GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Peptidylarginine deiminase 2 regulates expression of DGCR8 affecting miRNA biogenesis in gonadotrope cells.

In brief: DGCR8 microprocessor complex, which is important for miRNA biogenesis, is regulated by peptidylarginine deiminase 2 and expression fluctuates in gonadotrope cells across the mouse estrous cycle. Abstract: Canonical miRNA biogenesis requires DGCR8 microprocessor complex subunit, which helps cleave pri-miRNAs into pre-miRNAs. Previous studies found that inhibiting peptidylarginine deiminase (PAD) enzyme activity results in increased DGCR8 expression. PADs are expressed in mouse gonadotrope cells, which play a central role in reproduction by synthesizing and secreting the luteinizing and follicle stimulating hormones. Given this, we tested whether inhibiting PADs alters expression of DGCR8, DROSHA, and DICER in the gonadotrope-derived LßT2 cell line. To test this, LßT2 cells were treated with vehicle or 1 µM pan-PAD inhibitor for 12 h. Our results show that PAD inhibition leads to an increase in DGCR8 mRNA and protein. To corroborate our results, dispersed mouse pituitaries were also treated with 1 µM pan-PAD inhibitor for 12 h which increases DGCR8 expression in gonadotropes. Since PADs epigenetically regulate gene expression, we hypothesized that histone citrullination alters Dgcr8 expression thereby affecting miRNA biogenesis. LßT2 samples were subjected to ChIP using an antibody to citrullinated histone H3, which shows that citrullinated histones are directly associated with Dgcr8. Next, we found that when DGCR8 expression is elevated in LßT2 cells, pri-miR-132 and -212 are reduced, while mature miR-132 and -212 are increased suggesting heightened miRNA biogenesis. In mouse gonadotropes, DGCR8 expression is higher in diestrus as compared to estrus, which is the inverse of PAD2 expression. Supporting this idea, treatment of ovariectomized mice with 17ß-estradiol results in an increase in PAD2 expression in gonadotropes with a corresponding decrease in DGCR8. Collectively, our work suggests that PADs regulate DGCR8 expression leading to changes in miRNA biogenesis in gonadotropes.