Transient receptor potential canonical 5 channels plays an essential role in hepatic dyslipidemia associated with cholestasis.

Transient receptor potential canonical 5 (TRPC5), a calcium-permeable, non-selective cation channel is expressed in the periphery, but there is limited knowledge of its regulatory roles in vivo. Endogenous modulators of TRPC5 include a range of phospholipids that have an established role in liver disease, including lysophosphatidylcholine (LPC). Cholestasis is characterized by impairment of excretion of bile acids, leading to elevation of hepatic bile acids. We investigated the contribution of TRPC5 in a murine model of cholestasis. Wild-type (WT) and TRPC5 knock-out (KO) mice were fed a diet supplemented with 0.5% cholic acid (CA) for 21 days. CA-diet supplementation resulted in enlargement of the liver in WT mice, which was ameliorated in TRPC5 KO mice. Hepatic bile acid and lipid content was elevated in WT mice, with a reduction observed in TRPC5 KO mice. Consistently, liver enzymes were significantly increased in cholestatic WT mice and significantly blunted in TRPC5 KO mice. Localized dyslipidaemia, secondary to cholestasis, was investigated utilizing a selected lipid analysis. This revealed significant perturbations in the lipid profile following CA-diet feeding, with increased cholesterol, triglycerides and phospholipids, in WT, but not TRPC5 KO mice. Our results suggest that activation of TRPC5 contributes to the development of cholestasis and associated dyslipidemia. Modulation of TRPC5 activity may present as a novel therapeutic target for liver disease

Arachidonic acid-dependent gene regulation during preadipocyte differentiation controls adipocyte potential

Arachidonic acid (AA) is a major PUFA that has been implicated in the regulation of adipogenesis. We examined the effect of a short exposure to AA at different stages of 3T3-L1 adipocyte differentiation. AA caused the upregulation of fatty acid binding protein 4 (FABP4/aP2) following 24 h of differentiation. This was mediated by the prostaglandin F2α (PGF2α), as inhibition of cyclooxygenases or PGF2α receptor signaling counteracted the AA-mediated aP2 induction. In addition, calcium, protein kinase C, and ERK are all key elements of the pathway through which AA induces the expression of aP2. We also show that treatment with AA during the first 24 h of differentiation upregulates the expression of the transcription factor Fos-related antigen 1 (Fra-1) via the same pathway. Finally, treatment with AA for 24 h at the beginning of the adipocyte differentiation is sufficient to inhibit the late stages of adipogenesis through a Fra-1-dependent pathway, as Fra-1 knockdown rescued adipogenesis. Our data show that AA is able to program the differentiation potential of preadipocytes by regulating gene expression at the early stages of adipogenesis

Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis

Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis, it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification, and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and Lxrab-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol, and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis that is also necessary to protect against abnormalities in fetoplacental lipid homeostasis

Enhanced microbial bile acid deconjugation and impaired ileal uptake in pregnancy repress intestinal regulation of bile acid synthesis

Pregnancy is associated with progressive hypercholanemia, hypercholesterolemia, and hypertriglyceridemia, which can result in metabolic disease in susceptible women. Gut signals modify hepatic homeostatic pathways, linking intestinal content to metabolic activity. We sought to identify whether enteric endocrine signals contribute to raised serum bile acids observed in human and murine pregnancies, by measuring fibroblast growth factor (FGF) 19/15 protein and mRNA levels, and 7α-hydroxy-4-cholesten-3-one. Terminal ileal farnesoid X receptor (FXR)-mediated gene expression and apical sodium bile acid transporter (ASBT) protein concentration were measured by qPCR and western blotting. Shotgun whole-genome sequencing and ultra-performance liquid chromatography tandem mass spectrometry were used to determine the cecal microbiome and metabonome. Targeted and untargeted pathway analyses were performed to predict the systemic effects of the altered metagenome and metabolite profiles. Dietary CA supplementation was used to determine whether the observed alterations could be overcome by intestinal bile acids functioning as FXR agonists. Human and murine pregnancy were associated with reduced intestinal FXR signaling, with lower FGF19/15 and resultant increased hepatic bile acid synthesis. Terminal ileal ASBT protein was reduced in murine pregnancy. Cecal bile acid conjugation was reduced in pregnancy because of elevated bile salt hydrolase-producing Bacteroidetes. CA supplementation induced intestinal FXR signaling, which was not abrogated by pregnancy, with strikingly similar changes to the microbiota and metabonome as identified in pregnancy. Conclusion: The altered intestinal microbiota of pregnancy enhance bile acid deconjugation, reducing ileal bile acid uptake and lowering FXR induction in enterocytes. This exacerbates the effects mediated by reduced bile acid uptake transporters in pregnancy. Thus, in pregnant women and mice, there is reduced FGF19/15-mediated hepatic repression of hepatic bile acid synthesis, resulting in hypercholanemia

The effect of maternal undernutrition on the rat placental transcriptome: protein restriction up-regulates cholesterol transport

Fetal exposure to a maternal low protein diet during rat pregnancy is associated with hypertension, renal dysfunction and metabolic disturbance in adult life. These effects are present when dietary manipulations target only the first half of pregnancy. It was hypothesised that early gestation protein restriction would impact upon placental gene expression and that this may give clues to the mechanism which links maternal diet to later consequences. Pregnant rats were fed control or a low protein diet from conception to day 13 gestation. Placentas were collected and RNA Sequencing performed using the Illumina platform. Protein restriction down-regulated 67 genes and up-regulated 24 genes in the placenta. Ingenuity pathway analysis showed significant enrichment in pathways related to cholesterol and lipoprotein transport and metabolism, including atherosclerosis signalling, clathrin-mediated endocytosis, LXR/RXR and FXR/RXR activation. Genes at the centre of these processes included the apolipoproteins ApoB, ApoA2 and ApoC2, microsomal triglyceride transfer protein (Mttp), the clathrin-endocytosis receptor cubilin, the transcription factor retinol binding protein 4 (Rbp4) and transerythrin (Ttr; a retinol and thyroid hormone transporter). Real-time PCR measurements largely confirmed the findings of RNASeq and indicated that the impact of protein restriction was often striking (cubilin up-regulated 32-fold, apoC2 up-regulated 17.6-fold). The findings show that gene expression in specific pathways is modulated by maternal protein restriction in the day-13 rat placenta. Changes in cholesterol transport may contribute to altered tissue development in the fetus and hence programme risk of disease in later life

Ovarian cancer molecular pathology.

Peer reviewe

Steroid signalling in the human ovarian surface epithelium wound healing

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Steroid signalling in the human ovarian surface epithelium wound healing

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo