Cbfa1, an essential transcription factor for bone formation, is expressed in testis from the same promoter used in bone.

Cbfa1 is an essential transcription factor for bone formation, but its transcripts have also been detected in thymus and testis. To elucidate the expression of Cbfa1 in testis, we isolated Cbfa1 cDNA from mouse testis. First we examined the length of the transcripts expressed in mouse testis by Northern hybridization. Using a cDNA probe that can detect both Type-I and Type-II Cbfa1 (Type-I: originally reported as Pebp2alphaA by Ogawa et al.; Type-II: originally reported as til-1 by Stewart et al.), 1.8-kb transcripts were detected in testis, and larger transcripts (6.3 kb and 7.4 kb) in T-cell lines, thymus and bone. Using a Type-II specific probe (bone isoform-specific probe), surprisingly, 1.8-kb testis transcript(s) were detected as well as those (6.3 kb) found in bone. In addition, the transcription start site in mouse testis coincides with one of three start sites identified in bone. These results suggest that the testis transcript is generated from the same promoter of Type-II Cbfa1, which is thought to be active only in osteoblasts and some chondrocytes. Next, to define the difference in length of transcripts, we isolated the Cbfa1 cDNA from mouse testis. Sequence analysis of the cDNA showed that alternative splicing around exon 2 and poly-adenylation within exon 8 occurred in the testis isoform, which produce the smaller transcripts. These data revealed that testis Cbfa1 mRNA is transcribed from same promoter used in bones, but the post-transcriptional regulation is different between testis and skeletal tissues.

Isolation and characterization of the distal promoter region of mouse Cbfa1.

Cbfa1 is an essential transcription factor for bone formation, and as such little is known about the region responsible for the transcriptional regulation of this gene. Here we report the determination of the transcription start sites, isolation and partial characterization of distal promoter region of this gene. Three transcription start sites were identified by the 5'-Cap site method, recently invented for rapid examination of the 5'-end of genes of interest. A reporter construct containing 1.8 kb of 5' of transcription start sites had approximately 25-fold more luciferase activity than the promoter-less vector in osteoblastic cell lines. Deletion analysis of the reporter construct demonstrated that the minimal region to express promoter activity lies between bp -168 and -99, taking the most downstream transcription start site as +1. By Northern blot analysis, mRNA expression from the distal promoter was detected in the differentiated osteoblastic cell lines, UMR-106, ROS17/2.8 and MC3T3-E1, but not in cell lines of immature phenotype or originated from other organs. Luciferase activity was strongest in UMR-106 and ROS17/2.8, and weakest in COS-1 and HepG2, which are cell lines originating from other organs, corresponding to the level of mRNA expression. These results demonstrated that the distal promoter region examined here is important for tissue- and cell-type-specific gene expression of Cbfa1.

Cbfa1 isoforms exert functional differences in osteoblast differentiation.

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. We investigated functional differences among three isoforms of Cbfa1: Type I (originally reported as Pebp2alphaA by Ogawa et al. (Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863), Type II (originally reported as til-1 by Stewart et al. (Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter, E., Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8646-8651), and Type III (originally reported as Osf2/Cbfa1 by Ducy et al. (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). A reverse transcriptase-polymerase chain reaction analysis demonstrated that these isoforms were expressed in adult mouse bones. The transient transfection of Type I or Type II Cbfa1 in a mouse fibroblastic cell line, C3H10T1/2, induced the expression of alkaline phosphatase (ALP) activity. This induction was synergistically enhanced by the co-introduction of Xenopus BMP-4 cDNA. In contrast, the transient transfection of Type III cDNA induced no ALP activity. In C3H10T1/2 cells stably transfected with each isoform of Cbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type II Cbfa1 but not in cells with Type III Cbfa1. Osteocalcin, osteopontin,and type I collagen gene expressions were induced or up-regulated in all of the cells stably transfected with each isoform of Cbfa1, and Type II transfected cells exhibited the highest expression level of osteocalcin gene. A luciferase reporter gene assay using a 6XOSE2-SV40 promoter (6 tandem binding elements for Cbfa1 ligated in front of the SV40 promoter sequence), a mouse osteocalcin promoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by each Cbfa1 isoform with or without its beta-subunit. These results suggest that all three of the Cbfa1 isoforms used in the present study are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation.

[The relationship between body mass index and coronary risk factors].

We investigated the relationship between the body mass index and the prevalence of coronary risk factors, hypertension, hyperlipidemia, glucose intolerance and hyperuricemia, in middle-aged male workers. We found a positive correlation between the body mass index and the prevalence rate of these coronary risk factors. Mean values for serum total cholesterol, triglyceride, LDL-cholesterol and HbA1c increased with the increase in the body mass index, but decreased in HDL-cholesterol.

Cloning of mouse FGF10 and up-regulation of its gene expression during wound healing.

We cloned the mouse homolog of FGF10, which was recently reported as a new member of the FGF family. The predicted molecular mass of this molecule is 23.6 kDa, and both nucleotide and amino acid sequences show high degrees of similarity with those of the rat. Examination of mouse FGF10 mRNA expression in various tissues and developmental stages by Northern hybridization revealed tissue- and developmental stage-specific expression of the gene. Similarly to the rat counterpart, mouse FGF10 mRNA (4.5 kb) was expressed relatively abundantly in embryos and the lung, and at much lower levels in brain and heart. In addition, a shorter transcript (1.3 kb) is expressed only in testis. Considering the high similarity in primary structure between FGF10 and FGF7 (known as keratinocyte growth factor; KGF), we also examined the gene expression of FGF10 during wound healing using a mouse model. FGF10 mRNA was highly induced 1 day after injury and decreased rapidly by 3 days. This suggests that FGF10 is a primary factor in the process of wound healing similarly to other growth factors such as TGF alpha and FGF7.

Structure and expression of human fibroblast growth factor-10.

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately 19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.

Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family.

We isolated the cDNA encoding a novel member of the fibroblast growth factor (FGF) family from rat embryos by homology-based polymerase chain reaction. The FGF-related cDNA encodes a protein of 215 amino acids (approximately 24 kDa), which has a conserved approximately 120-amino acid core with approximately 30-60% amino acid sequence identity with the FGF family. This protein with a hydrophobic amino terminus appears to be a secreted protein. The cDNA was translated in a coupled in vitro transcription-translation system. The molecular mass of the translation product was observed to be approximately 26 kDa. The expression of the FGF-related mRNA in the rat embryo and adult tissues was determined by Northern analysis and in situ hybridization. The mRNA was expressed in several discrete regions of the embryo. In adult tissues, the mRNA was preferentially expressed in the lung. The expression profile of the FGF-related mRNA was different from those of other FGF family mRNAs. As this protein is the 10th documented protein related to FGFs, we tentatively term this protein FGF-10.

Localization of fibroblast growth factor-9 mRNA in the rat brain.

We examined the localization of fibroblast growth factor-9 (FGF-9) mRNA in the rat brain by in situ hybridization. FGF-9 mRNA was moderately or weakly expressed in widespread regions including the olfactory bulb, caudate putamen, cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, brainstem and cerebellum. However, FGF-9 mRNA was also strongly expressed in several specific nuclei including the red nucleus and oculomotor nucleus in the midbrain, the vestibular nucleus and facial nucleus in the brainstem and the medial cerebellar nucleus, interposed cerebellar nucleus and lateral cerebellar nucleus in the cerebellum. The cellular localization of FGF-9 mRNA indicated that the mRNA in the rat brain was expressed preferentially in neurons, although FGF-9 was originally isolated from human glioma cells. The localization profile of FGF-9 mRNA is different from those of aFGF, bFGF and FGF-5 mRNAs reported previously. The present findings indicate that FGF-9 has a unique role in the brain.

Rat FGF receptor-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus.

The fibroblast growth factor (FGF) receptor family consists of four members, FGFR-1, FGFR-2, FGFR-3 and FGFR-4, that are closely related receptor tyrosine kinases. We examined the expression of rat FGFR-4 mRNA in the brain by in situ hybridization and compared it with that of the mRNAs for other FGF receptors. In contrast with FGFR-1, FGFR-2 and FGFR-3 mRNAs which are expressed widely in the brain, the FGFR-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus neurons. The present finding indicates that FGFR-4 has a function specific to the medial habenular nucleus.