Competitive regulation of human intestinal ß-carotene 15,15'-monooxygenase 1 (BCMO1) gene expression by hepatocyte nuclear factor (HNF)-1α and HNF-4α.

AIM: Among the pro-vitamin A carotenoids, ß-carotene is an excellent source of vitamin A. ß-Carotene 15,15'-monooxygenase 1 (BCMO1) is a critical enzyme involved in the conversion of ß-carotene into vitamin A (retinal) in the small intestine of many vertebrates. In the present study, we investigated the regulation of human BCMO1 gene expression using human intestinal Caco-2 BBe cells. MAIN METHODS: We performed electrophoretic mobility shift assays and chromatin immunoprecipitation assays to investigate the binding properties of hepatocyte nuclear factor (HNF)-1α and HNF-4α to the proximal promoter of the human BCMO1 gene. Caco-2 BBe cells were also transfected with HNF-1α and HNF-4α siRNAs, and BCMO1 gene expression levels and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and luciferase reporter assays, respectively. KEY FINDINGS: We identified overlapping binding sites for HNF-1α and HNF-4α in the human BCMO1 gene proximal promoter. Endogenous nuclear HNF-1α and HNF-4α proteins competitively bound these sites in Caco-2 BBe cells. BCMO1 gene expression levels and promoter activity were significantly decreased in HNF-1α siRNA-transfected Caco-2 BBe cells. In contrast, HNF-4α siRNA-transfected cells exhibited a significant increase in BCMO1 gene expression and promoter activity. Mutation of these overlapping binding sites dramatically decreased BCMO1 promoter activity. SIGNIFICANCE: Our study indicates that the competitive actions of HNF-1α and HNF-4α on their overlapping binding sites in the human BCMO1 gene promoter oppositely regulate BCMO1 gene expression in the human small intestine.

Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome.

Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein α in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.

Hepatocyte nuclear factor-4alpha regulates human cellular retinol-binding protein type II gene expression in intestinal cells.

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4alpha expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4alpha significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4alpha protein levels by HNF-4alpha small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4alpha protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4alpha protein levels, HNF-4alpha protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4alpha is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4alpha in the regulation of human intestinal vitamin A absorption and metabolism.

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells.

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRalpha1 and TRbeta1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T3 treatment in medium supplemented with fetal bovine serum. Furthermore, T3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T3 is an important hormone for the regulation of vitamin A and beta-carotene metabolism-related gene expression in human small intestinal cells.

Developmental changes in gene expressions of beta-carotene cleavage enzyme and retinoic acid synthesizing enzymes in the chick duodenum.

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (BCMO1; EC 1.13.11.21). We previously reported that chick duodenal BCMO1 activity increased abruptly just after hatching. In this study, we further investigated mechanisms and physiological roles of the postnatal induction of BCMO1 expression in the chick duodenum. We showed that BCMO1 mRNA levels increased in the chick duodenum during postnatal period after hatching, but remain unchanged in the chick liver throughout the perinatal period. Serum hydrocortisone (HC) levels were also increased after hatching. Moreover, HC-administered chicks showed an enhancement of duodenal BCMO1 mRNA during the perinatal period. We further analyzed the developmental gene expression patterns of three types of retinoic acid (RA) synthesizing enzymes in the chick duodenum. Among them, retinal dehydrogenase 1 (RALDH1) mRNA levels in the chick duodenum increased during the postnatal period, indicating a similar developmental expression pattern to that of BCMO1. These results suggest that the postnatal induction of BCMO1 gene expression in the chick duodenum may be caused by the elevation of serum HC levels and may contribute to the RALDH1-mediated RA synthetic pathway.