Wdr59 promotes or inhibits TORC1 activity depending on cellular context.

Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. Here we report that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. We find that in the ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, our data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context.

Genetic and epigenetic changes in the eutopic endometrium of women with endometriosis: association with decreased endometrial αvß3 integrin expression.

About 40% of women with infertility and 70% of women with pelvic pain suffer from endometriosis. The pregnancy rate in women undergoing IVF with low endometrial integrin αvß3 (LEI) expression is significantly lower compared to the women with high endometrial integrin αvß3 (HEI). Mid-secretory eutopic endometrial biopsies were obtained from healthy controls (C; n=3), and women with HEI (n=4) and LEI (n=4) and endometriosis. Changes in gene expression were assessed using human gene arrays and DNA methylation data were derived using 385 K Two-Array Promoter Arrays. Transcriptional analysis revealed that LEI and C groups clustered separately with 396 differentially expressed genes (DEGs) (P<0.01: 275 up and 121 down) demonstrating that transcriptional and epigenetic changes are distinct in the LEI eutopic endometrium compared to the C and HEI group. In contrast, HEI vs C and HEI vs LEI comparisons only identified 83 and 45 DEGs, respectively. The methylation promoter array identified 1304 differentially methylated regions in the LEI vs C comparison. The overlap of gene and methylation array data identified 14 epigenetically dysregulated genes and quantitative RT-PCR analysis validated the transcriptomic findings. The analysis also revealed that aryl hydrocarbon receptor (AHR) was hypomethylated and significantly overexpressed in LEI samples compared to C. Further analysis validated that AHR transcript and protein expression are significantly (P<0.05) increased in LEI women compared to C. The increase in AHR, together with the altered methylation status of the 14 additional genes, may provide a diagnostic tool to identify the subset of women who have endometriosis-associated infertility.

Mineralocorticoid synthesis during the periovulatory interval in macaques.

Ovulation and luteal formation in primates are associated with the sustained synthesis of progesterone. The observed high intrafollicular concentrations of progesterone during the periovulatory interval raise the possibility that this steroid serves as a precursor for mineralocorticoids. The aim of this study was to determine if mineralocorticoids are synthesized by the luteinizing macaque follicle during controlled ovarian stimulation cycles in which follicular fluid and granulosa cell aspirates were obtained before or after an ovulatory hCG bolus. Follicular fluid concentrations of progesterone and 17alpha-hydroxyprogesterone increased within 3 h of an ovulatory hCG bolus. Their respective metabolites, 11-deoxycorticosterone (DOC) and 11-deoxycortisol, were not detectable before an ovulatory stimulus and increased starting at 6 h after hCG, while corticosterone and aldosterone were undetectable. Cortisol was present before and after hCG administration and had increased 2-fold at 24 h after an ovulatory stimulus. The expression of 21-hydroxylase (CYP21A2) mRNA increased within 3 h of hCG administration, while 11beta-hydroxylase-1 (CYP11B1) and 11beta-hydroxylase-2 (CYP11B2) mRNAs were not detectable. 11beta-Hydroxysteroid dehydrogenase-1 (HSD11B1) mRNA had increased at 12 h after hCG administration, and 11beta-hydroxysteroid dehydrogenase-2 (HSD11B2) had decreased by 3 h after hCG administration. Mineralocorticoid receptor mRNA levels did not change following hCG administration, while glucocorticoid receptor mRNA levels increased in response to an ovulatory stimulus. Treatment of granulosa cells with the mineralocorticoid receptor antagonist spironolactone blocked hCG-induced progesterone synthesis in vitro. These data indicate that macaque granulosa cells can synthesize mineralocorticoids in response to an ovulatory stimulus and that the mineralocorticoid receptor plays a key role in steroid synthesis associated with luteinization of macaque granulosa cells.