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Journal of Neuroinflammation Dec 2023Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression....
BACKGROUND
Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression. Neuroinflammation-induced hypothalamic dysfunction underlies behaviors associated with depression. The lipopolysaccharide (LPS)-induced mouse model of depression has been well-validated in numerous laboratories, including our own, and is widely used to investigate the relationship between neuroinflammation and depression. However, the sex-specific differences in metabolic alterations underlying depression-associated hypothalamic neuroinflammation remain unknown.
METHODS
Here, we employed the LPS-induced mouse model of depression to investigate hypothalamic metabolic changes in both male and female mice using a metabolomics approach. Through bioinformatics analysis, we confirmed the molecular pathways and biological processes associated with the identified metabolites. Furthermore, we employed quantitative real-time PCR, enzyme-linked immunosorbent assay, western blotting, and pharmacological interventions to further elucidate the underlying mechanisms.
RESULTS
A total of 124 and 61 differential metabolites (DMs) were detected in male and female mice with depressive-like behavior, respectively, compared to their respective sex-matched control groups. Moreover, a comparison between female and male model mice identified 37 DMs. We capitalized on biochemical clustering and functional enrichment analyses to define the major metabolic changes in these DMs. More than 55% of the DMs clustered into lipids and lipid-like molecules, and an imbalance in lipids metabolism was presented in the hypothalamus. Furthermore, steroidogenic pathway was confirmed as a potential sex-specific pathway in the hypothalamus of female mice with depression. Pregnenolone, an upstream component of the steroid hormone biosynthesis pathway, was downregulated in female mice with depressive-like phenotypes but not in males and had considerable relevance to depressive-like behaviors in females. Moreover, exogenous pregnenolone infusion reversed depressive-like behaviors in female mice with depression. The 5α-reductase type I (SRD5A1), a steroidogenic hub enzyme involved in pregnenolone metabolism, was increased in the hypothalamus of female mice with depression. Its inhibition increased hypothalamic pregnenolone levels and ameliorated depressive-like behaviors in female mice with depression.
CONCLUSIONS
Our study findings demonstrate a marked sexual dimorphism at the metabolic level in depression, particularly in hypothalamic steroidogenic metabolism, identifying a potential sex-specific pathway in female mice with depressive-like behaviors.
Topics: Humans; Mice; Male; Female; Animals; Depression; Neuroinflammatory Diseases; Lipopolysaccharides; Hypothalamus; Inflammation; Pregnenolone
PubMed: 38062440
DOI: 10.1186/s12974-023-02976-7 -
Biological Procedures Online Nov 2023Renal cancer therapies are challenging owing to the extensive spreading of this cancer to other organs and its ability to pose resistance to current medications....
BACKGROUND
Renal cancer therapies are challenging owing to the extensive spreading of this cancer to other organs and its ability to pose resistance to current medications. Therefore, drugs targeting novel targets are urgently required to overcome these challenges. The cholesterol side-chain cleavage enzyme (CYP11A1) is closely associated with steroidogenesis, and its downregulation is linked to adrenal dysfunction and several types of carcinoma. We previously found that overexpression of CYP11A1 inhibited epithelial-mesenchymal transition and induced G2/M arrest in the kidney cancer Caki-1 cell line. In this context, natural compounds that exhibit potent CYP11A1 stimulation activity can be promising therpaeutic agents for kidney cancer.
METHODS
We screened a panel of 1374 natural compounds in a wound-healing assay using CYP11A1-transfected Caki-1 cells. Of these, 167 promising biologically active compounds that inhibited cancer cell migration by more than 75% were selected, and their half-maximal inhibitory concentrations (IC) were determined. The IC of 159 compounds was determined and 38 compounds with IC values less than 50 µM were selected for further analysis. Steroid hormones (cholesterol and pregnenolone) levels in cells treated with the selected compounds were quantitated using LC-MS/MS to determine their effect on CYP11A1 activity. Western blotting for CYP11A1, autophagy signaling proteins, and ferroptosis regulators were performed to ivestigate the mechanisms underlying the action of the selected compounds.
RESULTS
We screened five promising natural lead compounds that inhibited cancer cell proliferation after three screening steps. The IC of these compounds was determined to be between 5.9 and 14.6 μM. These candidate compounds increased the expression of CYP11A1 and suppressed cholesterol levels while increasing pregnenolone levels, which is consistent with the activation of CYP11A1. Our results showed that CYP11A1 activation inhibited the migration of cancer cells, promoted ferroptosis, and triggered autophagy signaling.
CONCLUSIONS
This study indicates that the CYP11A1-overexpressing Caki-1 cell line is useful for screening drugs against kidney cancer. The two selected compounds could be utilized as lead compounds for anticancer drug discovery, and specifically for the development of antirenal cancer medication.
PubMed: 38036976
DOI: 10.1186/s12575-023-00225-y -
Frontiers in Immunology 2023Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of...
Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 ; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.
Topics: Pregnenolone; Th2 Cells; Molecular Dynamics Simulation; Steroids; Carrier Proteins
PubMed: 38022565
DOI: 10.3389/fimmu.2023.1229703 -
Cancer Reports (Hoboken, N.J.) Jan 2024Progesterone therapy is a relatively inexpensive treatment option for endometrial and breast cancers, with few side effects. Two signaling pathways usually mediate the...
BACKGROUND
Progesterone therapy is a relatively inexpensive treatment option for endometrial and breast cancers, with few side effects. Two signaling pathways usually mediate the physiological effects of progesterone, namely genomic and non-genomic actions. Genomic action occurs slowly via the nuclear progesterone receptor (PR), whereas the membrane progesterone receptor (mPR) induces rapid non-genomic action.
AIMS
We investigated the effects of progesterone and various PR agonists on ovarian cancer cells.
METHODS AND RESULTS
PR expression of six serous ovarian cancer cell lines was examined by western blotting, and mPR expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). PR-negative and mPR-positive ovarian cancer cells were exposed to progesterone and seven types of PR agonists (medroxyprogesterone acetate [MPA], dehydroepiandrosterone, dienogest, levonorgestrel, drospirenone, pregnenolone, and allopregnanolone) at 10-400 μM, and viable cell counts after exposure for 30 min were measured using the water-soluble tetrazolium (WST-1) assay. Ovarian cancer cell lines were exposed to 100 μM progesterone, and the expression of BAX, a pro-apoptotic protein, after 1-5 min was examined by western blotting. Western blotting detected no PR expression in the six serous ovarian cancer cell lines. In contrast, RT-qPCR detected mPR expression in all six serous ovarian cancer cell lines. Progesterone and MPA-induced cell death in all tested ovarian cancer cell lines in a concentration-dependent manner, whereas no effect was observed for other PR agonists. Western blotting revealed that pro-apoptotic protein BAX expression occurred 1 min after exposure to progesterone, suggesting that the cytocidal effects are mediated by rapid non-genomic action.
CONCLUSION
Progesterone and MPA exhibited a rapid cytocidal effect on PR-negative ovarian cancer cells through non-genomic action. Progesterone and MPA could be novel adjuvant therapies for ovarian cancer.
Topics: Female; Humans; Progesterone; Receptors, Progesterone; bcl-2-Associated X Protein; Progestins; Medroxyprogesterone Acetate; Ovarian Neoplasms; Genomics; Cell Death
PubMed: 38013666
DOI: 10.1002/cnr2.1934 -
The Journal of Biological Chemistry Jan 2024Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450) that removes six carbons of the side chain, the first and rate-limiting...
Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH) cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH) cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and k rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH) cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.
Topics: Humans; Adrenodoxin; Cholesterol; Cholesterol Side-Chain Cleavage Enzyme; Kinetics; Pregnenolone; Protein Binding; Oxidation-Reduction; Molecular Structure
PubMed: 38006947
DOI: 10.1016/j.jbc.2023.105495 -
Research Square Oct 2023Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a)...
Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 μM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.
PubMed: 37961499
DOI: 10.21203/rs.3.rs-3471389/v1 -
Advances in Drug and Alcohol Research 2023Despite the significant number of people who may be taking pregnenolone supplements while drinking alcohol (ethanol), the widely documented cerebrovascular actions of...
Despite the significant number of people who may be taking pregnenolone supplements while drinking alcohol (ethanol), the widely documented cerebrovascular actions of pregnenolone and ethanol, and the critical dependence of cerebrovascular function on cerebral artery diameter, there are no studies addressing the effect of pregnenolone + ethanol in combination on cerebral artery diameter. We investigated this by evaluating the effect of this combination on middle cerebral artery diameter in male and female C57BL/6J mice, both and . The use of de-endothelialized, pressurized middle cerebral artery segments allowed us to conduct a concentration-response study of constriction induced by pregnenolone ± ethanol, in which drug action could be evaluated independently of circulating and endothelial factors. In both male and female animals, pregnenolone at lower concentrations (≤1 μM) was found to synergize with 50 mM ethanol to cause vasoconstriction. In both sexes, this synergism was lost as one or both vasoconstrictors approached their maximally effective concentrations (75 mM and 10 μM for ethanol and pregnenolone, respectively), whether this was evaluated or using a cranial window. Vasoconstriction by pregnenolone + ethanol was abolished by 1 μM paxilline, indicating BK channel involvement. Moreover, cell-free recordings of BK channel activity in cerebral artery myocyte membranes showed that 10 μM pregnenolone and pregnenolone +50 mM ethanol reduced channel activity to an identical extent, suggesting that these drugs inhibit cerebrovascular BK channels a common mechanism or mechanisms. Indeed, pregnenolone was found to disrupt allosteric coupling to -driven gating, as previously reported for ethanol.
PubMed: 37846408
DOI: 10.3389/adar.2023.11735 -
Deubiquitinase UCHL1 regulates estradiol synthesis by stabilizing voltage-dependent anion channel 2.The Journal of Biological Chemistry Nov 2023Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The...
Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.
Topics: Animals; Female; Mice; Estradiol; Granulosa Cells; Ovarian Follicle; Swine; Ubiquitin Thiolesterase; Voltage-Dependent Anion Channel 2; Sus scrofa
PubMed: 37797697
DOI: 10.1016/j.jbc.2023.105316 -
Nature Plants Oct 2023Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules,...
Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and β-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.
Topics: Cardenolides; Plants; Digitalis; Pregnenolone
PubMed: 37723202
DOI: 10.1038/s41477-023-01515-9 -
Ecotoxicology and Environmental Safety Oct 2023Bisphenol A (BPA) is a chemical used in the production of certain plastics and resins. Recent research has found that BPA can inhibit the activity of 3β-hydroxysteroid...
Benzene ring bisphenol A substitutes potently inhibit human, rat, and mouse gonadal 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.
Bisphenol A (BPA) is a chemical used in the production of certain plastics and resins. Recent research has found that BPA can inhibit the activity of 3β-hydroxysteroid dehydrogenase/Δ-isomerases (3β-HSDs). Whether benzene ring BPA substitutes can inhibit human, rat, and mouse gonadal 3β-HSDs, the structure-activity relationship and the underlying mechanism remain unclear. In this study, we compared 6 benzene ring BPA substitutes to BPA in the inhibition of human, rat, and mouse gonadal 3β-HSDs and conducted structure-activity relationship and in silico docking analysis. The inhibitory activity (IC) of human 3β-HSD2 in KGN cells ranged from about 0.02 μM for bisphenol H to 8.75 μM for BPA, that of rat 3β-HSD1 in testicular microsomes ranged from 0.099 μM for bisphenol H to 31.32 μM for BPA, and that of mouse 3β-HSD6 ranged from 0.021 μM for BPH to ineffectiveness for 100 μM BPA. These compounds acted as mixed inhibitors with LogP inversely correlated with IC and ΔG positively correlated with IC value. Docking analysis showed that these compounds bind to the steroid active site of the 3β-HSD enzymes. In conclusion, some benzene ring BPA substitutes potently inhibit gonadal 3β-HSD in various species, and lipophilicity and binding affinity determine their inhibitory strength.
Topics: Rats; Mice; Humans; Animals; Benzene; Gonads; Benzhydryl Compounds; Structure-Activity Relationship
PubMed: 37703809
DOI: 10.1016/j.ecoenv.2023.115461