Inositol Requiring Enzyme 1 alpha (IRE) Regulates Granulosa Cell Steroidogenesis Independent of its Role in the Unfolded Protein Response

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 515-547-Female Reproductive Endocrinology
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-531
Larry Denner*1, Yvonne Bodenburg1, Jie Jiang1, Sigmund Haidacher1, Rebekah L Viner1, Rebecca A Chilvers1, John B Patterson2, Kevin P Rosenblatt3 and Randall J Urban1
1University of Texas Medical Branch, 2MannKind Corporation, 3University of Texas Health Science Center Houston
Many endocrine disorders affect fertility in women of reproductive age.  Cytochrome p450 side chain cleavage enzyme (CYP11A1, p450scc) is the rate-limiting enzyme in sex steroid hormone biosynthesis and is essential for the normal responses to gonadotropin hormones that maintain fertility and support pregnancy.  To identify kinases required for sex steroid biosynthesis, we performed a loss-of-function screen of a kinase siRNA library to all 626 human kinases in a high throughput assay measuring forskolin-stimulated progesterone production in the human KGN granulosa cell line.  We discovered that IRE (inositol-requiring enzyme 1 alpha) was required for steroid biosynthesis.  Knockdown of IRE, which decreased both the mRNA and protein, also decreased expression of CYP11A1 mRNA.  In addition, IRE expression was required for CYP11A1 transcription and progesterone production in response to the natural gonadotropins FSH in the KGN human granulosa cell line and LH in primary granulosa cells (HLGCs) from women undergoing assisted reproduction.  Immunocytochemistry showed that IRE was present in constitutive, large puncta colocalized with Nup98, a nuclear pore regulatory protein that is part of the nuclear pore transport machinery.  This localization was distinct from IRE in the ER that was identified by the ER markers GRP94 and calreticulin.  Subcellular fractionation confirmed expression of IRE in the nuclear membrane compartment.  Using reporter gene assays, we found that IRE directly regulates transcription of CYP11A1 through the proximal region of the promoter between -800 and -1200 bp. We previously showed that PSF binds to an insulin-like growth factor response element (IGFRE) in the proximal promoter of CYP11A1 to repress gene expression.  Now we have now found using RNA immunoprecipitation (RIP) that PSF also binds the CYP11A1 mRNA.  This interaction is mediated by IRE since PSF-CYP11A1mRNA binding is disrupted by knockdown of IRE.

 We found that IRE regulation of the sex steroid biosynthesis program was distinct from its canonical role in the unfolded protein response (UPR) mediating ER stress.  Thus, the prototypical inducers of the UPR, tunicamycin and thapsigargin, induced transautophosphorylation of S724 on IRE, BiP protein expression, dynamic clustering of IRE in the ER, increased phosphorylation of eIF2α and PERK, and degradation of ATF-6.  In addition, Xbp-u splicing to Xbp-s was induced by Tm and prevented by the IRE endoribonuclease inhibitor MK MKC4485 and 50mg MKC3946 (MannKind Corp.).  Thus, while the canonical UPR pathway is present and functional in granulosa cells, engagement of the steroidogenic program does not utilize this signaling machinery.  With global concerns increasing over declining fertility in humans, we present a novel molecular mechanism regulating sex steroid biosynthesis.

Nothing to Disclose: LD, YB, JJ, SH, RLV, RAC, JBP, KPR, RJU

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