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Many researches to elucidate the mechanism of gravity sense and its response in the living cells have been advanced. But it has not yet identified that key molecule or signal transduction pathway related to gravity sense and its response. Our goal is to clarify the mechanism of gravity sense, especially the point of gravity sense. First, we have investigated about differences of gene expression level (mRNA) of the endothelial cells cultivated under vector-averaged gravity condition (Clinorotation). The Differential Display pattern showed that expression level of several genes had changed between clinorotated condition and control. The homologues of these fragments were searched on the BLAST database. From BLAST database searching results, GEF and cell adhesion protein effected by clinorotaion. Moreover, morphological and immunological techniques data showed that the cytoskeletal formation of actin, tubulin, etc. or localization in cell of Rho protein were changed. These results suggested that signal transduction pathway through Rho played an important role in the gravity sense mechanism of endothelial cells. Furthermore, we are going to investigate relation between gene expression and morphological data.

Highlights: [•] BMP2 induces Y1 receptor expression in C2C12 cells. [•] Smad1/4 induces Y1 receptor expression in C2C12 cells. [•] Y1 gene promoter activity is regulated by BMP2 signaling. [•] Y1 receptor suppression by siRNA increases osteoblast-related gene expression. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, degradation of most intracellular proteins is carried out by the ubiquitin-proteasome pathway. Recent investigations suggest that bone metabolism is also regulated by this pathway. The clinical efficacy of bortezomib, a 26 S proteasome inhibitor used as an anticancer drug, has been linked to an increase in bone formation. In this study, we show that proteasome inhibitors induce expression of osteoblastic differentiation-related genes such as osteocalcin and alkaline phosphatase in C2 C12 cells. In contrast, myogenic differentiation is inhibited. Among the proteasome inhibitors tested, bortezomib induced the greatest increase in osteocalcin expression. Although these effects were similar to that of bone morphogenetic protein ( BMP) 2, proteasome inhibitors did not induce transcriptional activity of Smad1/4-dependent reporter or BMP2 signaling target gene expression. Transient transfection of osteocalcin promoter-luciferase constructs with bortezomib resulted in an increase in luciferase activity. Mutation of OSE2, but not OSE1, sites of the osteocalcin promoter diminished the bortezomib-induced activity. Also, Runx2 binding activity and protein levels were induced by bortezomib treatment. These results suggest that the bortezomib induces osteoblastic differentiation by modifying the activity of Runx2 and that the function of the proteasome in controlling degradation of differentiation-related transcription factors plays an important role in osteoblast differentiation. [ABSTRACT FROM AUTHOR]

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase ZL, which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase ZL cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ∼50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing. [ABSTRACT FROM AUTHOR]

Abstract: CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12–CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3β decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8kb fragment of the 5′-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as important post-transcriptional gene regulators. miR-206 is unique in that it is expressed only in skeletal muscle, including the myoblastic C2C12 cell line. In C2C12 cells, miR-206 expression was reduced dramatically after bone morphogenetic protein (BMP)-2 treatment. The down-regulation of miR-206 expression was also observed after co-transfection with constitutively-active Smad1 and Smad4, which are the intracellular signaling molecules of the BMP pathway. BMP-2 also reduced miR-206 expression in the presence of α-amanitin in a similar manner to that in the absence of α-amanitin. Moreover, the expression of pri-miR-206 was increased upon BMP-2 treatment for 6h compared to that in the absence of BMP-2. These results suggested that BMP-2 down-regulates miR-206 expression at the post-transcriptional level, by inhibiting the processing of pri-miR-206 into mature miR-206, and that BMP-2 could regulate miRNA biogenesis by a novel mechanism. [Copyright &y& Elsevier]