Abstract

1. SV40 SV40 SV40 luciferase luciferase luciferase SV40 luciferase Abstract Egr1 mRNA CSDAis an RNA binding protein expressed in LbT2 cells Csda siRNA reduces GnRH stimulation of LHB-luciferase AMD LbT2 cells were treated with 10 nM GnRH The gonadotropin Luteinizing Hormone (LH) is essential for reproductive function in mammals. LH is a heterodimeric glycoprotein secreted from the pituitary and is composed of two subunits: the common alpha subunit (α-GSU) and the unique beta subunit (LHβ). The genes that encode these subunits are tightly regulated by inputs from the hypothalamic-pituitary-gonadal axis. GnRH from the hypothalamus acts upon cells in the pituitary called gonadotropes to stimulate the expression of Lhb This stimulation is dependent upon proper induction and accumulation of the transcription factor Early growth response gene 1 (Egr1) by GnRH. The controlled expression and regulation of Egr1 is critical for LH production. Using the gonadotrope-derived LbT2 cell line, we have uncovered that a gene, Cold shock domain protein A (Csda) may be important for regulating Egr1 mRNA stability. When endogenous Csda expression is lowered by siRNA techniques there is a reduction in the amount of Egr1 and Lhb mRNA after treatment with GnRH. We hypothesize that CSDA may be acting through the untranslated region (UTR) of Egr1 to regulate its stability. Using a luciferase reporter vector, which contains different elements of the Egr1 3’-UTR, we have started to investigate the mechanisms that control Egr1 stability in LbT2 cells along with how CSDA may contribute to this stability. These studies will allow us to better understand the mechanisms of mRNA stability and to further our knowledge of how Egr1 and Lhb are controlled. • The 3’ untranslated region (UTR) of Egr1 is large and contains many elements that have the potential to regulate mRNA stability • LβT2 cells were transiently transfected with -779/+10 bovine LHB promoter-luciferase vector and 100 nM of non-targeting control interfering RNA (Con siRNA) or Csda interfering RNA (Csda siRNA) and subsequently treated with either vehicle or GnRH (100 nM). Data shown are the means ± SEM of three experiments. AMD + 100nM GnRH • Cold Shock Domain protein A (aka MSY 3/4) • Binds RNA and DNA • Contains a highly conserved 70 amino acid sequence (CSD) • Binds UCCAUCA Csd elements • Highly expressed in testes - controls protamine mRNA stability • Controls Vegf mRNA stability Time (hours): 0 0.5 1, 2, 3, 4, 5, 6, 8, 12 5’ UTR 3’ UTR 1896 1 295 3111 coding region Egr1 mRNA (NCBI RefSeq NM_007913) 16 e17.5 14 M 12 LbT2 S/L 10 Csda Tr C G Fold change 8 S2 6 Northern blot 4 pGL3 SV40 luciferase 2 Egr1 3’ UTR SV40 luciferase Csda siRNA reduces GnRH stimulation of endogenous Lhb The 3’ end of Egr1 enhances luciferase activity Egr1 3’ distal 0 Con siRNA + GnRH Csda siRNA + GnRH Con siRNA Csda siRNA • LβT2 cells • Represent early developing gonadotropes • Derived from a mouse pituitary at embryonic day 17.5 • Express all necessary transcription factors for Lhb expression • Can express Lhb Egr1 3’ proximal • LβT2 cells were transiently sham transfected or transfected with 100 nM of non-targeting control interfering RNA (Con siRNA) or Csda interfering RNA (Csda siRNA) and subsequently treated with either vehicle or GnRH (100 nM). RNA was isolated and Northern blot analysis was performed. LβT2 Sham Control siRNA Csda siRNA Time 0 1 2 4 8 24 0 1 2 4 8 24 0 1 2 4 8 24 Lhb Hpg Axis S2 • Hypothalamus – releases GnRH • Pituitary – composed of 5 cell types • Gonadotropes – secrete LH • thyrotropes – secrete TRH • Lactotropes – secrete Prolactin • Somatotropes – secrete GH • Corticotropes – secrete ACTH • Pulses of GnRH from the hypothalamus act upon gonadotrope cells in the pituitary control expression of and release of LH • LH acts on the testis and ovary and is necessary for reproduction Hypothalamus Relative activity in LbT2 cells 6 • GnRH + GnRH Fold Change 5 GnRH Gonadotropes pGL3 1.0 ± 0.07 6.7 ± 1.52 6.7 4 Pituitary Egr1 3’ UTR 1.9 ± 0.02 17.5 ± 0.95 9.1 AU rich regions and U rich regions Fold increase (Lhb/S2) 3 Egr1 3’ proximal UTR 5.3 ± 0.32 37.0 ± 2.25 6.8 LH 2 Egr1 3’ distal UTR 5.4 ± 0.46 30.2 ± 2.55 5.6 Studying gonadotropes using a cell line Polypyrimidine consensus Ovary 1 Testis The 3’ Csd consensus and CSDA protein modulate luciferase activity • Due to the complexity of the pituitary gland studying primary gonadotropes is very difficult • To study gonadotropes we utilize a cell line that has been developed polyadenylation signal 0 0 24 0 24 0 24 GnRH - + - + - + (100nM) Csd consensus • The proximal Csd sequence was mutated and used in transient transfections along with and 100 nM of non-targeting control interfering RNA (Control RNAi) or Csda interfering RNA (Csda RNAi) Sham Control siRNA Csda siRNA 100 GnRH regulates the transcription of Lhb and Egr1 AMD + GnRH y = 1.72 -.196x AMD + vehicle y = 1.99 -.225x 10 Relative Egr1 expression pGL3 SV40 luciferase • Pulses of GnRH control expression of Egr1 and Lhb • Proper expression of Lhb depends upon multiple transcription factors including SF1, EGR1, and PITX1 • The limiting transcription factor appears to be Egr1 which is induced by GnRH Egr1 3’ UTR SV40 luciferase 1 AMD + GnRH AMD + Vehicle Egr1 3’ proximal Egr1 3’ D proximal Hours 0 1 2 3 4 5 6 8 12 1 2 3 4 5 6 8 12 0.1 0 2 4 6 8 10 12 14 RNAi Relative Activity UTR fold-change RNAi % Loss Time (hours) pGL3 Control 1.14 ± 0.14 1X Csda 0.69 ± 0.18 39 Egr1 Egr1 proximal 3’ Control 23.10 ± 7.19 23X S2 Csda 6.39 ± 1.36 72 Egr1 is essential for fertility Egr1 D proximal 3’ Control 7.67 ± 2.55 7X Csda 4.71 ± 1.96 38 • Early Growth response protein 1 (Egr1) is a zinc-finger phosphoprotein which binds DNA in GC-rich sequences on the promoters of target genes. • Egr1 is considered to be an immediate early response gene • Egr1 is induced by GnRH in gonadotropes • Egr1 is necessary for Lhb expression and essential for fertility Post-transcriptional regulation of Egr1 in the gonadotrope is influenced by CSDATheodore R. Chauvin & John H. NilsonSchool of Molecular Biosciences, Washington State University, Pullman, WA, USA; Center for Reproductive Biology, Pullman WA, USA The effect of GnRH on Egr1 mRNA half-life RNA was isolated and Northern blot analysis performed • The 3’ UTR of Egr1 was inserted into a luciferase reporter vector and used in transient transfection assays in order to identify regions of the UTR which may be important for mRNA stability Conclusions • GnRH appears to have no effect on the half life of Egr1 mRNA • The 3’ UTR of Egr1 enhances luciferase activity in transfected LbT2 cells • The 3’ Csd sequence in the proximal region of the Egr1 UTR appears to modulate luciferase activity in transfected LbT2 cells • Reduction of CSDA by siRNA can also modulate activity of luciferase in transfected LbT2 cells • Reduction of CSDA by siRNA reduced GnRH stimulation of both a transfected LHB-luciferase construct and endogenous Lhb