Mineral composition is altered by osteoblast expression of an engineered G(s)-coupled receptor.

Activation of the G(s) G protein-coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive G(s) signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive G(s) activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRµCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive G(s) signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRµCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRµCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm(3) (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous G(s) activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients.

Expression of alternatively spliced isoforms of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger RNA in human kidney and bone cells.

Using a PCR-based strategy, two variants of the PTH/PTH-related peptide (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splicing of exons coding for the N-terminal portion of the receptor. In the S-N3-E2 isoform, the exon coding the signal peptide (S) is spliced to an alternative 3'-acceptor site, producing a product respecting the reading frame, but in which the E1 exon is replaced by 12 amino acids derived from the N3 intron. In the S-E2 isoform, in which the E1 exon is deleted by cassette exclusion, the reading frame is changed, but a truncated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese hamster ovary cells with the originally described S-E1-E2 isoform and the two splice variants, active transcription of PTH/PTH-rp receptor mRNA was detected by RT-PCR in all cases. Cell lines transfected with the S-E1-E2 and S-N3-E2 isoforms displayed a 15- to 25-fold and 2- to 3-fold increase, respectively, in cAMP content after stimulation with 2.4 x 10(-7) M human PTH(1-34), whereas cells transfected with the S-E2 isoform did not respond. PTH elicited an increase in intracellular calcium only in cells transfected with the S-E1-E2 isoform. Studies evaluating the surface expression of receptors using anti-human PTH/PTH-rp receptor antibodies and the ability of transfected cells to bind [125I]PTH-rp indicated that the low or absent responses to PTH stimulation resulted, at least in part, from low surface expression of the S-N3-E2 and S-E2 isoforms. These studies support the conclusion that exon E1 is extremely important in promoting surface expression of the PTH/PTH-rp receptor but indicate that isoforms lacking this exon can retain the ability to recognize PTH. The possible intracellular expression of these splice variants, which account for 15-20% of total PTH/PTH-rp receptor mRNA, needs to be evaluated.

Effect of age on circulating immunoreactive and bioactive parathyroid hormone levels in women.

Although levels of serum immunoreactive parathyroid hormone (iPTH) increase with age in women, this could be caused by retention of non-biologically active PTH fragments by the aging kidney. In 102 normal women, aged 30 to 89 yr, serum iPTH increased with age by 58% (r = 0.33, p less than 0.001) with antiserum GP-1M (which has midmolecule specificity) and 43% (r = 0.32, p less than 0.001) with antiserum CH-12M (which may have whole molecule specificity); urinary cAMP/GFR excretion increased by 29% (r = 0.22, p less than 0.05). The results of these assays were validated by comparison with serum levels of biologically active PTH (BioPTH) in immunoextracts of serum followed by renal adenylate cyclase assay in a selected subgroup of 25 of the women. Serum BioPTH correlated with serum iPTH assessed by antiserum GP-1M (r = 0.48, p less than 0.05) and antiserum CH-12M (r = 0.48, p less than 0.05) but not with urinary cAMP. The data are consistent with an increase of parathyroid function with aging: clearly, we do not find decreased parathyroid function as would be expected if age-related bone loss was not mediated, in part, by PTH.