miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD_J dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD_J TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

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The association of the cyclin D-Cdk (DC) complex with retinoblastoma protein (pRb) is required for the G1-S transition of the cell cycle. Cyclin synthesis, nuclear localization and degradation are control mechanisms for the transition, but regulation of the DC complex nuclear import also contributes to the transition. Analysis of the timing of the G1-S transition in mammalian cell lines revealed acceleration with overexpression of cyclin D2 and Cdk4. Immunolocalization assays revealed that cyclin D2 and Cdk4 formed a complex in the cytoplasm and approached the nucleus. They accumulated on the cytosolic surfaces of the nuclear pores and then were arrested at the nuclear membrane before the nucleus reached a critical size. Finally, the complex was released into the nucleus and colocalized with pRb there, which led to pRb phosphorylation and DNA synthesis. The translocalization depended on the G1-S transition. In contrast, a truncated cyclin D2 that was not able to fully associate with Cdk4 lost the ability for release into the nucleus. This pattern of translocalization suggests a spatial separation of the cyclin D-Cdk complex from pRb and DNA in the nucleus to regulate the G1-S transition.

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SNAREs (‘Soluble N-ethyl-maleimide sensitive factor attachment protein receptors’) play a critical role in the membrane fusion step of the vesicular transport system in eukaryotes. The number of the genes encoding SNARE proteins is estimated to be 64 in Arabidopsis thaliana. This number is much larger than those in other eukaryotes, suggesting a complex membrane trafficking in plants. The Arabidopsis SNAREs, the SYP7 group proteins, SYP71, SYP72, and SYP73, form a plant-specific SNARE subfamily with not-yet-identified functions. We have previously reported that the SYP7 subfamily proteins are predominantly localized to the endoplasmic reticulum in the Arabidopsis suspension cultured cells under transient expression condition. However, several proteomic analyzes indicated the plasma membrane localizations of one of SYP7 subfamily proteins, SYP71. In order to confirm the expression patterns and subcellular localization of SYP7, we performed combination analyses including promoter GUS analysis, a sucrose density gradient centrifugation analysis, as well as an observation on transgenic Arabidopsis plants expressing GFP-fused SYP71 under control of its native promoter. From these analyses, we concluded that one of the SYP7 subfamily proteins, SYP71, is predominantly expressed in all vegetative tissues and mainly localized to the plasma membrane. We also found that SYP71 is localized to the endoplasmic reticulum in the dividing cells of various types of tissues.

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We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5–2 nmol CTnT-siRNA towards 1–3×10⁶ cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

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In adult mouse, the mRNA corresponding to the alpha1 chain of type XII collagen (alpha 1(XII)) is predominantly detected in the bone. Additionally, murine osteoblastic cells, MC3T3-E1, increased the mRNA level of alpha 1(XII) response to the mechanical strain in the stretch culture system. Cyclic stretch stress resulted in a threefold increase in mRNA level of alpha 1(XII) as compared to the control experiment in MC3T3-E1. Transient transfection assays employing a reporter construct, together with site-directed mutagenesis studies, suggested that the AP-1 binding site in the first exon of mouse alpha 1(XII) gene is important for stretch stress-mediated upregulation of alpha 1(XII) expression. Electrophoretic mobility shift assay and associated antibody supershift experiments showed that stretch stress promotes the binding of c-Jun and JunD. Further chromatin immunoprecipitation experiments confirmed the participation of these transcription factors in the region. Also, the exogenous induction of the dominant negative form of c-Jun canceled the effect of stretch stress on the stimulation of the alpha 1(XII) gene. Here, we reported a potential responsive element to the stretch stress in mouse alpha 1(XII) gene. These data will provide new information on the mechanical strain-mediated transcriptional control of alpha 1(XII)-mediated fibrillogenesis in the bone.

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We investigated whether de-differentiated fat (DFAT) cells, a mature adipocyte-derived preadipocyte cell line, can be induced to trans-differentiate into osteoblasts in vitro and in vivo. All-trans retinoic acid (RA) induced expression of osteoblast-specific mRNAs encoding Cbfa1/Runx2, osterix, alkaline phosphatase, osteopontin, parathyroid hormone receptor, and osteocalcin in the DFAT cells, but did not induce the expression of adipocyte-specific mRNAs encoding PPARγ2, C/EBPα, and GLUT4. Moreover, alkaline phosphatase activity was expressed in DFAT cells and the cells underwent mineralization of the bone matrix in vitro. Furthermore, when DFAT cells were transplanted subcutaneously into C57BL/6N mice in diffusion chambers, these cells formed ectopic osteoid tissue without any host cell-invasion of the chambers. These results indicate that DFAT cells derived from mature adipocytes can be converted into fully differentiated osteoblasts in vitro and in vivo using RA. DFAT cells provide a unique model for studying the lineage commitment of the adipocytes and osteoblasts derived from mesenchymal stem cells. Identification of the pathways that regulate these processes could lead to the development of new therapeutic strategies for control of unwarranted growth of bone and adipose tissue.

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