Intrinsic mineralization defect in Hyp mouse osteoblasts. Am. J. Physiol. 275 (Endocrinol. Metab. 38): E700-E708, 1998.-X-linked hypophosphatemia (XLH) is caused by inactivating mutations of PEX, an endopeptidase of uncertain function. This defect is shared by Hyp mice, the murine homologue of the human disease, in which a 38 Pex deletion has been documented. In the present study, we report that immortalized osteoblasts derived from the simian virus 40 (SV40) transgenic Hyp mouse (TMOb-Hyp) have an impaired capacity to mineralize extracellular matrix in vitro. Compared with immortalized osteoblasts from the SV40 transgenic normal mouse (TMOb-Nl), osteoblast cultures from the SV40 Hyp mouse exhibit diminished 45Ca accumulation into extracellular matrix (37±6 vs. 1,484±68 counts·min-1 ·μg protein-1) and reduced formation of mineralization nodules. Moreover, in coculture experiments, we found evidence that osteoblasts from the SV40 Hyp mouse produce a diffusible factor that blocks mineralization of extracellular matrix in normal osteoblasts. Our findings indicate that abnormal PEX in osteoblasts is associated with the accumulation of a factor(s) that inhibits ineralization of extracellular matrix in vitro.

X-linked hypophosphatemia (XLH) and the Hyp-mouse disease homolog are caused by inactivating mutations of Phex which results in the local accumulation of an unknown autocrine/paracrine factor in bone that inhibits mineralization of extracellular matrix. In these studies, we evaluated whether the matrix phosphoglycoprotein MEPE, which is increased in calvaria from Hyp mice, is a substrate for Phex. Using recombinant full-length Phex (rPhexWT) produced in Sf9 cells, we failed to observe Phexdependent hydrolysis of recombinant human MEPE (rMEPE). Rather, we found that rPhex-WT inhibited cleavage of rMEPE by endogenous cathepsin-like enzyme activity present in Sf9 membrane. Sf9 membranes as well as purified cathepsin B cleaved MEPE into two major fragments of 50 and 42 kDa. rPhexWT protein in Sf9 membrane fractions, co-incubation of rPhexWT and cathepsin B, and pre-treatment of Sf9 membranes with leupeptin prevented the hydrolysis of MEPE in vitro. The C-terminal domain of Phex was required for inhibition of MEPE cleavage, since the C-terminal deletion mutant rPhex (1-433) [rPhex30M] failed to inhibit Sf9-dependent metabolism of MEPE. Phex-dependent inhibition of MEPE degradation, however, did not require Phex enzymatic activity, since EDTA, an inhibitor of rPhex, failed to block rPhexWT inhibition of MEPE cleavage by Sf9 membranes. Since we were unable to identify interactions of Phex with MEPE or actions of Phex to metabolize cathepsin B, Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. Although the molecular mechanism and biological relevance of non-enzymatic actions of Phex need to be established, these findings indicate that MEPE may be involved in the pathogenesis defective mineralization due to Phex deficiency in XLH and the Hyp-mouse.

Although the CBFA1 gene encodes an osteoblast-specific transcription factor that regulates osteoblast differentiation, uncertainty exists about the organization of its 5¾ end and the relevance of a novel N-terminal sequence identified in the mouse Cbfa1/Osf2 isoform. We found the novel 5'Cbfa1/Osf2 sequence is encoded by a previously unrecognized upstream exon, designated exon−1, which is highly conserved in mouse, rat and human. In addition, two splice donor sites may be utilized to generate Cbfa1/Osf2 cDNAs containing different N-terminal sequences. The first ATG and splice donor site in exon −1 is predicted to transcribe a cDNA containing the unique Osf2 5¾ sequence, whereas a second donor splice site gives rise to cDNAs that contain sequences encoding an 11 amino acid insert. In the human CBFA1 gene, an additional 2-bp nucleotide insert shifts the reading frame and results in stop codons in the cDNA sequence derived from exon−1. The 5'-most exon of the human CBFA1 gene, therefore, contains the 5'non-coding region rather than a human OSF2 homolog. The absence of a homologous OSF2 coding sequence in the human CBFA1 cDNA suggests that the novel mouse N-terminal Osf2 sequence is not essential for functioning of the CBFA1 gene product. In addition, multiple transcripts derived from a single CBFA1/Cbfa1 gene were detected in osteoblasts by Northern analysis and RT-PCR, including additional Cbfa1/Osf2 isoforms containing deletions of exons 1 and 4. Thus, the alternative use of transcription start sites and splicing leads to the genesis of CBFA1/Cbfa1 isoforms with possible differences in transactivation potentials.

Aims: To investigate the incidence of the CYP2C19 polymorphism in the Chinese Dai population. Methods: One hundred and ninety-three healthy Chinese Dai volunteers were identified with respect to CYP2C19 by genotype and phenotype analyses. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping procedures. The 4¢-hydroxymephenytoin (4¢-OH-MP) and S/R-mephenytoin (S/R-MP) excreted in the urine were determined by high-performance liquid chromatography and gas chromatography, respectively. Results: Eighteen subjects were identified as poor metabolisers (PMs). The frequency of PMs in the Chinese Dai subjects was 9.3% (95% confidence interval 5.2, 13.4), which is lowerthan that in the Chinese Han population (P<0.05). Chinese Dai subjects had a higher frequency of the mutant CYP2C19*2 allele (0.303) and a lowerfr equency of the mutant CYP2C19*3 allele (0.034). These two mutant alleles could explain all deficiencies of CYP2C19 activity in the Chinese Dai subjects. The frequency of the CYP2C19*3 allele is significantly lowerthan that in the Chinese Han population (P<0.05). The mean S/R ratio was lower in the homozygous extensive metabolisers (EMs) compared with that in heterozygous EMs (P<0.01), and the latter was lowerthan that in the PMs (P<0.01). Furthermore, the mean S/R ratio in CYP2C19*3/CYP2C19*2 heterozygous PMs was possibly lower than that in the CYP2C19*2/CYP2C19*2 homozygous PMs (P<0.05). Conclusion: The frequencies of PMs and CYP2C19*3 allele in the Chinese Dai population are significantly lowerthan those in the Han population. The CYP2C19 genotype analysis is largely consistent with the mephenytoin phenotype analysis. The variability of S/R ratios in EMs and PMs shows a gene–dosage effect.

Cbfa1 (or Runx2/AML-3/PEPB2a) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 50-

Our previous work has suggested that the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) can abolish the protective effect of ischemic preconditioning in the isolated rat heart. Therefore we tested the hypothesis that CGRP- or capsaicin-induced preconditioning protects against ischemia-reperfusion injury in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion caused a significant cardiac contractile dysfunction, ventricular arrhythmia, and an increased release of creatine phosphate kinase. Pretreatment with CGRP or capsaicin, which evokes release of CGRP from cardiac sensory nerves, for 5 min produced a significant improvement of cardiac function, a reduction in the incidence of ventricular arrhythmia, and a decrease in the release of creatine phosphate kinase. However, the cardioprotection provided by CGRP- or capsaicin-induced preconditioning was abolished by CGRP-(8-37) and ruthenium red, respectively. These findings suggest that CGRP- or capsaicin-induced preconditioning protects against ischemic myocardial injury. The present results also suggest that CGRP may be an endogenous myocardial protective substance in the rat.

Cbrαl基因编码一个成骨细胞特异性转录因子，调控成骨细胞发育、分化和骨的形成。Cbrαl基因含9个外显子，但它的多个外显子存在选择性剪接，产生多样Cbrαl的异构体，具有不同的转录激活潜能。多条信号传导通路如RasMAPK、cAMP、Smads DPC4通路等涉及到Cbrαl的活性或表达的调控。这些研究为药物调控成骨细胞分化和骨的形成及基因治疗骨质疏松症开拓新的思路。