction (PCR) with a pro alpha 2(I) specific primer as the 5' primer (20mer; corresponding to nucleotide positions 42-61 in the first exon of the murine pro alpha 2(I) collagen gene, COL1A2), and with the adapter sequence 5' to the dT17 as the 3' primer. The PCR conditions were optimized to allow amplification of the expected 4.0-5.0-kb product; a major 4.3-kb product was visualized by ethidium bromide, identified by in situ gel hybridization, and cloned. DNA sequencing determined that it contained the correct 5' sequence and the 3' end had a 68 basepair (bp) 3' untranslated region. The entire sequence that codes the amino-terminal propeptide domain has been determined and compared to the human sequence. The homology between human and mouse is less in the amino terminal propeptide than in the triple helical domain; exon 5 of murine COL1A2 codes for an additional six amino acids not found in human COL1A2.

Effects of etidronate-mediated suppression of bone remodeling on aluminum-induced de novo bone formation.

The mechanism by which aluminum chloride stimulates de novo bone formation is unknown. To evaluate the role of bone remodeling and mature osteoblastic function in aluminum-induced neoosteogenesis, we compared the osteogenic effects of aluminum in normal beagles to those in animals with low turnover osteomalacia induced by treatment with etidronate [1-hydroxyethane-1,1-diphosphoric acid (HEBP)]. As assessed by quantitative bone histomorphology, beagles treated with HEBP developed low turnover osteomalacia characterized by a 78% reduction in osteoblast number, a 5.5-fold increase in osteoid volume, complete absence of active mineralization, and diminished resorption surfaces compared to untreated controls. The iv administration of aluminum chloride to normal dogs generated new trabecular structures in the marrow cavity consistent with induction of de novo bone formation. This response consisted of increased trabecular bone volume and number, accumulation of woven osteoid, and increased number of bone-forming cells. The concomitant administration of HEBP failed to prevent induction of de novo bone formation by aluminum. Instead, the neoosteogenic process was superimposed on low turnover osteomalacia in HEBP-treated dogs. Serum aluminum concentrations were increased 2-fold, whereas bone aluminum accumulation was reduced by 58% in HEBP- and aluminum-treated dogs compared to that in aluminum-treated controls. These findings indicate that aluminum stimulation of neoosteogenesis in beagles is independent of mature osteoblast function, normal bone remodeling, and total bone aluminum accumulation. Rather, aluminum-induced de novo bone formation appears to result from stimulation of mesenchymal precursors to form a primitive type of bone which is distinct from coupled bone formation.

Cloning and characterization of the proximal murine Phex promoter.

Phex is an endopetidase that regulates systemic phosphate homeostasis. We investigated Phex gene transcription by cloning and performing functional analysis of the 2736 bp of the 5' flanking region of the mouse Phex gene containing its promoter. We identified a transcription start site, a consensus TATA-box, and multiple potential cis-acting regulator elements. To determine whether the promoter directs cell-type restricted Phex expression, we transfected full-length and 5'-deleted Phex luciferase reporter constructs into various cell lines. Phex-expressing C5.18 chondrocytes displayed the highest activity of the transfected Phex promoter constructs compared with non-Phex-expressing COS-7 cells, whereas promoter activity was intermediate in ROS 17/2.8 osteoblasts and maturation stage-dependent in MC3T3-E1 osteoblasts. Analysis of sequential 5'-deletion mutants of the Phex promoter in ROS 17/2.8 cells revealed bimodal activity, suggesting that both positive and negative cis-acting regions may be present. The chondrogenic factor SOX9 markedly stimulated Phex promoter activity, whereas Cbfa1, PTH, and 1,25(OH)(2)D(3) had no effect. Our findings are consistent with the predominant expression of Phex in bone and cartilage. Additional studies will be needed to confirm the regulatory regions in the Phex promoter that function in a cell-restricted manner.

Prostaglandin F2 alpha-induced mitogenesis in MC3T3-E1 osteoblasts: role of protein kinase-C-mediated tyrosine phosphorylation.

Prostaglandin F2 alpha (PGF2 alpha) stimulates DNA synthesis in osteoblasts through phospholipase-C-dependent increases in intracellular calcium and protein kinase-C (PKC) activity. We present evidence that stimulation of protein tyrosine phosphorylation by PKC is an additional component of the signaling pathways involved in PGF2 alpha-stimulated DNA synthesis in MC3T3-E1 osteoblast-like cells. Mitogenic doses of PGF2 alpha (42 nM) rapidly induced tyrosine phosphorylation of multiple substrates in these osteoblast-like cells. PGF2 alpha stimulated tyrosine phosphorylation of new proteins with apparent mol wt of 87, 80, 50, 47, 36, and 33 kilodaltons and up-regulated phosphorylation of preexisting tyrosine components with mol wt of 123, 112, 68, and 56 kilodaltons. Stimulation of PKC by 1.6 microM phorbol 12-myristate 13-acetate mimicked the pattern of PGF2 alpha-induced protein tyrosine phosphorylation, whereas PKC-deficient cells (induced by overnight pretreatment with 16 microM phorbol 12-myristate 13-acetate) were refractory to PGF2 alpha-stimulated protein tyrosine phosphorylation and DNA synthesis. The tyrosine kinase inhibitors tyrphostin and genistein blocked PGF2 alpha-stimulated DNA synthesis and protein tyrosine phosphorylation, and the tyrosine phosphatase inhibitor orthovanadate prolonged PGF2 alpha-stimulated tyrosine phosphorylation; these findings are consistent with activation of a putative tyrosine kinase. Calcium/calmodulin antagonists also inhibited PGF2 alpha-stimulated DNA synthesis, but the calcium-signaling pathway played no role in PGF2 alpha-induced tyrosine phosphorylation. Our findings suggest that cross-talk between receptor-mediated activation of PKC and protein tyrosine phosphorylation is an important distal signaling pathway necessary for PGF2 alpha-induced DNA synthesis in osteoblast-like cells.

Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts.

We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-dependent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrates cross-talk between G protein-coupled second messengers and protein tyrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate (PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increments in tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatment of osteoblasts, which raised intracellular cAMP levels and inhibited DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) as well as inhibited PKC-stimulated MAPK and Raf-1 activities. Despite this, PMA activated the intermediate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presence of a additional distal cAMP-dependent inhibitory mechanisms.

Rickets in cation-sensing receptor-deficient mice: an unexpected skeletal phenotype.

The hypothesis that local changes in extracellular calcium may serve a physiological role in regulating osteoblast, osteoclast, and cartilage function through the extracellular cation-sensing receptor, CasR, is gaining widespread support, but lacks definite proof. To examine the effects of CasR deficiency on the skeleton, we performed a detailed analysis of the skeleton in CasR knockout mice (CasR(-/-)) and wild-type littermates (CasR(+/+)). CasR ablation in the parathyroid glands of CasR(-/-) mice resulted in hyperparathyroidism, hypercalcemia, and hypophosphatemia. Except for dwarfism, the expected skeletal manifestations of PTH excess, namely chondrodysplasia and increased mineralized bone formation and resorption, were not the main skeletal features in CasR(-/-) mice. Rather, rickets was the predominant skeletal abnormality in these animals, as evidenced by a widened zone of hypertrophic chondrocytes, impaired growth plate calcification and disorderly deposition of mineral, excessive osteoid accumulation, and prolonged mineralization lag time in metaphyseal bone. CasR transcripts were identified in cartilage and bone marrow of CasR(+/+) mice, but not in mineralized bone containing mature osteoblasts and osteocytes. These findings indicate that a calcium-sensing receptor is present in the skeleton, and its absence results in defective mineralization of cartilage and bone by mechanisms that remain to be elucidated.

Aluminum-induced mitogenesis in MC3T3-E1 osteoblasts: potential mechanism underlying neoosteogenesis.

We recently reported that aluminum administration to beagles stimulates uncoupled bone formation in the marrow cavity which increases trabecular bone volume and generates new osseous networks within the axial skeleton. To investigate whether this osteogenic process results from direct stimulation of bone cell replication, we examined the mitogenic effects of aluminum on undifferentiated osteoblasts derived from the MC3T3-E1 clonal cell line. Addition of AlCl3 (1-50 microM) to serum-free culture medium of quiescent osteoblasts resulted in a dose-dependent increase in [3H]thymidine incorporation into DNA and a concordant increase in cell number to 48% of the density achieved at the maximum replicative rate induced by fetal bovine serum (FBS). The time course of aluminum-induced mitogenesis was similar to that of FBS, with onset of DNA synthesis detectable by 12 h and progressive increases in replicative rates observed over a 48-h study period. Moreover, maximal stimulation of DNA synthesis by AlCl3 and that by FBS were not additive, whereas aluminum exerted additional effects on cell replication when combined with low FBS concentrations. Analysis of cell cycle kinetics indicated that aluminum, analogous to FBS, influences the osteoblast replicative activity by inducing transition from the G0 to the S phase of the cell cycle. In addition, exposure of cells to aluminum resulted in rapid accumulation of c-fos mRNA by 30 min, indicating that aluminum, like fetal bovine serum, induced expression of growth-regulating genes. Deferoxamine mesylate, a chelator of aluminum, blocked the replicative actions of aluminum in a dose-dependent fashion. In contrast, pertussis toxin, a specific inhibitor of certain G-proteins, had no effect on the mitogenic effects of aluminum, indicating that aluminum-induced mitogenesis occurs by a pertussis toxin-insensitive pathway. Though the particular cellular pathway remains to be defined, these data provide initial evidence that aluminum-induced neoosteogenesis may depend upon direct stimulation of osteoblast replication.