Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension.

Introduction: Tetrandrine (Tet) is the main pharmacological component of Stephania tetrandra S. Moore, which is a well-documented traditional Chinese medicine known for its diuretic and antihypertensive properties. Unraveling the specific targets and mechanisms of Tet involved in inducing diuresis and mitigating hypertension can provide valuable insights into its therapeutic effects. This study aimed to explore the diuretic and antihypertensive targets and mechanisms of Tet using chemical biology coupled with activity analyses in vivo and in vitro. Methods: The diuretic effects of Tet were evaluated using a water-loaded mouse model. The direct target proteins for the diuretic and antihypertensive effects of Tet were determined using chemical biology. Furthermore, the molecular mechanism of Tet binding to target proteins was analyzed using a multidisciplinary approach based on the structure and function of the proteins. Finally, the effects of the Tet-targeted protein on downstream signaling pathways and blood pressure were evaluated in hypertensive model rats. Results: Tet exhibited significant antihypertensive and potassium-preserving diuretic effects. The mechanism underlying these effects involves the modulation of the enzyme activity by covalent binding of Tet to Cys423 of CYP11A1. This interaction alters the stability of heme within CYP11A1, subsequently impeding electron transfer and inhibiting aldosterone biosynthesis. Discussion: This study not only revealed the mechanism of the diuretic and antihypertensive effects of Tet but also discovered a novel covalent inhibitor of CYP11A1. These findings contribute significantly to our understanding of the therapeutic potential of Tet and provide a foundation for future research in the development of targeted treatments for hypertension.

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.

Expression levels of the selenium-uptake receptor LRP8, the antioxidant selenoprotein GPX1 and steroidogenic enzymes correlate in granulosa cells.

Abstract: Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1-8, CAT, SODs 1 and 2, PRDXs 1-6, GSR, TXN, TXNRDs 1-3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones. Lay summary: In the ovary, eggs are housed in follicles which contain the cells that produce oestrogen in the days leading up to ovulation of the egg. Oestrogen is produced by the action of enzymes. However, some of these enzymes also produce by-products called reactive oxygen species (ROS). These are harmful to eggs. Fortunately, cells have protective antioxidant enzymes that can neutralise ROS. This study was interested in which particular antioxidant enzyme(s) might be involved in neutralising the ROS in follicle cells. It was found that only one antioxidant enzyme, GPX1, appeared to be co-regulated with the enzymes that produce oestrogen and progesterone in the follicular cells. GPX1 contains the essential mineral selenium. In summary, this study has identified which antioxidant appears to be involved in neutralising ROS in the days leading to ovulation. It highlights the importance of selenium in the diet.

Placenta-specific CYP11A1 overexpression lead to autism-like symptom in offspring with altered steroid hormone biosynthesis in the placenta-brain axis and rescued by vitamin D intervention.

Alterations in steroid hormone regulation have been implicated in the etiology and progression of autism spectrum disorders (ASD), with the enzyme cytochrome P450 family 11 subfamily A member 1 (CYP11A1)-a key catalyst in cholesterol side-chain cleavage, prominently expressed in the adrenal glands, ovaries, testes, and placenta-standing at the forefront of these investigations. The potential link between aberrations in placental Cyp11a1 expression and the resultant neurodevelopmental disorders, along with the mechanisms underpinning such associations, remains inadequately delineated. In this study, we employed a placental trophoblast-specific Cyp11a1 Hipp11 (H11) knock-in murine model to dissect the phenotypic manifestations within the placenta and progeny, thereby elucidating the underlying mechanistic pathways. Behavioral analyses revealed a diminution in social interaction capabilities alongside an augmented anxiety phenotype, as evidenced by open field and elevated plus maze assessments; both phenotypes were ameliorated after vitamin D3 supplementation. Electrophysiological assays underscored the augmented inhibition of paired-pulse facilitation, indicating impaired neuroplasticity in Cyp11a1 H11-modified mice. An elevation in progesterone concentrations was noted, alongside a significant upregulation of Th1-related cytokines (IL-6 and TNFα) across the plasma, placental, and frontal cortex-a pathological state mitigable through vitamin D3 intervention. Western blotting revealed a vitamin D-mediated rectification of vitamin D receptor and PGC-1α expression dysregulations. Immunofluorescence assays revealed microglial activation in the knock-in model, which was reversible upon vitamin D3 treatment. In conclusion, Cyp11a1 overexpression in the placenta recapitulated an autism-like phenotype in murine models, and vitamin D3 administration effectively ameliorated the resultant neurobehavioral and neuroinflammatory derangements. This study substantiates the application of Cyp11a1 as a biomarker in prenatal diagnostics and posits that prenatal vitamin D3 supplementation is a viable prophylactic measure against perturbations in steroid hormone metabolism associated with ASD pathogenesis.

Steroidogenic cytochrome P450 enzymes as drug target.

Human cytochrome P450 (CYP) enzymes are composed of 57 individual enzymes that perform monooxygenase activities. They have diverse physiological roles in metabolizing xenobiotics and producing important endogenous compounds, such as steroid hormones and vitamins. At least seven CYP enzymes are involved in steroid biosynthesis. Steroidogenesis primarily occurs in the adrenal glands and gonads, connecting each reaction to substrates and products. Steroids are essential for maintaining life and significantly contribute to sexual differentiation and reproductive functions within the body. Disorders in steroid biosynthesis can frequently cause serious health problems and lead to the development of diseases, such as prostate cancer, breast cancer, and Cushing's syndrome. In this review, we provide current updated knowledge on the major CYP enzymes involved in the biosynthetic process of steroids, with respect to their enzymatic mechanisms and clinical implications for the development of new drug candidates.

Synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells.

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/ß-catenin signaling pathway, and ß-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the ß-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Gene editing in allergic diseases: Identification of novel pathways and impact of deleting allergen genes.

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

The association of CYP11A1 gene polymorphisms with the polycystic ovary syndrome patients

SUMMARY OBJECTIVE: The objective of this study was to investigate the allele frequencies of polymorphisms in genes CYP11A1 rs4886595 and CYP11A1 rs4887139 that are responsible for the steroidogenesis mechanism in polycystic ovary syndrome patients and control females. METHODS: Samples were obtained from the Department of Obstetrics and Gynecology in the Near East University Hospital from September 2019 to December 2019. Only the nonobese patients between the ages of 18-40 years were included in this study following informed consent. Obese patients and patients more than 40 years of age were excluded from the study. Nonobese women and normal ovulation were included in the control group. DNA was isolated from blood samples. Real-time polymerase chain reaction (PCR) was used to analyze single nucleotide polymorphisms (SNPs) in various genes linked to polycystic ovary syndrome. The studies were carried out using the samples obtained from 120 women, of whom 55 were nonobese and had normal ovulation, and 65 were polycystic ovary syndrome patients. The allelic frequencies of SNPs in genes linked to polycystic ovary syndrome were calculated using real-time PCR outcomes. RESULTS: The variation of the CYP11A1 rs4887139 G>A did not show any significance, while the variation of CYP11A1 rs4886595 C>A showed significant differences between the patient and the control groups (p=0.01), respectively. CONCLUSION: Future research ought to focus on elucidating the susceptible causes of polycystic ovary syndrome with a wide range of SNPs and more sample size. The genome-wide association studies in polycystic ovary syndrome patients of different origin will be important to identify candidate genes as well as proteins that are implied in polycystic ovary syndrome risk.

Association of CYP11A1 Polymorphisms with Recurrent Pregnancy Loss in the Female Population of Punjab.

Background: Recurrent pregnancy loss (RPL) is defined as the failure of two or more clinically recognised pregnancies before 20 weeks of gestation. The prevalence of clinically evident RPL is 1%-2% worldwide. The aetiologies of RPL include uterine anatomic anomalies, uncontrolled diabetes mellitus, untreated hypothyroidism, parental chromosomal abnormalities, antiphospholipid antibody syndrome, thrombophilia, genetic abnormalities and infections. Aims: This study was aimed at investigating the possible association between CYP11A1 (rs11632698) and (rs4077582) polymorphisms with RPL in the female population of Punjab. Settings and Design: The case- control study was conducted on 170 subjects, of which 80 RPL cases and 90 controls were analysed. Materials and Methods: Genotypic analysis was performed using the polymerase chain reaction - restriction fragment length polymorphism. Statistical Analysis Used: Pearson's Chi-square test was used. Results: The genotypic frequency of CYP11A1 (rs11632698) A > G polymorphism was statistically significantly different amongst cases and controls (P = 0.00001). It was observed that the presence of the G allele might increase the risk of RPL. A Chisquare analysis of CYP11A1 (rs4077582) (P = 0.01) indicated a significant difference amongst the genotypes of cases and controls of RPL. Conclusion: CYP11A1 variants (rs11632698 and rs4077582) may be useful markers in determining the genetic susceptibility to the pathogenesis of RPL. Keywords: CYP11A1, recurrent miscarriage, recurrent pregnancy loss, rs11632698, rs4077582, spontaneous abortion.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

Elemental evaluation, nutritional analysis, GC-MS analysis and ameliorative effects of Artocarpus communis J.R.Forst. & G.Forst. seeds' phytoconstituents on metabolic syndrome via in silico approach.

The nutritional as well as beneficial effects of the Artocarpus communis seed on metabolic syndrome complications have not been studied. In this research, the aim was to investigate the nutritional composition and beneficial effects of Artocarpus communis seeds' phytoconstituents on the p53 core, fat mass and obesity-associated (FTO) protein and cytochrome P450 CYP11A1 domains. The elements and phytochemicals in the seed were determined through atomic absorption spectroscopy assay and gas chromatography-mass spectrometry (GC-MS) analysis, respectively. Also, the compounds detected were docked to the p53 core, FTO protein and cytochrome P450 CYP11A1 domains protein. Artocarpus communis seed contains sodium (7.824 ± 0.0134 ppm), magnesium (10.187 ± 0.0239 ppm) and iron (1.924 ± 0.0017), while zinc and cadmium were undetected. Phenolics and flavonoids were the most abundant phytochemicals in the seed. Phytoconstituents, such as pentadecanoic acid, hexadecanoic acid and methyl ester, possessing different therapeutic effects were identified via GC-MS analysis. In A. communis seed, 3-methyl-4-nitro-5-(1-pyrazolyl) pyrazole and phenanthrene were able to bind more peculiarly and specifically to the p53 core, FTO protein and cytochrome P450 CYP11A1 domains. One of the important processes that were hypothesized for the recovery of metabolic syndrome in affected victims is shown by the molecular dynamics analysis, which shows that the binding of these chemicals to the targeted structure stabilized the proteins. Therefore, Artocarpus communis seeds could be a new strategy for the management of metabolic syndrome.Communicated by Ramaswamy H. Sarma.

The protective effects of date seeds, in either conventional or nanoformulation, against bisphenol A-induced testicular toxicity: involvement of testicular expression of CYP11A1, Nrf-2 and Bax/Bcl-2 ratio.

Background: Bisphenol A (BPA), an endocrine-disrupting chemical (EDC), is ubiquitous in our environment and poses a significant threat to male fertility. Date seeds (DSs) are used in folk medicine due to their antioxidant activity. Aim: The purpose of this study was to assess the beneficial effects of DSs, whether in powder or nanoparticle form, against BPA-induced testicular oxidative challenges and apoptosis, aided by inspection of specific genes linked to fertility, oxidative stress and intrinsic mitochondrial pathway of apoptosis. Methods: Thirty-five adult male albino rats were equally divided into 5 groups including control, BPA, BPA + date seeds powder "DSP", BPA + date seed nanoparticle 1/10 (DSNP 1/10) and BPA + DSNP 1/20 groups. Results: TEM showed that the ball-mill method was effective to form DSNP with an average size of 20 nm. BPA significantly impaired sperm motility, morphology, viability and concentration. It also reduced serum testosterone levels and evoked marked oxidative stress in the testes. Additionally, serum levels of triiodothyronine and thyroxine were extremely reduced. Moreover, testicular mRNA relative expression levels of CYP11A1 and Nrf-2 were markedly downregulated. Testicular apoptosis was also promoted whereas Bax/Bcl-2 ratio was profoundly elevated. Histological pictures of the testes, epididymis, seminal vesicles and prostate confirmed the unfavorable effects of BPA. Surprisingly, we first demonstrated that DSs, specifically the nanoparticle form, strongly alleviated all of BPA's negative effects, with DSNP 1/20 achieving the best results. Conclusion: Therefore, DSNP in both doses could be regarded as an ideal candidate for abating the male reproductive challenges caused by BPA.

Screening of Natural Compounds for CYP11A1 Stimulation Against Cell Renal Cell Carcinoma.

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 (IC50) were determined. The IC50 of 159 compounds was determined and 38 compounds with IC50 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 IC50 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.

Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization.

Six cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.

FXR Maintains the Intestinal Barrier and Stemness by Regulating CYP11A1-Mediated Corticosterone Synthesis in Biliary Obstruction Diseases.

Biliary obstruction diseases are often complicated by an impaired intestinal barrier, which aggravates liver injury. Treatment of the intestinal barrier is often neglected. To investigate the mechanism by which intestinal bile acid deficiency mediates intestinal barrier dysfunction after biliary obstruction and identify a potential therapeutic modality, we mainly used a bile duct ligation (BDL) mouse model to simulate biliary obstruction and determine the important role of the bile acid receptor FXR in maintaining intestinal barrier function and stemness. Through RNA-seq analysis of BDL and sham mouse crypts and qRT-PCR performed on intestinal epithelial-specific Fxr knockout (FxrΔIEC) and wild-type mouse crypts, we found that FXR might maintain intestinal stemness by regulating CYP11A1 expression. Given the key role of CYP11A1 during glucocorticoid production, we also found that FXR activation could promote intestinal corticosterone (CORT) synthesis by ELISA. Intestinal organoid culture showed that an FXR agonist or corticosterone increased crypt formation and organoid growth. Further animal experiments showed that corticosterone gavage treatment could maintain intestinal barrier function and stemness, decrease LPS translocation, and attenuate liver injury in BDL mice. Our study hopefully provides a new theoretical basis for the prevention of intestinal complications and alleviation of liver injury after biliary obstruction.

VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression.

BACKGROUND: The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis.VDR is widely expressed in testis cells such as Leydig cells, Sertoli cells, and sperm. The levels of steroids are critical for sexual development. In the early stage of steroidogenesis, cholesterol is converted to pregnenolone (precursor of most steroid hormones) by cholesterol side-chain lyase (CYP11A1), which eventually synthesizes the male hormone testosterone. OBJECTIVE: This study aims to reveal how VDR regulates CYP11A1 expression and affects testosterone synthesis in murine Leydig cells. METHODS: The levels of VDR, CYP11A1 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Targeted relationship between VDR and Cyp11a1 was evaluated by dual-luciferase reporter assay. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). RESULTS: Phylogenetic and motif analysis showed that the Cyp11a1 family had sequence loss, which may have special biological functions during evolution. The results of promoter prediction showed that vitamin D response element (VDRE) existed in the upstream promoter region of murine Cyp11a1. Dual-luciferase assay confirmed that VDR could bind candidate VDREs in upstream region of Cyp11a1, and enhance gene expression. Tissue distribution and localizatio analysis showed that Cyp11a1 was mainly expressed in testis, and dominantly existed in murine Leydig cells. Furthermore, over-expression VDR and CYP11A1 significantly increased testosterone synthesis in mice Leydig cells. CONCLUSIONS: Active vitamin D3 (VD3) and Vdr interference treatment showed that VD3/VDR had a positive regulatory effect on Cyp11a1 expression and testosterone secretion. VDR promotes testosterone synthesis in male mice by up-regulating Cyp11a1 expression, which played an important role for male reproduction.

Placental DNA methylation analysis of selective fetal growth restriction in monochorionic twins reveals aberrant methylated CYP11A1 gene for fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased susceptibility to perinatal morbidity and mortality. Evidence suggests that epigenetic changes play critical roles in the regulation of fetal growth. We sought to present a comprehensive analysis of the associations between placental DNA methylation and selective fetal growth restriction (sFGR), which is a severe complication of monochorionic twin pregnancies, characterized by one fetus experiencing restricted growth. Genome-wide methylation analysis was performed on 24 placental samples obtained from 12 monochorionic twins with sFGR (Cohort 1) using Illumina Infinium MethylationEPIC BeadChip. Integrative analysis of our EPIC data and two previous placental methylation studies of sFGR (a total of 30 placental samples from 15 sFGR twins) was used to identify convincing differential promoter methylation. Validation analysis was performed on the placentas from 15 sFGR twins (30 placental samples), 15 FGR singletons, and 14 control singletons (Cohort 2) using pyrosequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry (IHC). A globe shift toward hypomethylation was identified in the placentas of growth-restricted fetuses compared with the placentas of normal fetuses in monochorionic twins, including 5625 hypomethylated CpGs and 452 hypermethylated CpGs, especially in the regions of CpG islands, gene-body and promoters. The analysis of pathways revealed dysregulation primarily in steroid hormone biosynthesis, metabolism, cell adhesion, signaling transduction, and immune response. Integrative analysis revealed a differentially methylated promoter region in the CYP11A1 gene, encoding a rate-limiting enzyme of steroidogenesis converting cholesterol to pregnenolone. The CYP11A1 gene was validated to have hypomethylation and higher mRNA expression in sFGR twins and FGR singletons. In conclusion, our findings suggested that the changes in placental DNA methylation pattern in sFGR may have functional implications for differentially methylated genes and regulatory regions. The study provides reliable evidence for identifying abnormally methylated CYP11A1 gene in the placenta of sFGR.

Exploring the impact of CYP11A1's missense SNPs on the interaction between CYP11A1 and cholesterol: A comprehensive structural analysis and MD simulation study.

The process of steroidogenesis plays a vital role in human physiology as it governs the biosynthesis of mineralocorticoids, glucocorticoids, and androgens. These three classes of steroid hormones are primarily produced in the adrenal and gonadal glands through steroidogenesis pathways. Initiated by the side chain cleavage of cholesterol (CLR), this process leads to the conversion of cholesterol into pregnenolone and isocaproic aldehyde. The enzyme CYP11A1, encoded by the CYP11A1 gene, plays a key role in catalyzing the side chain cleavage of CLR. Several single nucleotide polymorphisms (SNPs) have been identified in the CYP11A1 gene, which may predispose carriers to disorders associated with abnormal steroidogenesis. Specifically, missense SNPs in the CYP11A1 gene have the potential to negatively impact the interaction between CYP11A1 and CLR, thus affecting the overall metabolome of steroid hormones. In this computational study, we focused on a specific set of missense SNPs reported in the CYP11A1 gene, aiming to identify variants that directly impact the interaction between CYP11A1 and CLR. The three-dimensional structure of the CYP11A1-CLR complex was obtained from the RCSB Protein Data Bank, while missense SNPs in the CYP11A1 gene were retrieved from Ensembl. To predict the most deleterious variants, we utilized the ConSurf server, SIFT, and PolyPhen. Furthermore, we assessed the impact of induced amino acid (AA) substitutions on the CYP11A1-CLR interaction using the PRODIGY server, PyMol, and Ligplot programs. Additionally, molecular dynamics (MD) simulations were conducted to analyze the effects of deleterious variants on the structural dynamics of the CYP11A1-CLR complex. Among the 8096 retrieved variants, we identified ten missense SNPs (E91K, W147G, R151W, R151Q, S391C, V392M, Q395K, Q416E, R460W, and R460Q) as deleterious for the interaction between CYP11A1 and CLR. MD simulations of the CYP11A1-CLR complexes carrying these deleterious AA substitutions revealed that Q416E, W147G, R460Q, and R460W had the most pronounced impacts on the structural dynamics of the complex. Consequently, these missense SNPs were considered the most deleterious ones. Further functional tests are recommended to assess the impact of these four missense SNPs on the enzymatic activity of CYP11A1. Moreover, Genome-Wide Association Studies (GWAS) should be conducted to determine the significance of their association with abnormal steroidogenesis diseases in various patient groups.