Biomedical subjects

Bryan Goodwin

Publications and source records attributed to Bryan Goodwin.

28 records · Page 2Linked to original sources

PubMed2002

Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha.

In rodent liver, transcription of the gene encoding cholesterol 7alpha-hydroxylase (CYP7A1), which catalyzes the rate-limiting step in the classic bile acid synthetic pathway, is stimulated by the liver X receptor alpha (LXRalpha), a nuclear receptor for oxysterol metabolites of cholesterol. This feed-forward regulatory loop provides a mechanism for the elimination of excess cholesterol from the body. In this report, we demonstrate that in primary cultures of human hepatocytes, activation of LXRalpha has the opposite effect, repressing CYP7A1 expression. This repression is mediated, at least in part, through induction of the orphan nuclear receptor, short heterodimer partner (SHP), which is also induced by bile acids. We demonstrate that SHP is regulated directly by LXRalpha through a DNA response element that overlaps with the previously characterized bile acid response element. Our data reveal a fundamental difference in the regulation of CYP7A1 in rodent and human hepatocytes and provide evidence that different species employ distinct molecular strategies to regulate cholesterol homeostasis.

Animals↗

PubMed2002

Regulation of hepatic drug metabolism: role of the nuclear receptors PXR and CAR.

Recent advances in the molecular biology of nuclear receptors have revealed that the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are able to act as sensors for lipophilic xenobiotics, including therapeutic drugs. These receptors in turn regulate enzymes and transporters involved in drug metabolism and disposition in an adaptive fashion. An unexpected finding was that the PXR was able to recognize bile acids; transgenic animals lacking this receptor are at increased risk of bile acid-induced liver injury. These findings provide new insights into hepatic drug metabolism as well as mechanisms regulating cholesterol and bile acid homeostasis.

Animals↗

PubMed2002

The CYP3A4*1B polymorphism has no functional significance and is not associated with risk of breast or ovarian cancer.

CYP3A4 is involved in the metabolism of endogenous steroids, and an allelic variant, CYP3A4*1B, consisting of an A to G polymorphism within the 5'-flanking region termed the nifedipine-specific response element (NFSE) has been associated with high grade and advanced stage of prostate cancers. Because steroid hormone exposure is known to influence breast and ovarian cancer risk, we conducted case-control studies to assess the relationship between CYP3A4*1B and risk of breast or ovarian cancer. CYP3A4 NFSE genotype was determined in 951 breast cancer cases and 500 controls frequency matched for age and 488 ovarian cancer cases and 276 controls of similar age distribution. Case-control analyses and comparisons of genotype distributions were conducted by unconditional logistic regression. In addition, the functional significance of the CYP3A4*1B polymorphism was assessed by analysis of CYP3A4-reporter gene constructs transiently transfected into liver-derived cell lines and primary cultures of well-differentiated rat hepatocytes. The GG genotype was rare in all groups (0-0.4%). There was no risk of cancer associated with the AG/GG genotypes combined, with an OR (95% CI) of 0.86 (0.54-1.33) for breast cancer (P = 0.5), and 1.51 (0.80-2.89) for ovarian cancer (P = 0.2). Analysis of CYP3A4-luciferase constructs showed that CYP3A4*1B did not consistently affect reporter gene activity. Our data suggest that the CYP3A4*1B polymorphism is not associated with risk of breast or ovarian cancer. In support of this negative finding, in-vitro functional studies indicate that NFSE genotype is not a critical factor in the transcriptional activity of the CYP3A4 5'-flanking region, and is thus unlikely to modulate CYP3A4-mediated metabolism of steroids.

5' Untranslated Regions↗

PubMed2002

Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor.

Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in adult human liver, is both constitutively expressed and transcriptionally activated by a variety of structurally diverse xenochemicals. In this study, we examined the role of the constitutive androstane receptor (CAR), a member of the steroid/retinoid/thyroid hormone receptor superfamily, in the transcriptional regulation of CYP3A4. Herein, we demonstrate that CAR is capable of trans-activating expression of the CYP3A4 gene, both in vitro and in vivo. Induction of CYP3A4 is dependent on cooperativity between elements within the promoter proximal region of the gene and the distal xenobiotic-responsive enhancer module. CAR responsiveness was shown to be primarily mediated by two high-affinity binding motifs located within the CYP3A4 gene 5'-flanking region, approximately 7720 and 150 bases upstream of the transcription initiation site. Importantly, the human CAR response elements also mediate trans-activation of CYP3A4 by the human pregnane X receptor, suggesting that interplay between these receptors is likely to be an important determinant of CYP3A4 expression.

Constitutive Androstane Receptor↗

PubMed2002

Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification.

The nuclear pregnane X receptor (PXR) and constitutive androstane receptor (CAR) play central roles in protecting the body against environmental chemicals (xenobiotics). PXR and CAR are activated by a wide range of xenobiotics and regulate cytochrome P450 and other genes whose products are involved in the detoxification of these chemicals. In this report, we have used receptor-selective agonists together with receptor-null mice to identify PXR and CAR target genes in the liver and small intestine. Our results demonstrate that PXR and CAR regulate overlapping but distinct sets of genes involved in all phases of xenobiotic metabolism, including oxidative metabolism, conjugation, and transport. Among the murine genes regulated by PXR were those encoding PXR and CAR. We provide evidence that PXR regulates a similar program of genes involved in xenobiotic metabolism in human liver. Among the genes regulated by PXR in primary human hepatocytes were the aryl hydrocarbon receptor and its target genes CYP1A1 and CYP1A2. These findings underscore the importance of these two nuclear receptors in defending the body against a broad array of potentially harmful xenobiotics.

Animals↗

PubMed2002

Regulation of cyp3a gene transcription by the pregnane x receptor.

The pregnane X receptor (PXR) is a promiscuous nuclear receptor that has evolved to protect the body from toxic chemicals. PXR is activated by a structurally diverse collection of xenobiotics, including several widely used prescription drugs. Various lipophilic compounds produced by the body, such as bile acids and steroids, also activate PXR. PXR stimulates the transcription of cytochrome P450 3A monooxygenases and other genes involved in the detoxification and elimination of these potentially harmful chemicals. Assays that detect PXR activation have important implications for the design of future drugs in two respects. On the one hand, PXR activation assays can be used to determine whether candidate drugs are likely to induce CYP3A gene expression and interact with other medicines. On the other hand, PXR agonists may prove useful in the treatment of diseases in which toxic metabolites accumulate, such as cholestatic liver disease.

Animals↗

PubMed2002

Nuclear receptors. I. Nuclear receptors and bile acid homeostasis.

Bile acids are required for the absorption of lipids and fat-soluble vitamins. The hepatic biosynthesis of bile acids is a major pathway for the catabolism and removal of cholesterol from the body. Because of their intrinsic toxicity, bile acid synthesis, transport, and metabolism must be tightly regulated. It is now apparent that members of the nuclear receptor family of lipid-activated transcription factors are key regulators of these physiological processes. A greater understanding of these receptors should afford novel opportunities for therapeutic intervention in chronic diseases such as cholestasis and dyslipidemia.

Animals↗

PubMed2002

The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism.

The nuclear pregnane X receptor (PXR; NR1I2) is an important component of the body's adaptive defense mechanism against toxic substances including foreign chemicals (xenobiotics). PXR is activated by a large number of endogenous and exogenous chemicals including steroids, antibiotics, antimycotics, bile acids, and the herbal antidepressant St. John's wort. Elucidation of the three-dimensional structure of the PXR ligand binding domain revealed that it has a large, spherical ligand binding cavity that allows it to interact with a wide range of hydrophobic chemicals. Thus, unlike other nuclear receptors that interact selectively with their physiological ligands, PXR serves as a generalized sensor of hydrophobic toxins. PXR binds as a heterodimer with the 9-cis retinoic acid receptor (NR2B) to DNA response elements in the regulatory regions of cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and elimination of xenobiotics from the body. Although PXR evolved to protect the body, its activation by a variety of prescription drugs represents the molecular basis for an important class of harmful drug-drug interactions. Thus, assays that detect PXR activity will be useful in developing safer prescription drugs.

Amino Acid Sequence↗

PubMed2002

Nuclear receptor regulation of genes involved in bile acid metabolism.

Over the past few decades, the nuclear receptor superfamily of transcription factors has provided many opportunities for therapeutic intervention in a variety of pathophysiological conditions. A major question now is how many additional drug targets can be identified within this class of proteins. Advances in differential gene expression (DGE) technologies allow powerful new approaches to answer this question. Although DGE analysis is but one of several modern approaches to target identification, it is especially pertinent for nuclear receptors because many of these proteins are ligand-regulated transcription factors that directly alter gene expression. This article will focus on recent DGE experiments that have elucidated the physiological role of nuclear receptors in regulation of the bile acid biosynthesis, transport, and metabolism in the liver. Of particular relevance is the role of nuclear receptors in regulating CYP7A1 because this gene encodes the enzyme controlling the first and rate-limiting step in cholesterol degradation to bile acids. Modulation of CYP7A1 expression could provide a therapeutic benefit for multiple disease conditions. This article will focus on the relative contributions of farnesoid X-activated receptor, liver receptor homologous protein-1, small heterodimer partner, pregnane X receptor, and liver X receptor to the regulation of this specific gene, and bile acid metabolism in general. These studies highlight the dynamic interplay between nuclear receptors in the regulation of specific metabolic pathways.

Bile Acids and Salts↗

PubMed2001

Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor.

The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi. We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4). MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands. In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels. The dexamethasone- and pregnenolone 16alpha-carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice. In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice. An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8). PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor alpha (RXRalpha, NR2B1). Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells. Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner. Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter. Mutation of the ER-8 element abolished the nuclear receptor response. These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5'-flanking region of this gene.

Animals↗