Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-6 subspecies was markedl inhibited, whereas cell lines expressing PKC-a, PKC-IH, and PKC-C subspecies were not sign tly affected. Flow cytometric analysis indicated that all cell les overexpressing PKC-8 subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrete cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-6 subspecies may play a role in the normal cell cycle progression.Protein kinase C (PKC) appears to play crucial roles in signal transduction leading to cell growth and differentiation. This enzyme was first shown to be activated by diacylglycerol generated by the receptor-mediated hydrolysis of inositol phospholipids and has been identified as a major receptor of tumor-promoting phorbol esters (1). Molecular cloning and biochemical studies have revealed that the PKC family is a large family, consisting ofat least eight subspecies, a, 3PI, /11, y, 8, E, A, and q (PKC-L) in mammalian'tissues (2-5). These subspecies show subtly different enzymological properties and distinct tissue distribution. Molecular genetic approaches have also been used to explore the role of this PKC family in cell proliferation by expressing defined enzyme subspecies, such as PKC-a, PKC-P, or PKC-y, in mammalian cells (6-11). In some cell lines stably overproducing PKC subspecies, growth promotion such as enhanced growth rates, increased saturation densities, and anchorageindependent growth has been observed (6,7,9,10 MATERIALS AND METHODS Construction of Expression Plasmids. cDNAs for rat PKC-a, -/311, -8, and -C subspecies were excised from plasmids pTB755, pTB708, pTB808, and pTB949, respectively (14-17), by complete or partial digestion with EcoP. These cDNAs were then separately inserted downstream of the Abelson murine leukemia virus long terminal repeat in plasmid pTB399 (18) by replacing the interleukin 2 sequence. The long terminal repeat-PKC cDNA segments between Sal I and Cla I sites ofthese plasmids were transferred to the same sites of plasmid pTB348 (18), which carried the hamster dihydrofolate reductase (DHFR) cDNA under the control of the simian virus 40 early promoter. The resulting expression plasmids for the a, . 3II,8, and ; subspecies are referred to as pTB789, pTB705, pTB1322, and pTB1324, respectively. All the procedures were done as described (19).Transfection, Selection, and Gene Amplification. DHFR-CHO cells (20) were transfected with each of the expression constructs by the calcium phosphate-DNA coprecipitation method (21). DHFR+ transformants were selected by colony formation in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 (vol/vol) dialyzed fetal bovine serum and proline (35 ,Ag/ml). Gene amplificatio...

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D 3 -bound [1,25(OH) 2 D 3 -bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH) 2 D 3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH) 2 D 3 -dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

Dynamical conductivity spectra [σ(ω)] have been measured for many heavy-fermion (HF) Ce and Yb compounds. A characteristic excitation peak has been observed in the infrared region of σ(ω) for all the compounds, and has been analyzed in terms of a simple model based on conduction (c)-f electron hybridized band. A universal scaling is found between the observed peak energies and the estimated c-f hybridization strengths of these HF compounds. This scaling demonstrates that the model of c-f hybridized band can generally and quantitatively describe the low-energy charge excitations in a wide range of HF compounds.

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin ␣ 3 as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor ␣ 3 ␤ 1 for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the ⌬N isoform on ITGA3. The high level ␣ 3 production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca 2؉ -induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.