We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1 m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1 m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1 m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1 m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblastrelated genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1 m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1 m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

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Runx2 (runt-related transcription factor 2) is a master regulator of skeletogenesis. Distinct promoters in the Runx2 gene transcribe the "bone-related" Runx2-II and non-osseous Runx2-I isoforms that differ only in their respective N termini. Existing mutant mouse models with both isoforms deleted exhibit an arrest of osteoblast and chondrocyte maturation and the complete absence of mineralized bone, but they do not distinguish the separate functions of the two N-terminal isoforms. To elucidate the function of the bone-related isoform, we generated selective Runx2-II-deficient mice by the targeted deletion of the distal promoter and exon 1. Homozygous Runx2-II-deficient (Runx2-II ؊/؊ ) mice unexpectedly formed axial, appendicular, and craniofacial bones derived from either intramembranous ossification or mesenchymal cells of the bone collar, but they failed to form the posterior cranium and other bones derived from endochondral ossification. Heterozygous Runx2-II-deficient mice had grossly normal skeletons, but were osteopenic. The commitment of mesenchymal cells ex vivo to the osteoblast lineage occurred in Runx2-II ؊/؊ mice, but osteoblastic gene expression was impaired. Chondrocyte maturation appeared normal, but the zone of hypertrophic chondrocytes was not transformed into metaphyseal bone, leading to widened growth plates in Runx2-II ؊/؊ mice. Compensatory increments in Runx2-I expression occurred in Runx2-II ؊/؊ mice but were not sufficient to normalize osteoblastic maturation or transcriptional activity. Our findings support distinct functions of Runx2-II and -I in the control of skeletogenesis. Runx2-I is sufficient for early osteoblastogenesis and intramembranous bone formation, whereas Runx2-II is necessary for complete osteoblastic maturation and endochondral bone formation.The Runx 1 family of transcription factors consists of three highly conserved mammalian genes, designated Runx1, Runx2, and Runx3 (1-5), that regulate, respectively, hematopoiesis (6), skeletogenesis (7-10), and neuronal development (11)(12). RUNX2 is the master transcription factor essential for skeletogenesis. Targeted disruption of the Runt domain of Runx2 results in the complete lack of both intramembranous and endochondral bone formation due to an arrest in osteoblast and chondrocyte maturation (8,10,(13)(14)(15)(16)(17)(18)(19). Haploinsufficiency of RUNX2 causes cleidocranial dysplasia in humans, and heterozygous Runx2 deficient mice show a similar phenotype, suggesting that the expression level of Runx2 influences the skeletal phenotype (10).Separate promoters control the expression of Runx2 isoforms with different 5Ј-UTRs and N-terminal sequences that bind to the same consensus DNA cis-acting elements, interact with similar co-factors, and demonstrate overlapping transactivation potential in vitro (17-24). The Runx2-II isoform (also called PEBP2␣A, major til-1 and Cbfa1.iso, Osf2) is derived from the distal "bone-related" promoter (P1) and encodes a 528-amino acid protein that begins with the 19 amino acids MASNSLFSAVTPCQQ...

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The 4'-hydroxylation of S-mephenytoin exhibits a polymorphism in humans, with the poor metabolizer phenotype exhibiting a lower frequency in white (3% to 5%) than in Oriental populations (13% to 23%). Two mutations in CYP2C19 (CYP2C19m1 and CYP2C19m2) have recently been described that account for approximately 85% of white and 100% of Japanese poor metabolizers. This study examines whether these mutations account for the poor metabolizer phenotype in the Chinese population. The metabolism of S-mephenytoin exhibited a bimodal distribution in 244 unrelated Chinese subjects, although the distribution of the two phenotypes overlapped. In 75 selected Chinese subjects, CYP2C19 genotype analysis predicted the phenotype with 100% accuracy. The frequency of the poor metabolizer phenotype was approximately 11% (95% confidence interval 7% to 15%). The frequency of the CYP2C19m1 allele was 0.289, whereas that of CYP2C19m2 was 0.044. Homozygous extensive metabolizers had slightly lower ratios of S/R-mephenytoin compared with heterozygous extensive metabolizers, showing a gene-dosage effect. These data show the advantages of genotype analysis in investigations of the mephenytoin phenotype in Oriental subjects.

With increasing life expectations, more and more patients suffer from fractures either induced by intensive sports or other bone-related diseases. The balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is the basis for maintaining bone health. Osterix (Osx) has long been known to be an essential transcription factor for the osteoblast differentiation and bone mineralization. Emerging evidence suggests that Osx not only plays an important role in intramembranous bone formation, but also affects endochondral ossification by participating in the terminal cartilage differentiation. Given its essentiality in skeletal development and bone formation, Osx has become a new research hotspot in recent years. In this review, we focus on the progress of Osx’s function and its regulation in osteoblast differentiation and bone mass. And the potential role of Osx in developing new therapeutic strategies for osteolytic diseases was discussed.

The P1 and P2 promoters of the Cbfa1/Runx2 gene produce Type I and II mRNAs with distinct complex 5'-untranslated regions, respectively designated UTR1 and UTR2. To evaluate whether the 5'-UTRs impart different translational efficiencies to the two isoforms, we created SV40 promoter-UTR-luciferase reporter (luc) constructs in which the translational potential of the 5'-UTR regions was assessed indirectly by measurement of luciferase activity in transfected cell lines in vitro. In MC3T3-E1 pre-osteoblasts, UTR2 was translated approximately twice as efficiently as the splice variants of UTR1, whereas translation of unspliced UTR1 was repressed. To determine if the UTRs conferred internal ribosome entry site (IRES)-dependent translation, we tested bicistronic SV40 promoter-Rluc-UTR-Fluc constructs in which Fluc is expressed only if the intercistronic UTR permits IRES-mediated translation. Transfection of bicistronic constructs into MC3T3-E1 osteoblasts demonstrated that both UTR2 and the spliced forms of UTR1 possess IRES activity. Similar to other cellular IRESs, activity increased with genotoxic stress induced by mitomycin C. In addition, we observed an osteoblastic maturation-dependent increase in IRES-mediated translation of both UTR2 and the spliced forms of UTR1. These findings suggest that Cbfa1 UTRs have IRES-dependent translational activities that may permit continued Cbfa1 expression under conditions that are not optimal for cap-dependent translation.

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