Development of novel highly sensitive methods to detect endogenous cGAMP in cells and tissue.

Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer.

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

Testis-specific protein on Y chromosome (TSPY) represses the activity of the androgen receptor in androgen-dependent testicular germ-cell tumors.

Testis-specific protein on Y chromosome (TSPY) is an ampliconic gene on the Y chromosome, and genetic interaction with gonadoblastoma has been clinically established. However, the function of the TSPY protein remains to be characterized in physiological and pathological settings. In the present study, we observed coexpression of TSPY and the androgen receptor (AR) in testicular germ-cell tumors (TGCTs) in patients as well as in model cell lines, but such coexpression was not seen in normal testis of humans or mice. TSPY was a repressor for androgen signaling because of its trapping of cytosolic AR even in the presence of androgen. Androgen treatment stimulated cell proliferation of a TGCT model cell line, and TSPY potently attenuated androgen-dependent cell growth. Together with the finding that TSPY expression is reduced in more malignant TGCTs in vivo, the present study suggests that TSPY serves as a repressor in androgen-induced tumor development in TGCTs and raises the possibility that TSPY could be used as a clinical marker to assess the malignancy of TGCTs.

Spermatogenesis-specific association of SMCY and MSH5.

The status of chromatin during spermatogenesis is dynamically regulated by specific histone codes or stage-specific histone changes. The functional links between such epigenetic regulation and proteins regulating meiosis are largely unknown. In mammals, genes encoded on the Y chromosome are thought to possess male-specific biological functions. While genes located within the azoospermia factor region (AZF) are known to be involved in spermatogenesis, the physiological function of individual genes is not known. SMCY is a gene mapped to the AZF, and in this report, we analyzed the function of SMCY protein during spermatogenesis. Biochemical identification of the proteins with which it interacted showed that SMCY formed a distinct complex with MSH5, a critical meiosis-regulatory protein in the human testicular germ cell line, NEC8. As anticipated, histone H3K4 demethylase activity was detected. Immunohistochemical analysis revealed the co-localization of SMCY with MSH5 at a specific stage of meiotic prophase progression during murine spermatogenesis. Our results suggest that SMCY may have a male-specific function as a histone H3K4 demethylase by recruiting a meiosis-regulatory protein to condensed DNA.

A reduction state potentiates the glucocorticoid response through receptor protein stabilization.

The intracellular redox state regulates all biological processes including gene expression. The glucocorticoid receptor (GR), a hormone-dependent transcription factor, is affected by the redox state. GR translocation from the cytoplasm to the nucleus is regulated by oxidative stress. The molecular mechanism of how the redox state affects GR transcriptional regulation, however, has not been clarified. We identified a deoxidizing agent, cobalt chloride (CoCl(2)), that potentiates the GR transcriptional effects by stabilizing endogenously expressed GR protein as well as exogenously over-expressed one without affecting GR mRNA level. Consequent GR protein stabilization enhanced co-factor recruitments on the target gene promoters. These results support the existence of a novel redox-dependent mechanism of GR transcriptional regulation mediated by receptor protein stabilization.

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs.

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.