Cloning and characterization of 5'-upstream sequence of the M32 gene for a mouse homologue of Drosophila heterochromatin protein 1 (HP1).

M32 [also termed chromatin modifier protein 2 (MOD2)] is a nuclear protein consisting of the condensed chromatin structure (heterochromatin) and considered one of the mammalian homologues of heterochromatin protein 1 (HP1), first isolated as one of the components of heterochromatin in Drosophila. This report presents the isolation and characterization of the 5'-upstream region of the mouse M32 gene containing a promoter region and 5'-untranslated region (5'-UTR) exon. The 5'-upstream region (approximately 0.27 kb starting from the 5' end of the 5'-UTR exon) of the M32 gene contained neither a TATA box nor a CCAAT box, but possessed potential binding sites for transcription factors such as Sp1, H4TF-1, PEA2, PEA3, GSG element and Egr-1, and was highly G/C-rich. The promoter activity of this 5'-upstream region was demonstrated by transfecting its fusion-construct with the E. coli beta-galactosidase gene into the F9 mouse teratocarcinoma cell line. The 5' ends of the mRNA were mapped to at least two positions in the 5'-upstream region. Interestingly, the 5'-upstream region exhibited a high degree of similarity to a portion of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 gene, which is thought to play a role in RNA processing, located in the reverse orientation to the M32 gene, and also to several known ESTs and cDNAs. These findings suggest that the 5'-upstream region of the M32 gene consists of a multiple regulatory complex which probably plays important roles in nuclear function such as chromatin organization and RNA processing.

Requirement of CD9 on the egg plasma membrane for fertilization.

CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, have been identified by their ability to induce cartilage and bone from nonskeletal cells and have been shown to act as a ventral morphogen in Xenopus mesoderm. We isolated a murine homeobox-containing gene, distal-less 5 (mDlx5), as a BMP-inducible gene in osteoblastic MC3T3-E1 cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA showed increase in various osteogenic markers, a fourfold increase in alkaline phosphatase activity, a sixfold increase in osteocalcin production, and appearance in mineralization of extracellular matrix. Furthermore, mDlx5 was induced orthotopically in mouse embryos treated with BMP-4 and in fractured bone of adult mice. Consistent with these observations, we also found that injection of mDlx5 mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos. These findings suggest that mDlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorsoventral patterning of embryonic axis.

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Nucleotide sequence of the ring3 gene in the class II region of the mouse MHC and its abundant expression in testicular germ cells.

The RING3 (NAT) gene is the first and only locus with no obvious function associated with the immune system in the class II region of the human major histocompatibility complex. This gene is a homologue of the Drosophila homeotic gene female sterile homeotic (fsh) and encodes a nuclear serine-threonine kinase. To study more about the physiological function of the RING3 gene, we isolated a mouse homologue from a genomic library, determined its gene structure, and investigated its expression profile. The mouse Ring3 gene spans approximately 8 kb and consists of 12 exons encoding a 798-amino-acid protein, sharing as high as 96% amino acid identity with the human RING3 protein. Northern hybridization revealed that the Ring3 gene abundantly produced 3.8- and 3.0-kb transcripts in the testis but was weakly expressed with 4.6- and 3.8-kb transcripts in somatic tissues. It appears that testis-specific 3.0-kb transcript gives rise to a smaller size Ring3 protein resulting from the usage of the second ATG codon for translational initiation compared to the almost ubiquitous 4.6-kb transcript. In RNAs isolated from fractionated testicular germ cells, the two testicular mRNAs were detected exclusively in the fractions containing a large population of round spermatids and pachytene spermatocytes. Furthermore, in situ hybridization on cross sections of seminiferous tubules in the testis showed that the expression of the Ring3 gene was initiated at the pachytene spermatocyte stage during meiosis and persisted throughout the round spermatid stage during spermiogenesis. These results suggest that the Ring3 gene plays an important role in spermatogenesis.

Aberrant regulation of bone trace elements in motheaten and osteopetrosis mutant mice.

Increasing numbers of genetic diseases involving bone development and models for these diseases have been identified recently. Analysis of these bone diseases have revealed that regulated action of multiple growth factors and subsequent signal transduction are essential for normal bone formation. In this paper, two murine mutant mice viable motheaten and osteopetrosis are analyzed. Mice with the recessive 'viable motheaten' mutation express a severe immunodeficiency syndrome and bone defects. Mutations at the motheaten locus were shown to be the result of aberrant splicing of the gene encoding hematopoietic cell phosphatase (Hcph). Mice homozygous for the osteopetrosis mutation develop congenital osteopetrosis due to a severe deficiency of osteoclasts. It has been recognized that bone trace element composition analysis helps to define bone-related physiological conditions. We have analyzed bone trace element composition in viable motheaten and osteopetrosis mutant animal models in this study. In order to gain insights into the effects of particular genetic defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in limb bones of viable motheaten and osteopetrosis mutant mice. An assessment of these trace element spectrum in the two mutant models with respect to each genetic defects are discussed in this paper.

Genomic structure of the human ribosomal protein L41 gene.

Using PCR technique, genomic DNA encoding the gene of the human ribosomal protein L41 was amplified. Subsequent sequencing showed that the gene was constituted with 4 exons and 3 introns. Because the amplified gene contained introns and whole the open reading frame, we concluded the fragment corresponded to the functional gene.

Mice lacking smooth muscle calponin display increased bone formation that is associated with enhancement of bone morphogenetic protein responses.

BACKGROUND: Calponin is a calmodulin-and actin-binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin-activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified. RESULTS: We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3-E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice. CONCLUSION: This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.

Dysregulation of trace element composition in ovariectomized cynomolgus monkey bones.

One of the challenging issues in modern biomedical science is the increasing number of osteoporosis patients due to the expansion of elderly populations. Among aging-related pathogenic changes, alterations in bone function and skeletal pathogenesis is a particularly important issue of concern. Osteoporosis is one of the most serious bone-related pathogenic states, as it causes serious loss of quality of life. Alterations in estrogen levels in accordance with aging are one of the key risk factors for osteoporosis. Complexed estrogen actions on bones can be traced by analyzing bone mineral components, as those elements accumulate as mineral complexes, reflecting the context of multiple cellular reactions such as bone resorption/osteogenesis. We have analyzed bone trace element composition in ovariectomized (OVX-treated) Cynomolgus monkey models in this study. In order to gain insights into the effects of such defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in vertebral bones of OVX-treated Cynomolgus monkeys. An assessment of these trace element spectra in OVX model animals is discussed. These results could provide useful markers for understanding the physiological states of bones in postmenopausal women.

Regulation of osteoblast-specific factor-1 (OSF-1) mRNA expression by dual promoters as revealed by RT-PCR.

OSF-1 (osteoblast-specific factor-1), which is also referred to as p18, HBBM, HB-GAM, HBGF-8, HARP, HBNF, and pleiotrophin, is a 121-amino acid polypeptide that can induce neurite outgrowth in vitro and is highly expressed in several tissues during fetal development but exhibits expression restricted to brain and bone tissues in adults. We have reported the genomic structure of mouse OSF-1 gene, in which the open reading frame spans four exons and at least two additional 5'-UTR exons (upstream exon U2 and downstream exon U1) exist. From analysis of isolated cDNAs, two types of cDNAs were identified: one has a sequence for U1 and U2 and the other has a sequence for an intron (present between U1 and U2) and U1. This suggests that the OSF-1 gene utilizes two alternative promoters, a distal and a proximal promoter, designated promoters II and I, respectively, for the translation initiation site (ATG). Promoter II is thought to exist upstream of the intron, while promoter I is present in the intron. RT-PCR was employed to examine which OSF-1 promoters are used during development and in various cell lines. In adult mice (aged 2 months), usage of promoter I was predominant, and OSF-1 mRNAs were expressed in many organs including brain and bone. At one fetal stage (E-19), promoter I was active in the major organs including brain, liver, kidney, and intestine, while promoter II was active only in the brain. In the cell lines examined, usage of promoter I was frequent, while promoter II was active only in a few cell lines such as MC3T3-E1 (cultured for 7 days) and C3H10T1/2. These findings suggest that OSF-1 may play fundamental roles in differentiation, growth and maintenance of adult organs as well as in embryogenesis, and indicate that the expression of OSF-1 is regulated, at least in part, by the usage of different promoters in the mouse.

Transformation-related expression of a low-molecular-mass tropomyosin isoform TM5/TM30nm in transformed rat fibroblastic cell lines.

We cloned a full-length rat TM5/TM30nm cDNA. Using this cDNA as a probe, we demonstrated that expression of TM5/TM30nm mRNA was higher in the tumorigenic rat fibroblastic cell lines SR-3Y1-2 and fos-SR-3Y1-202 than in the normal cell line 3Y1. High expression of TM5/TM30nm protein in SR-3Y1-2 and fos-SR-3Y1-202 cells was also detected by Western blot analysis using anti-TM5/TM30nm antiserum. In addition, the cellular localization of this protein differed between 3Y1 cells and tumorigenic ones. These findings suggest that TM5/TM30nm is involved in malignant transformation of rat fibroblastic cells.

Decreased expression of a single tropomyosin isoform, TM5/TM30nm, results in reduction in motility of highly metastatic B16-F10 mouse melanoma cells.

Tropomyosin is an actin-associated cytoskeletal protein expressed in muscle and non-muscle cells. There are several tropomyosin isoforms, and their cellular expression is known to be associated with transformation events caused by retroviral infection and chemical mutagens. We found that expression of a low-molecular weight tropomyosin isoform, TM5/TM30nm, was higher in a high-metastatic B16 mouse melanoma cell line, B16-F10, than in B16-F1, a low-metastatic mouse melanoma cell line. In order to determine whether this elevated level of TM5/TM30nm plays a role in malignant phenotype, B16-F10 cells were transfected with recombinant DNA containing antisense rat TM5/TM30nm cDNA linked to the human metallothioneinIIa promoter, which is inducible by heavy metals such as zinc and cadmium. When the stably transfected clones were treated with ZnSO4, decreased expression of TM5/TM30nm and reduction in cell motility, which is thought to be an indicator of cellular malignancy were observed. These findings suggest that TM5/TM30nm plays a fundamental role in regulating cell motility, which is essential for metastasis and invasion of tumor cells.

Isolation of a yeast tropomyosin-related cDNA clone that encodes a novel transmembrane protein having a C-terminal highly basic region.

A novel cDNA clone was isolated from a yeast Saccharomyces cerevisiae lambda gt11 cDNA library using rabbit anti-rat tropomyosin (TM30nm) polyclonal antibody (RTM8-2). It consists of an open reading frame of 951 bp, encoding 317 amino acid residues. The putative sequence recognized by RTM8-2 was present in Asn-235 to Thr-250. The deduced amino acid sequence and hydropathy plot suggested that this protein has a tropomyosin-homologous sequence, a predicted transmembrane, and a C-terminal basic region. A search of the data bases (EMBL and GenBank) revealed that the 128bp sequence in the 3' untranslated region (3'UTR) is almost identical (96.9%) to the human cDNA clone 54E05 sequence (EMBL accession number Z15978).

Effect of (-)-epigallocatechin gallate, the main constituent of green tea, on lung metastasis with mouse B16 melanoma cell lines.

(-)-Epigallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, inhibits tumor promotion and chemical carcinogenesis in animal experimental systems. Here we report that the peroral administration of EGCG inhibited metastasis of B16 melanoma cell lines, such as B16-F10 and BL6, in both experimental and spontaneous systems.