Dendritic Morphology of Developing Hippocampal Neurons in Cyp11a1 Null Mice.

INTRODUCTION: Neurosteroids have a variety of neurological functions, such as neurite growth, neuroprotection, myelination, and neurogenesis. P450scc, encoded by CYP11A1 gene, is the cholesterol side chain cleavage enzyme that catalyzes the first and rate-limiting step in steroidogenesis. In this study, we examine the dendritic morphology in developing hippocampal neurons of Cyp11a1 null mice at P15, a critical period for synapse formation and maturation. METHODS: Knockout mice were maintained until P15 with hormone administration. The Golgi-Cox method stained CA1 and CA3 pyramidal neurons in the hippocampus to reveal dendritic morphology. RESULTS: We demonstrated that Cyp11a1 null mice usually die within 7 days after birth and thus collected brain samples at postnatal day 5 (P5) for examination. There was significant shrinkage of dendrite size and diminishment of dendritic branching in CA1 and CA3 pyramidal neurons in the hippocampus of Cyp11a1 null mice, suggesting a developmental delay. We wonder if this delay may catch up later in life. Since the age of P15 is a critical period for synapse formation and maturation, the Cyp11a1 null mice were rescued by receiving hormone administration until P15 that the dendritic morphology in the developing hippocampal neurons could be examined. The results indicated that the total dendritic length, the number of dendritic branches, as well as dendritic arborization in the CA1 and CA3 pyramidal neurons are significantly decreased in P15 knockout mice when compared to the wild type. The spine densities were also significantly decreased. In addition, the Western blot analysis revealed decreased PSD-95 expression levels in the knockout mice compared to the wild type at P15. CONCLUSION: These results suggested that Cyp11a1 deficiency impairs the dendritic structures in the developing hippocampal pyramidal neurons.

Regulation of TLR4 signaling through the TRAF6/sNASP axis by reversible phosphorylation mediated by CK2 and PP4.

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor-associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor-associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4-infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.

The VraSR two-component signal transduction system contributes to the damage of blood-brain barrier during Streptococcus suis meningitis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause high morbidity and mortality in both humans and swine. As the most important life-threatening infection of the central nervous system (CNS), meningitis is an important syndrome of S. suis infection. The vancomycin resistance associated sensor/regulator (VraSR) is a critical two-component signal transduction system that affects the ability of S. suis to resist the host innate immune system and promotes its ability to adhere to brain microvascular endothelial cells (BMECs). Prior work also found mice infected with ΔvraSR had no obvious neurological symptoms, unlike mice infected with wild-type SC19. Whether and how VraSR participates in the development of S. suis meningitis remains unknown. Here, we found ΔvraSR-infected mice did not show obvious meningitis, compared with wild-type SC19-infected mice. Moreover, the proinflammatory cytokines and chemokines in serum and brains of ΔvraSR-infected mice, including IL-6, TNF-α, MCP-1 and IFN-γ, were significantly lower than wild-type infected group. Besides, blood-brain barrier (BBB) permeability also confirmed that the mutant had lower ability to disrupt BBB. Furthermore, in vivo and in vitro experiments showed that SC19 could increase BBB permeability by downregulating tight junction (TJ) proteins such as ZO-1, ß-Catenin, Occludin, and Clauidn-5, compared with mutant ΔvraSR. These findings provide new insight into the influence of S. suis VraSR on BBB disruption during the pathogenic process of streptococcal meningitis, thereby offering potential targets for future preventative and therapeutic strategies against this disease.

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium.

House dust mites (HDMs) are a common source of respiratory allergens responsible for allergic asthma and innate immune responses in human diseases. Since HDMs are critical factors in the triggering of allergen-induced airway mucosa from allergic asthma, we aimed to investigate the mechanisms of Toll-like receptors (TLR) in the signaling of the HDM extract that is involved in mucus hypersecretion and airway inflammation through the engagement of innate immunity. Previously, we reported that the somatic nuclear autoantigenic sperm protein (sNASP)/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis controls the initiation of TLRs to maintain the homeostasis of the innate immune response. The present study showed that the HDM extract stimulated the biogenesis of Mucin 5AC (MUC5AC) in bronchial epithelial cells via the TLR2/4 signaling pathway involving MyD88 and TRAF6. Specifically, sNASP binds to TRAF6 in unstimulated bronchial epithelial cells to prevent the activation of TRAF6-depenedent kinases. Upon on HDMs' stimulation, sNASP is phosphorylated, leading to the activation of TRAF6 downstream of the p38 MAPK and NF-κB signaling pathways. Further, NASP-knockdown enhanced TRAF6 signaling and MUC5AC biogenesis. In the HDM-induced mouse asthma model, we found that the HDM extract promoted airway hyperresponsiveness (AHR), MUC5AC, and allergen-specific IgE production as well as IL-5 and IL-13 for recruiting inflammatory cells. Treatment with the PEP-NASP peptide, a selective TRAF6-blocking peptide, ameliorated HDM-induced asthma in mice. In conclusion, this study indicated that the sNASP/TRAF6 axis plays a regulatory role in asthma by modulating mucus overproduction, and the PEP-NASP peptide might be a potential target for asthma treatment.

The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum.

WRKY transcription factors play a key role in the tolerance of biotic and abiotic stresses across various crop species, but the function of some WRKY genes, particularly in tomato, remains unexplored. Here, we characterize the roles of a previously unstudied WRKY gene, SlWRKY8, in the resistance to pathogen infection and the tolerance to drought and salt stresses. Expression of SlWRKY8 was up-regulated upon Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000), abiotic stresses such as drought, salt and cold, as well as ABA and SA treatments. The SlWRKY8 protein was localized to the nucleus with no transcription activation in yeast, but it could activate W-box-dependent transcription in plants. The overexpression of SlWRKY8 in tomato conferred a greater resistance to the pathogen Pst. DC3000 and resulted in the increased transcription levels of two pathogen-related genes SlPR1a1 and SlPR7. Moreover, transgenic plants displayed the alleviated wilting or chlorosis phenotype under drought and salt stresses, with higher levels of stress-induced osmotic substances like proline and higher transcript levels of the stress-responsive genes SlAREB, SlDREB2A and SlRD29. Stomatal aperature was smaller under drought stress in transgenic plants, maintaining higher water content in leaves compared with wild-type plants. The oxidative pressure, indicated by the concentration of hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA), was also reduced in transgenic plants, where we also observed higher levels of antioxidant enzyme activities under stress. Overall, our results suggest that SlWRKY8 functions as a positive regulator in plant immunity against pathogen infection as well as in plant responses to drought and salt stresses.

A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding.

As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation.

Local anesthetics induce apoptosis in human breast tumor cells.

BACKGROUND: Previous studies have shown that local anesthetics may induce apoptosis in some cell types. In this study, we investigated the apoptotic effects of local anesthetics in human breast tumor cells. METHODS: Human breast cancer (MCF-7) and mammary epithelial (MCF-10A) cell lines were treated with lidocaine and/or bupivacaine. Cell viability, DNA fragmentation, and annexin V immunofluorescence staining were assessed. The effects on apoptosis-related protein expression were investigated by Western blot analysis. The findings were extended to studies in an in vivo xenograft model. RESULTS: Treatment of breast tumor cells with lidocaine and bupivacaine resulted in inhibition of cell viability via induction of apoptosis. The effects were more prominent in MCF-7 cells than in MCF-10A cells. Treatment with local anesthetics induced caspase 7, 8, 9, and poly ADP-ribose polymerase cleavage. The cleavage of caspase 7 and poly ADP-ribose polymerase induced by local anesthetics were effectively blocked by caspase inhibitors. Furthermore, treatment of MCF-7 xenografts with local anesthetics resulted in higher expression of cleaved caspase 7 and an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. CONCLUSION: Lidocaine and bupivacaine induce apoptosis of breast tumor cells at clinically relevant concentrations. Our results reveal previously unrecognized beneficial actions of local anesthetics and call for further studies to assess the oncologic advantages of their use during breast cancer surgery.

5,6-Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) induces apoptosis in breast cancer cells through inhibiting of Mcl-1 expression.

The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells.

NCI677397 targeting USP24-mediated induction of lipid peroxidation induces ferroptosis in drug-resistant cancer cells.

Cancer represents a profound challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major problem in cancer therapy and can result in progression of the disease. In our previous studies, we developed small-molecule inhibitors targeting ubiquitin-specific peptidase 24 (USP24) to combat drug-resistant lung cancer. Recently, we found that the USP24 inhibitor NCI677397 induced ferroptosis, a type of programmed cell death, in drug-resistant cancer cells by increasing lipid reactive oxygen species (ROS) levels. In the present study, we investigated the molecular mechanisms and found that the targeting of USP24 by NCI677397 increased gene expression of most lipogenesis-related genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), and activated autophagy. In addition, the activity of several antioxidant enzymes, such as glutathione peroxidase 4 (GPX4) and dihydrofolate reductase (DHFR), was inhibited by NCI677397 treatment via an increase in protein degradation, thereby inducing lipid ROS production and lipid peroxidation. In summary, we demonstrated that NCI677397 induced a marked increase in lipid ROS levels, subsequently causing lipid peroxidation and leading to the ferroptotic death of drug-resistant cancer cells. Our study provides new insights into the clinical use of USP24 inhibitors as ferroptosis inducers (FINs) to block drug resistance during chemotherapy.

Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1.

Liver receptor homologue-1 (LRH-1) is a member of the nuclear receptor superfamily. We characterized two functional nuclear localization signals (NLSs) in LRH-1. NLS1 (residues 117-168) overlaps the second zinc finger in the DNA binding domain. Mutagenesis showed that the zinc finger structure and two basic clusters on either side of the zinc finger loop are critical for nuclear import of NLS1. NLS2 (residues 169-204) is located in the Ftz-F1 box that contains a bipartite signal. In full-length LRH-1, mutation of either NLS1 or NLS2 had no effect on nuclear localization, but disruption of both NLS1 and NLS2 resulted in the cytoplasmic accumulation of LRH-1. Either NLS1 or NLS2 alone was sufficient to target LRH-1 to the nucleus. Both NLS1 and NLS2 mediate nuclear transport by a mechanism involving importin α/ß. Finally, we showed that three crucial basic clusters in the NLSs are involved in the DNA binding and transcriptional activities of LRH-1.

PEP-sNASP Peptide Alleviates LPS-Induced Acute Lung Injury Through the TLR4/TRAF6 Axis.

Acute lung injury (ALI) is a severe inflammatory lung disease associated with macrophages. Somatic nuclear autoantigenic sperm protein (sNASP) is a negative regulator of Toll-like receptor (TLR) signaling that targets tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in macrophages, which is required to maintain homeostasis of the innate immune response. In the present study, we generated a cell permeable PEP-sNASP peptide using the sNASP protein N-terminal domain, and examined its potential therapeutic effect in a mouse model of ALI induced by the intranasal administration of lipopolysaccharide (LPS) and elucidated the underlying molecular mechanisms in RAW 264.7 cells. In vivo, PEP-sNASP peptide treatment markedly ameliorated pathological injury, reduced the wet/dry (W/D) weight ratio of the lungs and the production of proinflammatory cytokines (interleukin (IL)-1ß, IL-6, and TNF-α). In vitro, we demonstrated that when the PEP-sNASP peptide was transduced into RAW 264.7 cells, it bound to TRAF6, which markedly decreased LPS-induced proinflammatory cytokines by inhibiting TRAF6 autoubiquitination, nuclear factor (NF)-κB activation, reactive oxygen species (ROS) and cellular nitric oxide (NO) levels. Furthermore, the PEP-sNASP peptide also inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Our results therefore suggest that the PEP-sNASP may provide a potential protein therapy against oxidative stress and pulmonary inflammation via selective TRAF6 signaling.

PIASy inhibits LRH-1-dependent CYP11A1 expression by competing for SRC-1 binding.

The orphan nuclear receptor LRH-1 (liver receptor homologue-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. In the present study, we investigated the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) y], the shortest member of the PIAS family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO (small ubiquitin-related modifier) ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 SUMOylation status. In addition, histone deacetylase activity was not involved in the inhibition of LRH-1 by PIASy. Immunoprecipitation and mammalian two-hybrid analyses indicated that PIASy interacted with LRH-1 through the C-terminal region, including the AF-2 (activation function-2) motif, which was also involved in the interaction between LRH-1 and the co-activator SRC-1 (steroid receptor co-activator-1). PIASy inhibited the binding of SRC-1 to LRH-1, although overexpression of SRC-1 partially overcame the PIASy inhibition of LRH-1 induction of the CYP11A1 promoter. The results of the present study suggest that competition with co-activators may be an important mechanism underlying the PIASy repression of LRH-1-mediated transactivation.

Correction: miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate cell proliferation and apoptosis in NSCLC.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate cell proliferation and apoptosis in NSCLC.

Non-small cell lung cancer (NSCLC) is one of the most common aggressive malignancies. miRNAs have been identified as important biomarkers and regulators of NSCLC. However, the functional contributions of miR-1260b to NSCLC cell proliferation and apoptosis have not been studied. In this study, miR-1260b was upregulated in NSCLC plasma, tissues, and cell lines, and its high expression was correlated with tumor size and progression. Functionally, miR-1260b overexpression promoted cell proliferation and cell cycle, conversely inhibited cell apoptosis and senescence. Mechanically, miR-1260b negatively regulated SOCS6 by directly binding to its 3'-UTR. Furthermore, miR-1260b-mediated suppression of SOCS6 activated KIT signaling. Moreover, YY1 was an upstream regulator of miR-1260b. This study is the first to illustrate that miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate NSCLC cell proliferation and apoptosis, and is a potential biomarker and therapeutic target for NSCLC. In sum, our work provides new insights into the molecular mechanisms of NSCLC involved in cell proliferation and apoptosis.

sNASP inhibits TLR signaling to regulate immune response in sepsis.

Many Toll-like receptors (TLRs) signal through TNF receptor-associated factor 6 (TRAF6) to activate innate immune responses. Here, we show that somatic nuclear autoantigenic sperm protein (sNASP) binds to TRAF6 to prevent TRAF6 autoubiquitination in unstimulated macrophages. Following LPS stimulation, a complex consisting of sNASP, TRAF6, IRAK4, and casein kinase 2 (CK2) is formed. CK2 phosphorylates sNASP at serine 158, allowing sNASP to dissociate from TRAF6. Free TRAF6 is then autoubiquitinated, followed by activation of downstream signaling pathways. In sNasp S158A knockin (S158A-KI) mice, LPS-treated macrophages could not phosphorylate sNASP, which remained bound to TRAF6. S158A-KI mice were more susceptible to sepsis due to a marked reduction in IL-1ß, TNF-α, and IFN-γ production accompanied by an inability to clear bacteria and recruit leukocytes. Furthermore, phosphorylation-regulated release of sNASP from TRAF6 is observed following activation of TLR-1, -2, -4, -5, and -6. Thus, sNASP is a negative regulator of TLR signaling to modulate the innate immune response.

SENP1 regulates PTEN stability to dictate prostate cancer development.

SUMO protease SENP1 is elevated in multiple carcinomas including prostate cancer (PCa). SENP1 exhibits carcinogenic properties; it promotes androgen receptor-dependent and -independent cell proliferation, stabilizes HIF1α, increases VEGF, and supports angiogenesis. However, mice expressing an androgen-responsive promoter driven SENP1-transgene (SENP1-Tg) develop high-grade prostatic intraepithelial neoplasia, but not carcinoma. We now show that tumor suppressive PTEN signaling is induced in SENP1-Tg to enhance prostate epithelial cell apoptosis. SENP1 blocks SUMO1-dependent ubiquitylation and degradation of PTEN. In the absence of SENP1, SUMO1-modified PTEN is sequestered in the cytosol, where binding to ubiquitin-E3 ligase WWP2 occurs. Concurrently, WWP2 is also SUMOylated, which potentiates its interaction with PTEN. Thus, SENP1 directs ubiquitin-E3-substrate association to control PTEN stability. PTEN serves as a barrier for SENP1-mediated prostate carcinogenesis as SENP1-Tg mice develop invasive carcinomas only after PTEN reduction. Hence, SENP1 modulates multiple facets of carcinogenesis and may serve as a target specifically for aggressive PTEN-deficient PCa.

Docosahexaenoic acid induces proteasome-dependent degradation of estrogen receptor alpha and inhibits the downstream signaling target in MCF-7 breast cancer cells.

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor alpha (ERalpha). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERalpha distribution. MCF-7 cells, an ERalpha-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 microM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERalpha expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERalpha expression resulted from proteasome-dependent degradation and not from decreased ERalpha mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERalpha, reduced cyclin D1 expression and inhibition of MAPK signaling.

Liver receptor homolog-1 localization in the nuclear body is regulated by sumoylation and cAMP signaling in rat granulosa cells.

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 SUMO-1 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.