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GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells

ABSTRACT: Abstract: Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. It is well established that a gonadotropin releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope derived LBT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterize the temporal dynamics of this modification. Our results show that actin and tubulin are rapidly citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. Data shows that 10 nM GnRHa induces citrullination of beta-actin with maximal levels occurring at 10 minutes. The level of beta-actin citrullination is reduced in the presence of the pan-PAD inhibitor bi-phenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa induced actin reorganization in dispersed mouse gonadotrope cells. GnRHa induces citrullination of beta-tubulin with elevated levels occurring at 30 minutes; similarly to beta-actin, this response is attenuated in the presence of BB-ClA. To examine the functional consequence of beta-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 LBT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that MT average lifetime increases following 30 minutes 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. Keywords: gonadotrope, peptidylarginine deiminase, citrullination, cytoskeleton, beta tubulin, beta actin, microtubules

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Brian D Cherrington

PROVIDER: MSV000093973 | MassIVE | Wed Jan 31 18:03:00 GMT 2024

SECONDARY ACCESSION(S): PXD049056

REPOSITORIES: MassIVE

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Project description:Diaphanous-related formin-3 (DIAPH3), a cytoskeletal regulator of the formin family, is lost at high frequency in metastatic breast and prostate cancers and has characteristics of a non-canonical metastasis suppressor. We previously demonstrated that DIAPH3 silencing evokes transition to an “amoeboid” phenotype, characterized by rapid motility, RhoA/ROCK and MEK/ERK pathway activation, dynamic membrane blebbing, and increased shedding of microvesicles. Here we report that low DIAPH3 mRNA expression correlates with reduced survival in multiple cancer patient cohorts. In line with induction of amoeboid behavior, traction force microscopy (TFM) revealed that polarized force generation, contractility, and response to substrate stiffness are reduced by DIAPH3 loss. Proteomic analyses revealed that DIAPH3 precipitates with tubulins, and reciprocal co-immunoprecipitation analyses revealed greater binding of DIAPH3 to MT polymers than soluble tubulin. DIAPH3 also bound to acetylated-tubulin (Ac-tubulin), suggesting association with a stable MT population. DIAPH3 silencing reduced Ac-tubulin and increased the cellular content of dynamic MT, as indicated by TFM and live cell imaging. DIAPH3 silencing also increased responsiveness to MT-disrupting agents. Treatment with the taxanes paclitaxel and docetaxel, and the non-taxane epothilone B, induced a greater change in Ac-tubulin when cells lacked DIAPH3. Intracellular accumulation of Oregon Green 488-paclitaxel was also increased by DIAPH3 loss, and accordingly, DIAPH3 silencing enhanced cytotoxicity by paclitaxel, docetaxel, or epothilone B. Gene expression profiles of breast cancer patients treated with chemotherapeutic regimens containing taxanes revealed an association of low DIAPH3 expression with improved relapse-free survival. Collectively, these results suggest that regulation of MT stability/dynamics by DIAPH3 enhances amoeboid tumor cell susceptibility to microtubule poisons. These findings have potential therapeutic implications.

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GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells

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