Liver receptor homolog-1 localization in the nuclear body is regulated by sumoylation and cAMP signaling in rat granulosa cells Feng-Ming Yang, Chien-Ting Pan, Huei-Man Tsai, Tai-Wei Chiu, Mei-Ling Wu and Meng-Chun Hu Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan Keywords cAMP signaling; CYP11A1; granulosa cells; liver receptor homolog-1; sumoylation Correspondence M.-C Hu, Graduate Institute of Physiology, National Taiwan University College of Medicine, No 1, Jen-Ai Road, 1st Section, Taipei 100, Taiwan Fax: +886 23964350 Tel: +886 23123456 ext 88239 E-mail: mengchun@ntu.edu.tw (Received July 2008, revised November 2008, accepted 10 November 2008) doi:10.1111/j.1742-4658.2008.06785.x Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the nuclear receptor superfamily, mainly expressed in endoderm-derived tissues and in the ovary In ovarian granulosa and luteal cells, LRH-1 regulates the expression of genes associated with ovarian steroidogenesis LRH-1 can be transported to transcriptionally inactive nuclear bodies after conjugation with small ubiquitin-related modifier (SUMO) In the present study, we investigated the effects of SUMO modification at five lysine residues of LRH-1 in rat granulosa cells Lysine 289 could be conjugated with SUMO1 in vitro, and the mutation K289R increased transcriptional activity of LRH-1, suggesting that SUMO conjugation is associated with transcription repression Coexpression of SUMO-1 targets LRH-1 to the dot-like nuclear bodies, but the effect of lysine mutations on blocking subnuclear localization depended on the cell type In COS-7 cells, mutation of either K173 or K289 prevented SUMO-1-mediated translocation of LRH-1 into nuclear bodies and also reduced the conjugation by SUMO-1, suggesting that K289 and K173 are two important sites involved in SUMO-1 modification In granulosa cells, three or more altered lysine residues were required for nucleoplasm retention This result suggests that multiple lysine residues are targets for SUMO conjugation in vivo and granulosa cells are more sensitive to SUMO-1-mediated LRH-1 localization to nuclear bodies Nuclear body localization of LRH-1 was suppressed by forskolin and cholera toxin Forskolin treatment obviously influences the expression of members involved in the SUMO pathway The results obtained in the present study suggest that cAMP signaling could change the dynamic process of sumoylation and repress LRH-1 targeting to nuclear speckles in rat granulosa cells Liver receptor homolog-1 (LRH-1; NR5A2), a member of the nuclear hormone receptor NR5A subfamily, was originally identified to be the mammalian homolog of the Drosophila fushi tarazu factor (Ftz-F1) LRH-1 is mainly expressed in tissues derived from the gut endoderm, including the liver, pancreas and intestine, and high levels of LRH-1 are also found in the ovary [1,2] LRH-1 knockout mice die at embryonic day 7.5, indicating that LRH-1 plays a crucial role in development [3] In enterohepatic tissues, LRH-1 is involved in cholesterol and bile acid homeostasis by regulating several essential genes in the reverse cholesterol transport and bile acid synthesis pathways [4–7] Abbreviations CYP11A1, cholesterol side-chain cleavage cytochrome P450; Ftz-F1, Drosophila fushi tarazu factor 1; GFP, green fluorescence protein; HESC, human endometrial stromal cells; LRH-1, liver receptor homolog-1; PIAS, protein inhibitor of activated STAT; PMA, 4b-phorbol 12-myristate 13-acetate; SENP, SUMO-specific protease; SF-1, steroidogenic factor-1; SUMO, small ubiquitin-related modifier FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS 425 LRH-1 subnuclear localization in granulosa cells F.-M Yang et al In the ovary, LRH-1 is abundantly expressed in granulosa and luteal cells [8,9] LRH-1 stimulates the promoter activity of several steroidogenic genes [10,11] in ovarian granulosa cells, demonstrating that LRH-1 is a potent regulator of ovarian steroidogenesis Mice with granulosa-specific knockout of the LRH-1 are sterile and exhibit deficiencies in ovulation, progesterone production and corpus luteum formation [12] This indicates that LRH-1 is essential for female reproductive function In addition, LRH-1 enhances aromatase promoter II expression in breast adipose and may play a role in breast tumor development [13,14] Nuclear hormone receptors are typically ligand-activated transcription factors and their activities are modulated by coactivators and corepressors [15] The physiological agonists for LRH-1 have yet to be defined Several coregulators such as multiprotein bridging factor [16], short heterodimer partner [6] and the dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, gene-1 [11,17] have been reported to interact with LRH-1 and to affect LRH-1-mediated gene activation In addition, the activity of LRH-1 is modulated by post-translational modification The transcriptional activity of LRH-1 is stimulated by phosphorylation following 4b-phorbol 12-myristate 13-acetate (PMA) induction [18], and serine residues 238 and 243 in the hinge region of human LRH-1 are important in this PMA-dependent transactivation Recently, sumoylation was reported to comprise another important post-translational regulatory mechanism The small ubiquitin-related modifier (SUMO) is covalently linked to lysines in target proteins through sequential enzymatic reactions that are similar to ubiquitination and require an E1-activation enzyme (i.e a heterodimer of SAE1 and SAE2) and the E2-conjugating enzyme UBC9 [19] Several E3 ligases, such as protein inhibitor of activated STAT (PIAS) family proteins, have been described to promote the transfer of SUMO from E2 to target proteins in vivo The attachment of SUMO may result in diverse effects on the substrate protein, including cellular and subnuclear localization [20,21], changes in stability [22] and transcriptional activity [23,24] SUMO conjugation is dynamic and reversible SUMO-specific proteases (SENPs) remove SUMO conjugates from substrates [25], and several SENPs have been identified in mammals [25–27] These SENP enzymes have distinct subcellular localizations and a substrate preference [27–30] SENP1 and SENP2 can deconjugate all SUMO substrates, and modulate the function of several transcription factors [31] 426 LRH-1 is a target protein of SUMO modification [32,33] The hinge region lysine, residue 289 in the mouse and 224 in the human LRH-1, was the major SUMO conjugation site Sumoylation of LRH-1 is associated with the repression of transcriptional activity and localization in promyelocytic leukemia protein nuclear bodies [32] Desumoylation results in the release of LRH-1 from transcriptionally inert promyelocytic leukemia protein nuclear bodies and access to active chromatin domains As with most SUMO target proteins, the regulation of LRH-1 sumoylation is not understood In the present study, we examined five potential target lysines for SUMO modification and identified additional sites that could be sumoylated in vivo Mutation of either lysine 173 (K173R) or 289 (K289R) indicated that lysine 173 and 289 were crucial for the transactivation of LRH-1 and SUMO-1-mediated localization to subnuclear speckles Furthermore, we demonstrated that no single lysine residue was absolutely required to target LRH-1 to nuclear bodies in rat granulosa cells that expressed endogenous LRH-1 Forskolin and cholera toxin reduced the accumulation of LRH-1 in nuclear bodies Forskolin also affected the expression of several sumoylation enzymes These results suggest that cAMP could be an important signaling for regulating the dynamics of the sumoylation pathway and LRH-1 activity in granulosa cells Results LRH-1 is modified by SUMO-1 in vitro To examine whether LRH-1 can be modified with SUMO, 35S-labeled LRH-1 was synthesized in vitro and incubated with recombinant SUMO-1 protein, E1 (SAE1 ⁄ SAE2) and E2 (UBC9) enzymes Upon separation of the reaction products by SDS ⁄ PAGE, a slowermigrating band at approximately 110 kDa was observed in addition to the major LRH-1 band (Fig 1A, lane 2), which was not present in the absence of SUMO-1, E1 or E2 (Fig 1A, lanes 3–5), suggesting that the slow-migrating band represented the SUMOconjugated form of LRH-1 SUMO is known to be covalently attached to a lysine residue with a consensus sequence WKX(E ⁄ D), in which W represents a large hydrophobic residue [34] We found five lysine residues in the WKX(E ⁄ D) consensus sequence at amino acids 173, 213, 289, 329 and 389 To determine the lysine residues of LRH-1 responsible for SUMO attachment, we mutated these five potential target lysines to arginine (K173R, K213R, K289R, K329R and K389R) and analyzed the effect of these mutations FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS F.-M Yang et al LRH-1 subnuclear localization in granulosa cells A Fig LRH-1 conjugates with SUMO-1 in vitro (A) 35S-labeled LRH-1 was in vitro synthesized and subjected to in vitro sumoylation reactions Proteins SUMO-1, E1 and UBC9 are required for the in vitro sumoylation of LRH-1 (B) Showing in vitro sumoylation reactions of in vitro translated, 35 S-labeled wild-type LRH-1 and five lysine substitution mutants The reaction products were separated on 10% SDS ⁄ PAGE and detected by autoradiography B 118 118 85 85 Sumoylation site lysine 289 negatively regulates LRH-1-mediated transcription To evaluate the effect of sumoylation on LRH-1 transcriptional activity, wild-type or lysine mutants of LRH-1 were transfected into COS-7 cells with cholesterol side-chain cleavage cytochrome P450 (CYP11A1) promoter-linked luciferase As shown in Fig 2, LRH-1 strongly enhanced the activity of the 4.4 kb CYP11A1 2.5 Relative activity ** 1.5 0.5 LRH-1 – Wt 47 47 on in vitro sumoylation As shown in Fig 1B, the SUMO-1-modified products were generated from K173R, K213R, K329R and K389R mutants, as well as from wild-type LRH-1 By contrast, the sumoylated form of LRH-1 was not found in the K289R mutant, suggesting that lysine 289 could be the major site for SUMO-1 conjugation in vitro Sumoylation K173R K213R K289R K329R K389R Fig Mutation at lysine 289 enhances LRH-1 transactivation The CYP11A1 promoter-luciferase construct was cotransfected in COS-7 cells with an empty expression vector (–) or expression vectors for wild-type (Wt) or mutated LRH-1 at lysine site as indicated Cells were lysed and assayed for luciferase activity after transfection for 24 h The relative luciferase activity of each cotransfectant was compared to wild-type construct (arbitrarily set to 1) Data are expressed as the mean ± SE of three independent experiments Mutation at lysine 289 of LRH-1 significantly enhanced the transactivation (**P < 0.01) promoter in COS-7 cells The sumoylation-deficient mutant K289R had two-fold higher transcriptional activity compared to wild-type LRH-1, whereas mutants K173R, K213R, K329R and K389R had similar activity to the wild-type (Fig 2) This result is consistent with the earlier notion that sumoylation mutants have higher transcriptional activities [32,33] These results suggest that the region around the SUMO conjugation site functions as a repression domain, which may influence the transcriptional activity of LRH-1 via sumoylation Lysine 173 and 289 are required for SUMO-1mediated localization in nuclear bodies in COS-7 SUMO modification regulates the subcellular localization of many target proteins [35] We thus examined whether SUMO modification affects the localization of LRH-1 COS-7 cells were cotransfected with expression plasmids encoding LRH-1 fused to enhanced green fluorescence protein (GFP-LRH1) and DsRed-tagged SUMO-1 In cells transfected with GFP-LRH1 alone, GFP-LRH1 was diffusely distributed throughout the nucleus (data not shown) However, in the presence of SUMO-1, wild-type LRH-1 was strongly colocalized with SUMO-1 in the dot-like nuclear bodies (Fig 3A,a) To determine whether SUMO-1 conjugation is required for LRH-1 localization to nuclear bodies, GFP-tagged LRH-1 was cotransfected with the conjugation-deficient SUMO-1 mutant, DsRedSUMO-1 AA [36] into COS-7 cells As shown in Fig 3A,g, both LRH-1 and SUMO-1 AA failed to target the nuclear bodies, confirming that sumoylation is required for specific subnuclear localization of LRH-1 Subsequently, we set out to determine which particular lysine sumoylation site of LRH-1 is involved in the SUMO-1-mediated subnuclear localization Cotransfection analysis revealed that the localization pattern of mutants K213R, K329R and K389R was similar to the wild-type with distinct nuclear bodies (Fig 3A, FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS 427 LRH-1 subnuclear localization in granulosa cells F.-M Yang et al A a b h c i d j e k f B g l C Fig Lysines 173 and 289 are essential for SUMO-1-mediated LRH-1 subnuclear localization in COS-7 cells (A) EGFP-LRH1 wild-type or lysine mutants were cotransfected with DsRed-SUMO-1 (a–f) or conjugation-deficient SUMO-1 mutant DsRed-SUMO-1 AA (g–l) into COS-7 cells After 24 h, cells were fixed and detected by direct fluorescence Merged images demonstrating co-localization of proteins (yellow) are shown on the right Scale bar = 25 lm (B) COS-7 cells were cotransfected with the CYP11A1 promoter-luciferase reporter plasmid and an empty expression vector (–) or expression vectors for wild-type (Wt) or mutated FLAG-LRH-1 at lysine site Cells were lysed and assayed for luciferase activity after transfection for 24 h Relative activity is presented as the ratio of luciferase activity of the wild-type construct Data are expressed as the mean ± SE values of three independent experiments (**P < 0.01) (C) Wild-type or mutated Myc-LRH-1 was cotransfected with DsRed-SUMO-1 and FLAG-PIAS3 in COS-7 cells The nuclear extracts were analyzed by immunoblotting with anti-Myc 428 FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS F.-M Yang et al c,e,f), whereas sumoylation-defective mutant K289R was dispersed across the nucleoplasm of COS-7 cells (Fig 3A,d) Interestingly, mutant K173R also displayed the same diffuse nuclear distribution as mutant K289R (Fig 3A,b) The result implicates lysine 173 as a potential sumoylation site in COS-7 cells Arginine substitution at either lysine residue 173 or 289 resulted in the loss of subnuclear localization, demonstrating that both lysine residues were necessary for complete recruitment of LRH-1 to SUMO-1 formed nuclear bodies in COS-7 cells Although mutation of lysine 173 had little effect on the LRH-1-mediated transcription (Fig 2), the double mutant K173 ⁄ 289R had a further enhanced activity compared to the K289R mutant (Fig 3B) To demonstrate that LRH-1 was modified by SUMO in cells, COS-7 cells were cotransfected with Myc-tagged LRH-1 and SUMO-1 in the presence or absence of an E3 ligase PIAS3 A fraction of slowermigrating bands could be detected by immunoblotting with an anti-Myc in the presence of PIAS3 (Fig 3C, lane 3), indicating that PIAS3 promotes LRH-1 sumoylation in cells Mutation at lysine 289 dramatically resulted in the loss of SUMO-modified forms (Fig 3C, lane 5) The reduction of SUMO-conjugated LRH-1 was also found in mutant K173R, but not in the K389R mutant (Fig 3C, lane and 6) Mutation of both lysine 173 and 289 appeared to abolish sumoylation completely (Fig 3C, lane 7) Collectively, these results demonstrate that lysine 173 and 289 are involved in the SUMO-1 modification in cells and that lysine 289 is the major target for SUMO-1 conjugation Multiple SUMO conjugation is associated with the targeting of LRH-1 into nuclear bodies that is inhibited by cAMP signaling Because the ovary expresses LRH-1, we further examined the effect of sumoylation site mutation on the nuclear localization of LRH-1 in primary rat granulosa cells By contrast to the results obtained with COS-7 cells, the mutant K289R that could not be sumoylated in vitro still showed significant subnuclear distribution patterns when expressed together with SUMO-1 in rat granulosa cells (Fig 4D) In addition, mutation at lysine residues 173, 213, 329 and 389 did not disrupt the localization of LRH-1 into nuclear bodies with SUMO-1 (Fig 4B,C,E,F) Moreover, cotransfection with DsRed-SUMO-1 AA led to a loss of subnuclear localization in both wild-type and mutants (Fig 4G– L), indicating that SUMO conjugation is necessary for LRH-1 localization to the nuclear bodies in rat granulosa cells Therefore, none of these five lysine residues LRH-1 subnuclear localization in granulosa cells alone was essential for SUMO-1-mediated localization of LRH-1 to nuclear bodies in rat granulosa cells To test whether multiple lysine residues could efficiently serve as the acceptor sites for SUMO-1 conjugation, we introduced mutations at lysine residues 173, 213, 329 or 389 into the mutant K289R, respectively, to generate four double mutants When cotransfected with SUMO-1 into granulosa cells, all of the double mutants yielded a similar speckled nuclear distribution pattern as the wild-type (Fig 5A–C; data for mutants K213 ⁄ 289R and K289 ⁄ 329R not shown) However, triple-mutations with three arginine substitutions at 173, 213 and 289 or 289, 329 and 389 resulted in some LRH-1 localization in the nucleoplasm and a reduced concentration in nuclear bodies (Fig 5D,E) Furthermore, the five lysine mutant (GFP-LRH1-5KR) was even more diffusely distributed throughout the nucleus (Fig 5F) Taken together, these data suggest that multiple lysine residues in LRH-1 could be targets for SUMO-1 conjugation in rat granulosa cells Mutation of three or more sites abolished the accumulation of LRH-1 in nuclear bodies It is well known that the control of ovarian function and steroid production by gonadotrophins is mainly through the activation of the cAMP ⁄ protein kinase A pathway LRH-1 is an important regulator of ovarian steroidogenesis The release of LRH-1 from nuclear bodies increases its transcriptional activity [32] Therefore, we tested whether cAMP signaling could affect the subnuclear localization of LRH-1 in granulosa cells The primary rat granulosa cells cotransfected with LRH-1 and SUMO-1 were treated with forskolin to increase cAMP levels As shown in Fig 6A, LRH-1 is present in the nuclear speckles when SUMO-1 was present Forskolin treatment reduced the targeting of LRH-1 to the nuclear bodies and redistributed LRH-1 to the nucleoplasm (Fig 6B) A similar result was also observed when cells were treated with cholera toxin, an activator of cAMP synthesis (Fig 6C) However, addition of the protein kinase C activator PMA to granulosa cells had little effect on the localization of LRH-1 into nuclear bodies (Fig 6D) These results suggest that cAMP signaling could prevent LRH-1 localization to nuclear bodies of rat granulosa cells cAMP signaling pathway regulates the expression of sumoylation-related enzymes Because LRH-1 localization in the nuclear speckles is associated with sumoylation, we therefore determine whether cAMP signaling affects the SUMO modification pathway We used RT-PCR to study the effects of forskolin on the expression of genes We first analyzed FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS 429 LRH-1 subnuclear localization in granulosa cells LRH-1 SUMO-1 F.-M Yang et al Merged LRH-1 SUMO-1AA Merged G A GFP-LRH1 H B GFP-LRH1K173R C I GFP-LRH1K213R J D GFP-LRH1K289R E K GFP-LRH1K329R L F GFP-LRH1K389R Fig No single lysine is essential for SUMO-1-mediated LRH-1 subnuclear localization in rat granulosa cells EGFP-LRH1 wild-type or lysine mutants were cotransfected with DsRed-SUMO-1 (A–F) or conjugation-deficient SUMO-1 mutant DsRed-SUMO-1 AA (G–L) into primary rat granulosa cells After 24 h, cells were fixed and detected by direct fluorescence Merged images demonstrating the co-localization of proteins (yellow) are shown on the right Scale bar = 25 lm the expression of LRH-1 target gene CYP11A1, a cAMP-responsive steroidogenic gene, to confirm the function of forskolin in rat granulosa cells Forskolin strongly stimulated CYP11A1 expression in a timedependent manner (Fig 7A) The mRNA levels of Cpy11a1 were 14-, 50- and 263-fold higher after 3, 12 and 24 h, respectively To determine the effect of cAMP on sumoylation pathway, the expression of the genes for SUMO E1 subunit SAE1 and E2 UBC9 was examined As shown in Fig 7B, SAE1 mRNA levels increased to approximately 500% of control, whereas UBC9 mRNA levels decreased by four-fold after forskolin treatment (Fig 7C) In addition, forskolin caused a marked reduction in the mRNA levels of two SUMO E3 ligases: PIAS3 (by approximately 90%) and PIASxb (by 50%) 430 (Fig 7D,F) By contrast, compared to control cells, the mRNA level of PIASxa was almost completely unaffected by forskolin treatment (Fig 7E) To further examine the expression of SUMO-specific protease, which is capable of reversing sumoylation, we observed that forskolin resulted in a two-fold increase of SENP2 mRNA levels (Fig 7G) Down regulation of UBC9 and PIAS3 proteins was further confirmed by western blot analysis (Fig 7H) Taken together, these data show that cAMP signaling induced a significant change in expression of several sumoylation-related genes Discussion SUMO modification has recently been recognized as an important post-translational mechanism regulating FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS F.-M Yang et al LRH-1 LRH-1 subnuclear localization in granulosa cells LRH-1 SUMO-1 A SUMO-1 A Vehicle GFP-LRH1 B B GFP-LRH1K173/289R Forskolin C C GFP-LRH1K289/389R CTX D D GFP-LRH1K173/213/289R E GFP-LRH1K289/329/389R PMA Fig The cAMP pathway attenuates SUMO-1-mediated LRH-1 subnuclear localization in rat granulosa cells EGFP-LRH1 wild-type was cotransfected with DsRed-SUMO-1 into primary rat granulosa cells After 24 h, cells were treated with dimethylsulfoxide (vehicle control), forskolin, cholera toxin (CTX) or PMA for 24 h, before fixing for fluorescence microscopy Scale bar = 25 lm F GFP-LRH15KR Fig Multiple sites are involved in the SUMO-1-mediated LRH-1 subnuclear localization in rat granulosa cells EGFP-LRH1 wild-type (A) or multiple lysine mutants (B–F) were cotransfected with DsRed-SUMO-1 into primary rat granulosa cells After 24 h, cells were fixed and detected by direct fluorescence 5KR, five mutations at lysine residues 173, 213, 289, 329 and 389 Scale bar = 25 lm the activity and subcellular localization of transcriptional factors [37] In the present study, we mutated five lysine residues in consensus SUMO sites and demonstrated that lysine 289 was most efficiently conjugated by SUMO-1 in vitro Similar results were also observed with steroidogenic factor-1 (SF-1; NR5A1), the closest mammalian homolog of LRH-1, where mutation at the major sumoylation site lysine 194 augments SF-1 activity [36,38] SUMO modification enables the regulation of subcellular localization of various proteins, such as HIPK2 and SF-1 [36,39] In the present study, we demonstrated that SUMO conjugation is required for the subnuclear localization of LRH-1 We showed that, in COS-7 cells, both lysine 173 and 289 were essential for the localization of LRH-1 to nuclear bodies and associated with the repression in transcriptional activity, whereas, in rat granulosa cells, triple mutation of the sumoylation sites was required to prevent LRH-1 localization to nuclear bodies Differences in the presence of cellular factors could account for the cell type-specific effects observed in present study Some other factors may also enhance SUMO modification of LRH-1 in vivo, such as E3 proteins, or promote the targeting of sumoylated LRH-1 into nuclear bodies The expression of these putative factors could differ between cells Whether coordinated SUMO modifications of these sites are required for the correct regulation of LRH-1 function in granulosa cells remains to be determined Taken together, in addition to lysine 289, LRH-1 possesses several potential sumoylation target sites that have not been described previously The differential effects of sumoylation of target sites between COS-7 and granulosa cells suggest that the in vivo regulation of SUMO conjugation is likely to be complex FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS 431 LRH-1 subnuclear localization in granulosa cells E ** 20 000 B C % of control 800 12 h ** 400 100 50 24 h ** 200 C 3h 3h 12 h 24 h PIASxβ 100 ** 50 24 h 75 50 ** ** 3h 12 h 25 C C 3h G 300 UBC9 D ** ** ** 12 h 24 h H SENP2 ** 200 ** ** 100 24 h PIAS3 C 3h 12 h 24 h a UBC9 100 Actin 50 b ** C ** 3h 12 h 24 h PIAS3 ** Actin C We found that SUMO-1-mediated accumulation of LRH-1 in nuclear bodies was suppressed by forskolin treatment, suggesting that activation of the cAMP pathway may have a positive regulatory effect on LRH-1 activity In response to pituitary gonadotropins, the cAMP signaling pathway is the most important pathway in the stimulation of gonadal steroidogenesis [40] In addition, LRH-1 is a potent trans-activator for ovarian steroidogenesis [9,11,41] It therefore implicated that cAMP signaling could indirectly modulate LRH-1 activity by keeping LRH-1 away from transcriptionally inert subnuclear locations to stimulate the expression of steroidogenic genes The modification of proteins by SUMO is a highly dynamic process, and the balance between SUMO conjugation and deconjugation can be shifted after exposure of cells to different stimuli For example, SUMO conjugation is increased in response to heat shock or oxidative stress [42,43] By contrast, a low concentration of hydrogen peroxide induces protein desumoylation by inhibition of conjugating enzymes activity through the formation of a disulfide bridge in 432 12 h C 100 PIASxα 150 F ** C % of control 3h SAE1 600 % of control ** ** % of control 10 000 % of control Cyp11a1 30 000 % of control % of control A F.-M Yang et al h 12 h 24 h Fig The cAMP pathway regulates the expression of SUMO pathway genes Primary rat granulosa cells were treated with dimethylsulfoxide (vehicle control, C) for 24 h or forskolin for 3, 12 or 24 h (A–G) RNA was extracted and then quantified by real-time RT-PCR Data are expressed as the mean ± SE relative to control of three separate experiments **P < 0.01 compared to control (H) Protein levels of UBC9 and PIAS3 were analyzed by western blotting E1 and E2 [44] In the present study, we observed that activation of the cAMP pathway caused a significant change in the sumoylation machinery The mRNA levels of the E1 activating subunit SAE1 and the SUMO specific protease SENP2 were increased, whereas expression of E2 UBC9 and E3 ligases PIAS3 and PIASxb were reduced upon treatment with forskolin in rat granulosa cells for 24 h The changes of SUMO pathway enzymes by cAMP were also described in human endometrial stromal cell (HESC) differentiation [45] The most significant changes were observed in the expression of E3-SUMO ligases and SENPs during HESC differentiation However, the altered patterns of these enzymes in HESCs are not completely consistent with our data For example, the level of PIAS3 is increased in HESCs, whereas it is decreased in granulosa cells In addition, cAMP has little effect on the expression levels of E1, E2 UBC9 and SUMO-1 in HESCs However, our data show that the expression of both E1 and E2 is regulated by cAMP signaling in granulosa cells and Shao et al [46] demonstrated that SUMO-1 expression is decreased after gonadotropin FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS F.-M Yang et al treatment in mouse granulosa cells in vivo These results suggest that the effect of cAMP signaling on SUMO pathway is complex and cell type dependent Notwithstanding, the up regulation of E1, the down regulation of E2 or E3 and the increased expression of SENP2 lead to attenuated SUMO conjugation In addition, SUMO-1 expression is reduced upon cAMP activation in mouse granulosa cells [46] Therefore, whether cAMP signaling results in a global hyposumoylation of most substrates, including LRH-1, requires further investigation However, this may explain why forskolin could abolish the localization of LRH-1 to nuclear bodies in granulosa cells Attenuated sumoylation in response to cAMP has been identified in HESCs [45] A dramatic reduction in the mRNA levels of PIAS3 was noted in the present study Our data also show that PIAS3 promotes LRH-1 sumoylation in COS-7 cells This suggests that PIAS3 may serve as an E3 ligase and be involved in the regulation of LRH-1 sumoylation cAMP is a key signaling pathway for the stimulation of CYP11A1 expression in steroidogenic tissues [47] Multiple and divergent downstream signaling cascades are activated by cAMP in immature and mature granulosa cells [40] The altered expression of SUMO conjugating enzymes by cAMP indicates that the SUMO pathway could be one of the mechanisms by which cAMP regulates CYP11A1 expression In summary, our data show that LRH-1 may contain multiple sumoylation targets and that sumoylation regulates the transcriptional activity and subnuclear localization of LRH-1 In rat granulosa cells, SUMO1-mediated subnuclear localization is attenuated by the cAMP signaling pathway Our studies further indicate a novel regulatory mechanism of cAMP signaling for altering the sumoylation cycle, involving modulating the expression of sumoylation pathway-related genes in rat granulosa cells Experimental procedures Cell isolation and culture All procedures were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee Primary granulosa cells were isolated from Wistar rats purchased from the Laboratory Animal Center, National Taiwan University College of Medicine (Taipei, Taiwan) Immature female rats (21–24 days) were injected subcutaneously with 15 IU of equine chorionic gonadotrophin (Sigma, St Louis, MO, USA) After 48 h, ovaries were excised and placed in DMEM ⁄ F12 medium (Gibco BRL, Gaithersburg, MD, USA) containing 20 mm Hepes and LRH-1 subnuclear localization in granulosa cells antibiotics Granulosa cells were released by puncturing the large- and medium-sized follicles with a 25-gauge needle Granulosa cells were washed and harvested by brief centrifugation and then cultured in DMEM ⁄ F12 medium supplemented with 10% fetal bovine serum and antibiotics COS-7 cells were maintained in DMEM ⁄ high glucose (3%), supplemented with 10% fetal bovine serum Plasmid constructs Plasmid pCMX-mLRH-1 was generously provided by D J Mangelsdorf (Howard Hughes Medical Institute, University of California, Los Angeles, CA, USA) [6] The full coding sequence of LRH-1 was digested with EcoRI and BamHI from pCMX-mLRH-1 and then subcloned into the pFlag-CMV2 (Sigma), pEGFP-C1 (Clontech, Palo Alto, CA, USA) and pGEM-7ZF (Promega, Madison, WI, USA) vectors Arginine-substitution LRH-1 mutants (K173R, K213R, K289R, K329R and K389R) were generated based on pFLAG-LRH1 or pGEM-LRH1 by PCR-based sitedirected mutagenesis [48] and then subcloned into different vectors The single mutants LRH-1 K329R and K389R were then used for the template to create double mutations K289 ⁄ 329R and K289 ⁄ 389R, respectively, by site-directed mutagenesis All mutations were verified by DNA sequencing LRH-1 double mutations K173 ⁄ 289R and K213 ⁄ 289R were obtained by subcloning the EcoRI-StuI fragment containing K173R or K213R into the EcoRI ⁄ StuI site of single mutant K289R Myc-tagged wild-type or mutant LRH-1 was generated by inserting the corresponding fragments into pcDNA-Tag3A (Stratagene, La Jolla, CA, USA) cleaved with EcoRI and HindIII The BsrGI-BamHI fragment from pEGFP-LRH1-K213 ⁄ 289R was inserted into the same sites of pEGFP-LRH1-K173R to generate the triple mutant pEGFP-LRH1-K173 ⁄ 213 ⁄ 289R The BlpI-BamHI fragment from pEGFP-LRH1-K389R was inserted into the same sites of pEGFP-LRH1-K289 ⁄ 329R to generate the triple mutant pEGFP-LRH1-K289 ⁄ 329 ⁄ 389R pEGFP-LRH1-5KR was constructed by the ligation of a BstXI-BamHI fragment from pEGFP-LRH1-K289 ⁄ 329 ⁄ 389R into the BstXI ⁄ BamHI site of pEGFP-LRH1-K173 ⁄ 213 ⁄ 289R The luciferase reporter pSCC4.4-Luc was produced by cloning of upstream regions (–4400 to +55) of the human CYP11A1 into pGL3-Basic vector (Promega) The constructs pDsRedSUMO-1 and pDsRed-SUMO-1 AA have been described previously [36] and were kindly provided by B.-C Chung (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan) The plasmid pFLAG-PIAS3, which encodes FLAG-tagged mouse PIAS3, was a gift from K Shuai (University of California, Los Angeles, CA, USA) In vitro sumoylation assay The 35S-labeled protein was in vitro transcribed ⁄ translated using the TNT Coupled Reticulocyte Lysate System FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ª 2008 FEBS 433 LRH-1 subnuclear localization in granulosa cells F.-M Yang et al (Promega) In vitro sumoylation reactions were performed by incubating lL of in vitro 35S-labeled product with lg of Aos1 ⁄ Uba2, 150 ng of UBC9 and lg of SUMO-1 in sumoylation buffer (20 mm Hepes, pH 7.5, mm MgCl2 and mm ATP) SAE1 ⁄ SAE2, UBC9 and SUMO-1 were obtained from Corgen Inc (Guilford, CT, USA) After 30 of incubation at 37 °C, the reaction products were separated on 10% SDS ⁄ PAGE and then analyzed by autoradiography Kyoto, Japan) The signal was detected using the Immobilon Western HRP system (Millipore, Billerica, MA, USA) Fluorescence microscopy Twenty-four hours before transfection, cells were subcultured onto 24-well plates at a density 105 cells ⁄ well Cells were transfected with 150 ng of LRH-1 expression plasmids, 50 ng of reporter pSCC4.4-Luc and ng of control reporter phRLuc (Biosignal Packard, Perkin Elmer, Waltham, MA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) After 24 h, luciferase activities were determined using the Dual-GloÔ Luciferase Reporter System (Promega) Reporter activities were normalized to internal Renilla luciferase activities Data from three independent experiments were analyzed using a one-way analysis of variance Twenty-four hours before transfection, cells were plated on coverslips in 24-well culture plates Transfections used Lipofectamine with Plus reagent (Invitrogen) for COS-7 cells or FuGENE6 (Roche Diagnostics, Basel, Switzerland) for primary rat granulosa cells in accordance with the manufacturer’s instructions Each well received 200 ng of pEGFP-LRH1 wild-type or mutant constructs and 400 ng of pDsRed-SUMO-1 or pDsRed-SUMO-1 AA as indicated After 24 h, cells were fixed in 4% paraformaldehyde for 10 and then mounted For reagent treatments, 24 h after the transfection, media were replaced with fresh media and then 100 lm forskolin (Sigma) in dimethylsulfoxide, 200 ngỈmL)1 cholera toxin (Sigma) or 100 nm 4b-phorbol 12-myristate 13-acetate (Sigma) in dimethylsulfoxide were added Cells were cultured for 24 h before being fixed in 4% paraformaldehyde for 10 and mounted Images were obtained with a Zeiss Axioskop2 microscope (Carl Zeiss, Jena, Germany) Electroporation and western blotting RNA extraction and RT-PCR To detect the sumoylated forms of LRH-1 in COS-7, 2.5 · 106 cells in 100 lL of serum-free medium were placed in a mm cuvette (Bio-Rad, Hercules, CA, USA) A total amount of lg of DNA (2 lg of Myc-tagged LRH-1 expression plasmid, 1.5 lg of pDsRed-SUMO-1 and 1.5 lg of pFLAG-PIAS3) were added and incubated on ice for 10 Cells were electroporated (150 V, 40 ms, three pulses) using a ECM 830 Electroporation Generator (Harvard Apparatus; BTX, Holliston, MA, USA) and placed on ice for 10 The electroporated cells were then transferred to a 100 mm dish and cultured for 48 h in complete medium supplemented with 10% fetal bovine serum After washing with NaCl ⁄ Pi, cells were lysed in 500 lL of lysis buffer (10 mm Hepes, pH 7.9, 10 mm KCl, mm EDTA, 0.5% NP-40, 20 mm N-ethylmaleimide, 10 mm iodoacetamine, mm dithiothreitol, mm phenylmethanesulfonyl fluoride and 10 lgỈmL)1 of leupeptin) and incubated for 15 on ice The nuclear fraction was recovered by centrifugation at 9000 g at °C for and resuspended in 150 lL of extraction buffer (20 mm Hepes, pH 7.9, 420 mm NaCl, mm EDTA, 20 mm N-ethylmaleimide, 10 mm iodoacetamine, mm dithiothreitol, mm phenylmethanesulfonyl fluoride and 10 lgỈmL)1 leupeptin) After incubation for h on ice, the nuclei were lysed by sonication Equal amounts of total protein (20 lg) were subjected to an 8% SDS ⁄ PAGE and analyzed by western blotting using anti-Myc (Upstate Biotechnology, Lake Placid, NY, USA) treated with Signal Enhancer HIKARI (Nacalai Tesque, Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions RNA (2 lg) was reverse transcribed with oligo(dT) using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) in 20 lL reaction volume, before performing real-time PCR using the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems) The PCR reaction mixture consisted of 0.5 lL of RT product, 10 lL of Power SYBR Green PCR Master Mix (Applied Biosystems) and 125 or 250 nm specific primer pairs in a final reaction volume of 20 lL The sequence information for rat sumoylation-related genes was obtained from the NCBI database, and the specific primer pairs, designed using primer express software (Applied Biosystems), were: CYP11A1 (sense, 5¢-GAGAATCCAGCTTCTTTCCC-3¢ and antisense, 5¢-GGCGACACTGTATGAATTGC-3¢); SENP2 (sense, 5¢-AACAGTCTCTACAATGCGGCCA-3¢ and antisense, 5¢-CCGTGTTCCATTACAAGCAGAA-3¢); SAE1 (sense, 5¢-GACCTGCTTCCCGATGACTTT-3¢ and antisense, 5¢-TTCCTCCAACCACAGCACATAC-3¢); UBC9 (sense, 5¢-CAACAAAGAACCCTGATGGCACGA-3¢ and antisense, 5¢-GCATCCGTAGCTTGAACAAGCCTC-3¢); PIAS3 (sense, 5¢-ACTGCAGGGACCCTGCTACA-3¢ and antisense, 5¢-CTTGATCAGTGCTCGGGAATG-3¢); PIASxa (sense, 5¢-TGCACCTCATTCACCGTCAT-3¢ and antisense, 5¢-CTCAAACGTGGGCTTAGTGTCTT-3¢); PIASxb (sense, 5¢-CCTTCTACTTCCATTGCACCTCAT-3¢ and antisense, 5¢-AAACGTGGGCTTAGTGTCTTGAA-3¢); b-actin Transfection and reporter assays 434 FEBS Journal 276 (2009) 425–436 ª 2008 The Authors Journal compilation ê 2008 FEBS F.-M Yang et al (sense, 5Â-GGGAAATCGTGCGTGAC-3Â and antisense, 5¢-CAAGAAGGAAGGCTGGAA-3¢) The PCR conditions were 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 56 °C for All reactions were performed in triplicate Relative quantification of mRNA levels was determined by the 2)DDCt method and normalized to b-actin in accordance with the manufacturer’s instructions (Applied Biosystems) Data were obtained from three independent experiments in triplicate and presented as mean ± SE One-way analysis of variance was performed 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L), indicating that SUMO conjugation is necessary for LRH-1 localization to the nuclear bodies in rat granulosa cells Therefore, none of these five lysine residues LRH-1 subnuclear localization in. .. whether cAMP signaling could affect the subnuclear localization of LRH-1 in granulosa cells The primary rat granulosa cells cotransfected with LRH-1 and SUMO-1 were treated with forskolin to increase