The ubiquitin ligase subunit ß-TrCP in Sertoli cells is essential for spermatogenesis in mice.

ß-TrCP is the substrate recognition subunit of an SCF-type ubiquitin ligase. We recently showed that deletion of the genes for both ß-TrCP1 and ß-TrCP2 paralogs in germ cells of male mice resulted in accumulation of the transcription factor DMRT1 and spermatogenic failure, whereas systemic ß-TrCP1 knockout combined with ß-TrCP2 knockdown had previously been shown to lead to disruption of testicular organization and accumulation of the transcription factor SNAIL. Here we investigated ß-TrCP function in Sertoli cells by generating mice with targeted deletion of the ß-TrCP2 gene in Sertoli cells on a background of whole-body ß-TrCP1 knockout. Loss of ß-TrCP in Sertoli cells caused infertility due to a reduction in the number of mature sperm. Whereas spermatogonia were not affected, male germ cells entered meiosis prematurely and the number of round spermatids was reduced in the mutant mice. Extracts of Sertoli cells and of the testis from the mutant mice manifested accumulation of SNAIL, and expression of the SNAIL target gene for E-cadherin was down-regulated in Sertoli cells from these animals. Our results indicate that ß-TrCP in Sertoli cells regulates Sertoli cell-germ cell interaction through degradation of SNAIL, with such regulation being critical for sperm development.

Regulation of mitosis-meiosis transition by the ubiquitin ligase ß-TrCP in male germ cells.

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that ß-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of ß-TrCP2 in male germ cells of ß-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The ß-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in ß-TrCP-deficient testes. DMRT1 contains a consensus ß-TrCP degron sequence that was found to bind ß-TrCP. Overexpression of ß-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in ß-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that ß-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation.

Deleted in Esophageal Cancer 1(DEC1) is down-regulated and contributes to migration in head and neck squamous cell carcinoma cell lines.

BACKGROUND: Previous studies have shown that the expression of Deleted in Esophageal Cancer 1 (DEC1) is significantly reduced in esophageal squamous cell carcinoma. Patients with head and neck squamous cell carcinoma (HNSCC) often develop esophageal carcinomas. MATERIALS AND METHODS: We analyzed the expression of DEC1 and histone modifications in HNSCC cell lines. The motility and invasive ability of the HNSCC cell lines were also studied. RESULTS: Of 18 cell lines, 12 (66.7%) showed down-regulation of DEC1. Chromatin immunoprecipitation assays indicated that H3 K27 trimethylation levels in the DEC1-down-regulated cell lines were greater than that in the DEC1-expressed cell lines. Migration assays showed that the DEC1-down-regulated cell lines tended to be more motile than the DEC1-expressed cell lines. CONCLUSION: DEC1 is down-regulated and tends to contribute to the migration ability of HNSCC cell lines. In addition, H3 K27 trimethylation potentially plays an important role in the regulation of DEC1 expression.

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway.

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self-renewal capacity from those of ES cells. All three EC cell lines maintain self-renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self-renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self-renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells-derived conditioned medium may be responsible for the self-renewal capacity of EC and ES cells independently of LIF signaling.

Discovery of Novel Indazole Derivatives as Orally Available ß3-Adrenergic Receptor Agonists Lacking Off-Target-Based Cardiovascular Side Effects.

We previously discovered that indazole derivative 8 was a highly selective ß3-adrenergic receptor (ß3-AR) agonist, but it appeared to be metabolically unstable. To improve metabolic stability, further optimization of this scaffold was carried out. We focused on the sulfonamide moiety of this scaffold, which resulted in the discovery of compound 15 as a highly potent ß3-AR agonist (EC50 = 18 nM) being inactive to ß1-, ß2-, and α1A-AR (ß1/ß3, ß2/ß3, and α1A/ß3 > 556-fold). Compound 15 showed dose-dependent ß3-AR-mediated responses in marmoset urinary bladder smooth muscle, had a desirable metabolic stability and pharmacokinetic profile (Cmax and AUC), and did not obviously affect heart rate or mean blood pressure when administered intravenously (3 mg/kg) to anesthetized rats. Thus, compound 15 is a highly potent, selective, and orally available ß3-AR agonist, which may serve as a candidate drug for the treatment of overactive bladder without off-target-based cardiovascular side effects.

Discovery of Novel Indazole Derivatives as Highly Potent and Selective Human ß3-Adrenergic Receptor Agonists with the Possibility of Having No Cardiovascular Side Effects.

Novel indazole derivatives were prepared and evaluated for their biological activity and cardiovascular safety profile as human ß3-adrenergic receptor (AR) agonists. Although the initial hit compound 5 exhibited significant ß3-AR agonistic activity (EC50 = 21 nM), it also exhibited agonistic activity at the α1A-AR (EC50 = 219 nM, selectivity: α1A/ß3 = 10-fold). The major metabolite of 5, which was an oxidative product at the indazole 3-methyl moiety, gave a clue to a strategy for improvement of the selectivity for ß3-AR agonistic activity versus α1A-AR agonistic activity. Thus, modification of the 3-substituent of the indazole moiety effectively improved the selectivity to develop compound 11 with potent ß3-AR agonistic activity (EC50 = 13 nM) and high selectivity (α1A/ß3 = >769-fold). Compound 11 was also inactive toward ß1 and ß2-ARs and showed dose dependent ß3-AR mediated relaxation of marmoset urinary bladder smooth muscle, while it did not obviously affect heart rate or blood pressure (iv, 3 mg/kg) in anesthetized rats.

Zn-promoted regioselective and sequence-selective one-pot joining reaction of three components: alkyl iodides, alpha,beta-unsaturated esters (or nitriles), and acylating agents.

One-pot treatment of alkyl iodides, alpha,beta-unsaturated esters (or nitriles), and acylating agents such as nitriles or acid anhydrides in the presence of Zn metal at room temperature in the same reaction system brought about a regioselective and sequence-selective three-component joining reaction involving first C-alkylation at the beta-position and second C-acylation at the alpha-position of alpha,beta-unsaturated esters (or nitriles) to afford the corresponding alpha,alpha-dialkylketoesters (or alpha,alpha-dialkylketonitriles) in moderate to good yields. [reaction: see text]

Expression profile of LIT1/KCNQ1OT1 and epigenetic status at the KvDMR1 in colorectal cancers.

The human chromosome region 11p15.5 contains a number of maternally and paternally imprinted genes, and the LIT1/KCNQ1OT1 locus acts as an imprinting center in the proximal domain of 11p15.5. Loss of imprinting (LOI) of LIT1 and its correlation with methylation status at a differentially methylated region, the KvDMR1, were investigated in 69 colorectal cancer tissue specimens. LIT1 expression profiles were also examined by RNA-fluorescence in situ hybridization in 13 colorectal cancer cell lines. In 69 colorectal cancer tissue specimens, LOI of LIT1 was observed in nine of the 17 (53%) informative cases. Moreover, LOI of LIT1 was only observed in tumor samples. In the cell lines, methylation status at the KvDMR1 correlated well with LIT1 expression profiles. Loss of expression of LIT1 also correlated with enrichment of H3 lysine 9 (H3-K9) dimethylation and reduction of H3 lysine 4 (H3-K4) dimethylation. Thus, LIT1 expression appears to be controlled by epigenetic modifications at the KvDMR1, although CDKN1C expression, which is considered to be controlled by LIT1, was not associated with epigenetic status at the KvDMR1 in some colorectal cancer cell lines. Therefore, these findings suggest that LOI of LIT1 via epigenetic disruption plays an important role in colorectal carcinogenesis, but it is not necessarily associated with CDKN1C expression.