Inhaled corticosteroids and the increased risk of pulmonary tuberculosis: a population-based case-control study.

AIMS: The association between inhaled corticosteroid (ICS) use and pulmonary tuberculosis (TB) development is uncertain. We conducted a population-based case-control study to investigate whether ICS use increases the risk of developing TB. METHODS: Tuberculosis patients aged 18 years and older were identified using the National Health Insurance Research Database (NHIRD) in Taiwan between 2002 and 2010. Each TB patient was frequency matched to four control patients according to age, sex and index year. We retrospectively followed up the medications and comorbid medical conditions for the 5 years prior to the index date. We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) of TB development using multiple logistic regression models. RESULTS: Most of the study participants were men (68.7%), and the mean age among the 8091 TB patients and 32,364 comparison participants was 61.3 ± 18.6 years. After adjusting for potential covariates, ICS use caused a 2.04-fold increased risk of developing TB (adjusted OR: 2.04, 95% CI: 1.78-2.33). When considering dose-response and adjusting for potential covariates, ICS and oral corticosteroids (OCS) use remained independent risk factors and exhibited a dose-response relationship of TB development. The multiplicative increased risk of TB was also significant in patients using ICS and OCS compared with patients not using ICS and OCS (adjusted OR: 4.31, 95% CI: 3.39-5.49). Previous TB history exhibited the greatest risk of TB development among the comorbidities (adjusted OR: 8.50, 95% CI: 7.52-9.61). CONCLUSION: Long-term ICS use may increase the risk of TB.

Complementary and alternative medicine (CAM) usage in Singaporean adult cancer patients.

BACKGROUND: In multiracial and multicultural Singapore, patients are exposed to complementary and alternative medicine (CAM) from both eastern and western cultures. Although studies have shown that CAM usage is highly prevalent among cancer patients, no study on the prevalence of CAM in Singaporean adult cancer patients had been published. PATIENTS AND METHODS: 403 adult cancer patients treated at the Ambulatory Treatment Unit of National Cancer Centre Singapore completed an interviewer-administered questionnaire. RESULTS: Median age of patients was 56 years old (range 22-84). Fifty-six percent of patients reported CAM usage and the most commonly used CAM include Traditional Chinese Medicine, bird's nest and special diet. CAM use was found to be associated with race, education level and prior CAM use before cancer diagnosis. Fifty-four percent of respondents informed their oncologists regarding CAM usage and 66.4% of oncologists were agreeable for CAM usage. However, most patients (63%) did not verify information on CAM before usage and a majority of patients taking CAM felt it was effective. CONCLUSION: Majority of adult cancer patients used CAM and it is important for health-care professionals to keep abreast of research on CAM, to actively illicit information regarding usage and to provide appropriate advice and counseling.

Bile synthesis in rat models of inflammatory bowel diseases.

BACKGROUND: A broad spectrum of hepatobiliary disorders are found in patients with inflammatory bowel diseases. The aim of the present work was to study interactions between gut and liver in experimental rat models of colitis and small bowel inflammation. MATERIALS AND METHODS: Colitis was induced either by trinitrobenzene sulphonic acid or dextran sodium sulphate. Small-bowel inflammation was induced by indomethacin. Bile acid secretion, bile acid pool, and cholesterol 7-alpha hydroxylase were studied. Cholesterol 7-alpha hydroxylase protein expression was analysed in the microsomal liver fraction. As portal mediators released form the inflamed gut we measured lipopolysaccharide, tumour necrosis factor-alpha and interleukin-1beta in portal serum. The hepatic inflammatory response was evaluated by binding activity of nuclear factor-kappaB, activator protein-1 and alpha-2-macroglobulin. RESULTS: Increased bile acid secretion, total bile acid content in gut and liver (bile acid pool size), and hepatic cholesterol 7-alpha hydroxylase protein and mRNA levels were found in the two colitis models associated with only a minor hepatic acute phase and cytokine response. In contrast, during indomethacin-induced small-bowel inflammation bile acid secretion, pool size, and cholesterol 7-alpha hydroxylase decreased in parallel to a strong hepatic cytokine and acute phase response. CONCLUSIONS: Colitis without portal cytokine release and acute phase reaction shows an induction of bile acid secretion, pool size, and cholesterol 7-alpha hydroxylase. In contrast, intestinal inflammation after indomethacin treatment is associated with an acute phase response and a repression of bile acid synthesis.

A pollen-specific polygalacturonase from lily is related to major grass pollen allergens.

A pollen-specific gene from lily (Lilium longiflorum Thunb. cv. Snow Queen), designated LLP-PG, was characterized. Southern blots of lily genomic DNA indicated that LLP-PG is a member of a small gene family. A thorough sequence analysis revealed that the LLP-PG gene is interrupted by two introns and encodes a protein of 413 amino acids, with a calculated molecular mass of 44 kDa, and a pI of 8.1. Evaluation of the hydropathy profile showed that the protein has a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. A sequence similarity search showed a significant homology of the encoded protein to pollen polygalacturonases (PGs) from various plant species and to an important group (group 13) of grass pollen allergens. The LLP-PG transcript is pollen-specific and it accumulates only at the latest stage during pollen development, in the mature pollen. In contrast to other "late genes" LLP-PG transcript can neither be induced by abscisic acid (ABA) nor by dehydration. Immunoblot analyses of pollen protein extracts from lily, timothy grass and tobacco with IgG antibodies directed against LLP-PG and against the timothy grass pollen allergen, Phl p 13, indicated that lily LLP-PG shares surface-exposed epitopes with pollen PGs from monocotyledonous and dicotyledonous plants. Enzyme-linked immunosorbent assay (ELISA) analyses and inhibition ELISA assays with patients' IgE demonstrated a very low IgE reactivity of lily rLLP-PG and a lack of cross-reactivity between rLLP-PG and the timothy grass pollen allergen, rPhl p 13. These data demonstrated that despite the significant sequence homology and the conserved surface-exposed epitopes LLP-PG represents a low-allergenic member of pollen PGs.

Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression.

Sterol 12alpha-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5'-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from -57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7alpha-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4alpha- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4alpha-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4alpha. Mammalian two-hybrid assay confirmed that HNF4alpha interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4alpha in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4alpha gene transcription. In summary, our data revealed the critical roles HNF4alpha play on CYP8B1 transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4alpha through interaction of HNF4alpha with SHP and reduction of HNF4alpha expression in the liver.

Nuclear receptor-mediated repression of human cholesterol 7alpha-hydroxylase gene transcription by bile acids.

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated alpha-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species.

Transcriptional regulation of human oxysterol 7 alpha-hydroxylase gene (CYP7B1) by Sp1.

Oxysterol 7 alpha-hydroxylase catalyzes hydroxylation of oxysterols and neurosterols and plays a role in the alternative bile acid synthesis pathway. This gene is widely expressed in many organs and peripheral tissues and may protect tissues from the toxicity of oxysterols. Mutation in CYP7B1 caused neonatal cholestasis. To examine the regulatory mechanisms governing CYP7B1 expression, the 5' flanking sequence of the CYP7B1 was analyzed and revealed a CpG island of about 1.2 kb. Transient transfection assays of deletion mutants of the CYP7B1 promoter-luciferase reporter gene in human liver-derived HepG2, fibroblast NT1088, and human embryonic kidney 293 cell lines revealed that the region from -291 to +189 was critical for gene transcription. Three GC box sequences located between -25 and +10 were essential for basal transcription because mutations of these sequences markedly reduced promoter activity. Sp1 and Sp3 bound to these sequences as demonstrated by DNase I footprinting assays and electrophoretic mobility shift assay. Thus, regulation of CYP7B1 transcription by Sp1 may play a pivotal role in regulating oxysterol levels, which regulate cholesterol metabolism.

Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha).

The cholesterol 7alpha-hydroxylase gene (CYP7A1) plays an important role in regulation of bile acid biosynthesis and cholesterol homeostasis. Oxysterol receptor, LXR, stimulates, whereas the bile acid receptor, FXR, inhibits CYP7A1 transcription. The goal of this study was to investigate the role of LXRalpha on the regulation of rat, human and hamster CYP7A1 transcription in its native promoter and cellular context. Cotransfection with LXRalpha and RXRalpha expression plasmids strongly stimulated rat CYP7A1/luciferase reporter activity in HepG2 cells and oxysterol was not required. However, LXRalpha had much less effect on hamster and no significant effect on human CYP7A1 promoter activity in HepG2 cells. In Chinese hamster ovary cells, cotransfection with LXRalpha stimulated reporter activity by less than 2-fold and addition of 22(R)-hydroxycholesterol caused a small but significant stimulation of rat, human and hamster CYP7A1 promoter activity. At least two direct repeats of AGGTCA-like sequences with 4-base spacing (DR4) and five-base spacing (DR5), in previously identified bile acid response elements of the rat CYP7A1 were able to bind LXRalpha/RXRalpha and confer LXRalpha stimulation. However, LXRalpha did not bind to the corresponding sequences of the human gene and bound weakly to hamster and mouse DR4 sequences. Therefore, rats and mice have the unusual capacity to convert cholesterol to bile acids by LXRalpha-mediated stimulation of CYP7A1 transcription, whereas other species do not respond to cholesterol and develop hypercholesterolemia on a diet high in cholesterol.

Peroxisome proliferator-activated receptor alpha (PPARalpha) and agonist inhibit cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription.

Fibrates are widely used hypolipidemic drugs that regulate the expression of many genes involved in lipid metabolism by activating the peroxisome proliferator-activated receptor alpha (PPARalpha). The objective of this study was to investigate the mechanism of action of peroxisome proliferators and PPARalpha on the transcription of cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. When cotransfected with the expression vectors for PPARalpha and RXRalpha, Wy14,643 reduced human and rat cholesterol 7alpha-hydroxylase gene (CYP7A1)/luciferase reporter activities by 88% and 43%, respectively, in HepG2 cells, but not in CV-1 or CHO cells. We have mapped the peroxisome proliferator response element (PPRE) to a conserved sequence containing the canonical AGGTCA direct repeats separated by one nucleotide (DR1). This DR1 sequence was mapped previously as a binding site for the hepatocyte nuclear factor 4 (HNF-4) which stimulates CYP7A1 transcription. Electrophoretic mobility shift assay (EMSA) showed no direct binding of in vitro synthesized PPARalpha/RXRalpha heterodimer to the DR1 sequence. PPARalpha and Wy14,643 did not affect HNF-4 binding to the DR1. However, Wy14,643 and PPARalpha/RXRalpha significantly reduced HNF-4 expression in HepG2 cells. These results suggest that PPARalpha and agonist repress cholesterol 7alpha-hydroxylase activity by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore attenuates the transactivation of CYP7A1 by HNF-4.

HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1).

The gene for cholesterol 7alpha-hydroxylase (CYP7A1) contains a sequence at nt -149 to -118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter. Hepatocyte nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) synergistically activate transcription of the CYP7A1 promoter. Transactivation of CYP7A1 by HNF4 in the human hepatoma cell line, HepG2, was enhanced by cotransfection with COUP-TFII or the basal transcription element binding protein (BTEB). HNF4 prepared from rat liver nuclear extracts bound to oligomers homologous to the nt -146 to -134 sequences in electrophoretic mobility shift assays (EMSA), which corresponded to a conserved region containing a direct repeat of hormone response elements spaced by one nucleotide (DR1). The sequences surrounding this DR1 were found to be essential for the HNF4 transactivation. In vitro-translated COUP-TFII was found to bind the adjacent sequences from nt -139 to -128 (DR0), but COUP-TFII interacted with this region at a much lower affinity than to the COUP-TFII-site at nt -72 to -57 (DR4). Mutations at nt -139 to -128 or nt -72 to -57 reduced the COUP-TFII and HNF4 synergy; however, these COUP-TFII-binding sequences were not absolutely required for the cooperative effect of HNF4 and COUP-TFII on transactivation. These results indicated that the observed transactivation was the result of protein/protein interactions facilitated by the juxtaposition of the binding elements.

Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription.

Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

Structure and functions of human oxysterol 7alpha-hydroxylase cDNAs and gene CYP7B1.

Oxysterol 7alpha-hydroxylase has broad substrate specificity for sterol metabolites and may be involved in many metabolic processes including bile acid synthesis and neurosteroid metabolism. The cloned human oxysterol 7alpha-hydroxylase (CYP7B1) cDNA encodes a polypeptide of 506 amino acid residues that shares 40% sequence identity to human cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In contrast to the liver-specific expression of CYP7A1, CYP7B1 mRNA transcripts were detected in human tissues involved in steroid genesis (brain, testes, ovary, and prostate) and in bile acid synthesis (liver) and reabsorption (colon, kidney, and small intestine). The human oxysterol 7alpha-hydroxylase transiently expressed in 293/T cells was able to catalyze 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone (DHEA). The human CYP7A1 and CYP7B1 both contain six exons and five introns. However, CYP7B1 spans at least 65 kb of the genome and is about 6-fold longer than CYP7A1. The transcription start site (+1) was localized 204 bp upstream of the initiation codon. No TATA box-like sequence was found near the transcription start site. Transient transfection assays of CYP7B1 promoter/luciferase reporter constructs in HepG2 cells revealed that the promoter was highly active. The 5' upstream region from nt -83 to +189 is the core promoter of the gene.

Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.

The gene encoding cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146- TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.

Regulation of bile acid synthesis.

Bile acids are important physiological agents required for disposal of cholesterol and absorption of vitamins and fats. Bile acids are synthesized from cholesterol in the liver. Enterohepatic circulation of bile acids is very efficient and plays an important physiological role in lipid absorption and secretion, and regulation of bile acid biosynthesis and cholesterol homeostasis. Conversion of cholesterol to bile acids requires 15 different enzymatic steps. Four cytochrome P450 enzymes play important roles in bile acid biosynthesis. The classic bile acid biosynthesis pathway starts with modification of the sterol ring and followed by side chain cleavage reactions to synthesize cholic acid (CA) and chenodeoxycholic acid (CDCA), the primary bile acids in most species. The first and rate-limiting enzyme in this pathway is cholesterol 7alpha -hydroxylase, a microsomal cytochrome P450, CYP7A. Another microsomal cytochrome P450 sterol 12alpha-hydroxylase (CYP12) is required for the synthesis of cholic acid. Mitochondrial cytochrome P450 sterol 27-hydroxylase (CYP27) catalyzes sterol side chain oxidation to convert C27 sterol to C24 bile acids. An alternative bile acid biosynthesis pathway (acidic) has been known for sometime but only recently has attracted much attention. In this pathway, side chain oxidation precedes modification of the sterol ring. Mitochondrial sterol 27-hydroxylase (CYP27) catalyzes the first reaction and followed by 7alpha-hydroxylation catalyzed by a microsomal oxysterol 7alpha-hydroxylase (CYP7B). Recent advances in purification and cloning of these major enzymes in the pathways have led to better understanding the molecular basis of regulation of bile acid synthesis and physiological role of the alternative pathways.

Basic transcription element binding protein (BTEB) transactivates the cholesterol 7 alpha-hydroxylase gene (CYP7A).

Cholesterol 7 alpha-hydroxylase catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in the liver. Previously, we have identified two bile acid response elements located in nt -74 to -54 (BARE-I) and -148 to -118 (BARE-II) regions. The nucleotide sequences in these BAREs are highly conserved and shared a novel sequence, AGTTCAAG. To identify and isolate nuclear protein factors that bind to these BAREs, we have screened a human liver cDNA expression library with oligonucleotide probes containing the sequence from nt -149 to -127. Twenty positive clones were selected and purified. Partial nucleotide sequences of these clones were determined. Nucleotide homology search of DNA databases of the sequences of these clones revealed that sequence of one clone, G13, is identical to basic transcription element binding protein (BTEB), a GC box-binding protein of Sp1 family transcription factors known to regulate many cytochrome P450 genes. Electrophoretic mobility shift assays have identified a basic transcription element (BTE) in BARE-II and a Sp1 binding site located in the nt -100/-82 region of the CYP7A promoter. Transient transfection assays have confirmed that BTEB was able to transactivate the CYP7A promoter/luciferase chimergic gene.

Identification of a bile acid response element in the cholesterol 7 alpha-hydroxylase gene CYP7A.

The transcriptional activity of the cholesterol 7 alpha-hydroxylase gene CYP7A is repressed by bile acids. Taurine conjugates of chenodeoxycholate and deoxycholate, but not cholate and ursodeoxycholate, inhibited the CYP7A promoter/luciferase reporter activity in transient transfection assays in Hep G2 cells. A region from nucleotide (nt) -74 to -55 was found to mediate bile acid response. However, deletion of this bile acid response element (BARE-I) enhanced reporter activity but did not eliminate the bile acid response. This is due to the presence of another BARE-II located in a conserved region between nt -149 and -128. Deletion or mutations of these sequences reduced promoter activity and abolished bile acid repression. This BARE-II shares an identical AGTTCAAG core sequence with BARE-I. Electrophoretic mobility shift assays of BARE-I and BARE-II probes using Hep G2 nuclear extract and the partially purified binding activity of nt -65/-54 DNA-affinity column revealed that the same or a similar nuclear protein might bind to both BAREs. BARE-II is the major BARE involved in the transcriptional repression of the CYP7A gene by hydrophobic bile acids.

Orphan receptors chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and retinoid X receptor (RXR) activate and bind the rat cholesterol 7alpha-hydroxylase gene (CYP7A).

The cholesterol 7alpha-hydroxylase gene (CYP7A) is transcriptionally regulated by a number of factors, including hormones, bile acids, and diurnal rhythm. Previous studies have identified a region from nucleotides (nt) -74 to -55 of the rat CYP7A promoter that enhanced bile acid repression of the SV40 early promoter, as assayed with a luciferase reporter gene in transiently transfected HepG2 cells. The rat CYP7A promoter/reporter activity was strongly stimulated by cotransfection with an expression plasmid encoding the nuclear hormone receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in a dose-dependent manner. Site-directed mutagenesis in the region of nt -74 to -55 altered this stimulation. Recombinant COUP-TFII expressed in HepG2 or COS-1 cells were found to bind to nt -74 -55 and nt -149 -128 probes by electrophoretic mobility shift assay (EMSA) and by supershifting the corresponding band with COUP-TFII-specific antibodies. The region of nt -176 -117 was previously mapped as a retinoic acid response region and was found to bind retinoid X receptor (RXR). EMSA supershift assays of wild-type and mutant oligomers using antibody against RXR revealed that the sequences between nt -145 and -134 were important for RXR binding. We conclude that COUP-TFII stimulates the transcriptional activity of the rat CYP7A promoter by binding to the sequences between nt -74 to -54 and nt -149 to -128. RXR may stimulate CYP7A gene transcription by binding to a direct repeat of the hormone response element separated by one nucleotide located at nt -146 -134.

Hormonal regulation of cholesterol 7alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat.

In primary cultures of rat hepatocytes, transcription of the cholesterol 7alpha-hydroxylase gene is induced synergistically by glucocorticoid and thyroid hormones. The objective of the present study was to evaluate the role of endogenous glucocorticoid and thyroid hormones in the maintenance of cholesterol 7alpha-hydroxylase gene expression in vivo. Male Sprague-Dawley rats underwent adrenalectomy (A), thyroidectomy (T), adrenalectomy + thyroidectomy (A + T), hypophysectomy (H), or sham surgery (paired controls). Ten days post surgery, livers were harvested and choles terol 7alpha-hydroxylase specific activity, steady-state mRNA levels, and transcriptional activity were determined. Serum corticosterone levels were <2% of paired controls in A, A + T, and H rats. Free thyroxine index was <32% of paired controls in rats with T and H. When compared to sham-operated controls, A + T and H led to decreases in cholesterol 7alpha-hydroxylase specific activities of 44 +/- 8% and 57 +/- 3%, respectively (P < 0.03 and < 0.05). Similar changes were observed in cholesterol 7alpha-hydroxylase steady-state mRNA levels, which decreased by 43 +/- 10% (P < 0.001) and 56 +/- 19% (P < 0.05), respectively. Cholesterol 7alpha-hydroxylase transcriptional activity in A + T and H rats decreased by 34 +/- 11% (P < 0.01) and 61 +/- 4% (P < 0.001), respectively. The observed decreases were greater after H than after A + T, suggesting the possibility that another pituitary hormone plays a role in regulation of cholesterol 7alpha-hydroxylase. Thyroidectomy alone led to a decrease in cholesterol 7alpha-hydroxylase specific activity of 37 +/- 7% (P < 0.05) and a trend toward decreased steady-state mRNA levels (21 +/- 12%; P = ns). Adrenalectomy did not significantly decrease cholesterol 7alpha-hydroxylase specific activity or mRNA levels. Neither thyroidectomy nor adrenalectomy alone affected transcriptional activity. We conclude that under physiologic circumstances, full expression of the cholesterol 7alpha-hydroxylase gene requires synergistic action of glucocorticoids and thyroid hormone.

Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation.

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.

Regulation of the hamster cholesterol 7 alpha-hydroxylase gene (CYP7A): prevalence of negative over positive transcriptional control.

Cholesterol 7 alpha-hydroxylase plays a crucial role in cholesterol homeostasis. We investigated the regulation of this enzyme in the hamster, a suitable animal model for studying cholesterol metabolism. DNase I hypersensitivity assay revealed the presence of a hypersensitive region in the proximal promoter. Both negative (bile acids, phorbol esters and insulin) and positive (glucocorticoid hormones) effects were mediated through sequences in the region 318 bp upstream of the ATG codon. All-trans-retinoic acid, cAMP, and LDL did not affect transcriptional activity. These findings show that the hamster cholesterol 7 alpha-hydroxylase gene undergoes a predominant negative regulation, as opposed to the rat CYP7A homologous gene.