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 the 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 were 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, 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 decrease 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.

HSD3B1 Expression Is Upregulated by Interleukin 4 in HT-29 Colon Cancer Cells via Multiple Signaling Pathways.

3ß-Hydroxysteroid dehydrogenase/isomerase is essential for the synthesis of active steroid hormones. Interleukin 4 (IL4) induces the expression of HSD3B1 in various human cancer cell lines. Here, we demonstrated that administration of IL4 to an HT-29 colon cancer cell line induced high expression of HSD3B1 at the mRNA and protein levels. In the HT-29 cells, IL4 stimulated the activity of signal transducer and activator of transcription 6 (STAT6) and promoted its binding to the STAT6-binding site in the HSD3B1 promoter. The STAT6 inhibitor significantly suppressed HSD3B1 induction by IL4 in a dose-dependent manner. Moreover, inhibition of the PI3-kinase/AKT pathway strongly suppressed the IL4-induced HSD3B1 expression. Glycogen synthase kinase 3 (GSK3), a downstream target of AKT, had a stimulatory effect on the IL4-induced HSD3B1 expression. However, IL4 stimulated the phosphorylation of AKT, which inhibited the GSK3 activity at the early stage. Hence, GSK3 potentiated the HSD3B1 levels at the late stage of the IL4 stimulation. Additionally, inhibitors of mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, but not of JNK, partly reduced the HSD3B1 expression following the IL4 stimulation. We further demonstrated that IL4 potently promoted steroid synthesis. Our results indicate that IL4 induces HSD3B1 expression via multiple signaling pathways in HT-29 cells and may play a role in the regulation of steroid synthesis.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria.

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and ß subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and ß subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

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.

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.

Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals.

CYP11A1 encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression of CYP11A1 in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controlling CYP11A1 expression is steroidogenic factor-1 (SF-1), but the transcriptional regulation of CYP11A1 in the brain is unclear. We recently used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional-specific expression of Cre reporter in the SCC-Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF-1 binding site did not affect brain CYP11A1 promoter activity. The upstream SF-1 binding site, however, is required for CYP11A1 promoter function in the embryonic adrenals. The 3.8-kb promoter, like the 4.4-kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7-kb promoter was only observed in the caudal part of midbrain. This suggests that the 5'-flanking region between 3.8 and 2.7 kb contains a crucial element for activation of CYP11A1 promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that control CYP11A1 expression in the brain and embryonic adrenals.

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.

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy.

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.

Regulation of liver receptor homologue-1 by DDB2 E3 ligase activity is critical for hepatic glucose metabolism.

Liver receptor homologue-1 (LRH-1) plays a critical role in hepatic metabolism and disease. Here we show that LRH-1 protein stability is regulated by the cullin 4 (CUL4) E3 ubiquitin ligase complex. We found that DNA damage-binding protein 2 (DDB2) directly interacts with LRH-1 and functions as a substrate recognition component of CUL4-DDB1 to promote LRH-1 ubiquitination and proteasomal degradation. In human hepatoma (HepG2) cells, we observed that protein levels of endogenous LRH-1 are increased by insulin without a change in mRNA levels of LRH-1. However, overexpression of DDB2 impaired the insulin-stimulated increase in LRH-1 levels. In addition, DDB2 overexpression decreased LRH-1 transcriptional activation and expression of target genes, such as glucokinase, whereas knockdown of DDB2 increased the expression of glucokinase. Finally, we demonstrated that DDB2 knockdown increases glucose uptake and intracellular levels of glucose-6-phosphate in HepG2 cells. Our study reveals a novel regulatory mechanism of LRH-1 activity and suggests a role for DDB2 in hepatic glucose metabolism.

Bisphenol A disrupts steroidogenesis and induces a sex hormone imbalance through c-Jun phosphorylation in Leydig cells.

Bisphenol A (BPA) is a well-known endocrine disrupting chemical (EDC) that is used to manufacture plastic consumer products. It is well known that exposure to BPA can induce defects in gonad development and negatively influences reproductive function in both males and females. In this study, we assessed the effects of BPA on hormone production in Leydig cells, which secrete hormones in the testes and support male fertility. We examined two steroidogenic enzymes, CYP11A1 and CYP19 that involved in sex hormone synthesis in mouse MA-10 Leydig cells. We found that BPA activated CYP gene in both mRNA and protein levels then resulted in alteration of the normal sex hormone ratio. Furthermore, we found that BPA induced c-Jun phosphorylation and contributed to CYP gene expression. Similar results were observed in an animal study. In conclusion, BPA disrupts the hormone environment in testis via steroidogenic gene activation through the JNK/c-Jun signaling pathway.

Proximal GATA-binding sites are essential for human HSD3B1 gene transcription in the placenta.

The enzyme 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD) is involved in the synthesis of active steroid hormones. Two human 3ß-HSD isoforms are expressed in a tissue-specific pattern. HSD3B1 (type I) expression is essential to produce progesterone for pregnancy maintenance. To understand the mechanisms of human HSD3B1 activation in the placenta, 2.2 kb of 5'-flanking sequence and 5'-deletions were fused to the luciferase reporter gene and transfected into human JEG-3 cells. The proximal -238/+337 sequence had the highest promoter activity. Two GATA elements were identified at -106/-99 and -52/-45. Mutations of either sites greatly reduced promoter activity in JEG-3 cells, demonstrating the importance of GATA sites. EMSA revealed the specific binding of GATA2 and GATA3 to the GATA sequences at -106/-99 and -52/-45. ChIP assays demonstrated the association of GATA2 but not GATA3 with the GATA-binding regions of the HSD3B1 promoter in JEG-3 cells. GATA2 knockdown significantly reduced HSD3B1 expression in JEG-3 cells; however, GATA3 knockdown increased HSD3B1 expression. Western blot analysis revealed high levels of GATA2 but not GATA3 in human placental tissues. This study identified GATA motifs as essential control elements for HSD3B1 transcription and GATA2 as a novel transcriptional regulator of HSD3B1 expression in the human placenta.

Steroidogenesis in zebrafish and mouse models.

Steroid hormones regulate physiological homeostasis for salt, sugar, and sex differentiation. All steroids are synthesized from a common precursor, cholesterol, in a step that converts cholesterol to pregnenolone. The enzyme carrying out this first conversion step is CYP11A1. To further investigate the importance of steroid biosynthesis, animal models with defects in the Cyp11a1 gene are used. Mice with targeted disruption of the Cyp11a1 gene produce no steroids with severe adrenal defects. These mice survive during embryogenesis, but die after birth. Zebrafish with a block in cyp11a1 gene function has an earlier defect, presumably because it lacks adequate maternal steroid supply. When cyp11a1 activity was compensated by the injection of antisense morpholino oligos, the embryos have shortened axis and a defect of epibolic cell movement during early embryogenesis. The discovery of steroid function in cell movement is novel, and should provide new insights into our understanding of diverse functions of steroids.

Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1.

Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.