Project 1 Pharmacology and physiology of retinoids in vivo. 1. Defective retinoic acid metabolism in the livers of AHR-/- mice. We found that increased levels of hepatic RA in the livers of AHR-/- mice are due to decreased cytochrome P450-dependent metabolism of RA to 4-hydroxy-RA. To identify the P450 isoforms involved in RA metabolism in mouse, liver microsomes from AHR-null and wild-type mice were subjected to western blotting . Signal intensity in western blots probed with anti-rat CYP2C6 antibody correlated with levels of RA 4-hydroxylation. Anti-CYP2C6-class antibody inhibited RA 4-hydroxylase activity in microsomes prepared from wild type, but not AHR-null, mouse liver. Analysis of five recombinant mouse CYP2C P450s revealed that only CYP2C39 catalyzed RA 4-hydroxylation. Consistent with the fact that hepatic RA-4 hydroxylation is not induced by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse line, CYP2C39 mRNA was expressed at only mildly elevated levels in wild-type mice treated with TCDD and was not reduced in the absence of AHR. Therefore, CYP2C39 protein, but not mRNA, is reduced in AHR-null mouse liver. These data strongly suggest that CYP2C39 is an important mediator of hepatic RA homeostasis that is indirectly controlled by AHR status. 2. Reversal of the AHR-/- phenotype by nutritional vitamin A deficiency.AHR-null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, TGaseII activity, TGF over expression and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR-null mice a vitamin A deficient diet with the purpose to reverse the phenotype. Liver retinoid content fell sharply within the first few weeks on the deficient diet. Analysis of TGF 1, 2, and 3 expression revealed a reduction to control levels in the AHR-/- mice. Similarly, the observed increase in TGF -RI and -RII and Smad4 was reduced to normal wild-type mouse levels in AHR-/- mice fed the retinoid-deficient diet. The livers from these mice showed a parallel decrease in collagen deposition and a reversal of liver fibrosis. Taken together, these data support the hypothesis that an imbalanced cross-talk between TGF and the retinoid signaling pathways is responsible for triggering fibrogenesis in AHR -/- mice. Project 2. Influence of retinoids on normal and malignant human cells 1.Differences in lecithin retinol acyl transferase (LRAT) between normal and malignant human breast cells. We have previously reported a reduced retinyl ester synthesis by human breast adenocarcinoma MCF-7 cells compared to normal human breast epithelial cells (HMEC).We have now shown that two additional breast adenocarcinoma cell lines (MB-231 and MB-453) fail to synthesize retinyl esters, suggesting this as a general phenomenon associated with breast cancer cells. We addressed possible mechanisms responsible for this observed decrease in retinyl ester synthesis. Sequence analysis of the LRAT gene between HMEC and MCF-7 cells showed several polymorphisms, but no mutations with functional significance could be detected in the exonic regions between the two cell lines. RT-PCR studies using oligonucleotide primers designed to amplify the coding sequence of the LRAT from the two cell lines showed that, in sharp contrast to the HMEC cells, no amplified bad could be detected from the MCF-7 cells. This suggests that these cells may use alternative splicing mechanisms to possibly generate a non-functional LRAT. Similar studies showed very low amplification in MB-231 and no amplification in MB-453 cells. We asked as to whether LRAT deficiency is common to other human carcinoma cells. Lung cancer A549 and HT-177 cells did not esterify retinol, in contrast to normal human lung epithelial cell (NHBE-31), which were very active in retinyl ester synthesis. Further, isolated proliferating epidermal melanocytes (HFSC/2) esterified retinol, whereas cultured human melanoma cells with fibroblastoid morphology showed negligible retinyl ester synthesis. However, melanoma cells with epithelioid morphology were capable of retinol esterification, demonstrating that retinyl esterification is not impaired in all tumor cells. 2. Defective retinoid signaling in human breast carcinoma MB-231 cells. We tried to define mechanisms responsible for the loss of RA responsiveness in malignant breast cancer cells. Three human breast cell lines were used: normal human mammary epithelial cells (HMEC), malignant estrogen receptor (ER) positive MCF7 and highly malignant ER negative MDA-MB-231. First, we compared protein expression by western blot with specific antibody. The amount of RAR and RXR expression in highly malignant MB-231 and HMEC were almost the same. Next, subcellular localization of RARa and RXRa was studied by immunofluorescence and transfection of GFP-RARa and RXRa. RARa predominantly localized in the nucleus of all cell lines and its localization did not change during malignant progression. RXRa was distributed uniformly throughout the nucleus in HMEC and MCF-7 cells. In sharp contrast to its uniform nuclear distribution in HMEC and MCF-7 cells, RXRa showed a punctate pattern in the nuclear speckle in highly malignant MDA-MB-231 cells. Interestingly, RXRa remained in the nuclear speckle when the cells were treated with ligand, agonist and trichostatin A (TSA) to facilitate transcription. Overexpressed C and N terminus GFP-, N terminus Flag-, and C terminus HA-RXRa did not localize to the nuclear speckle, however, non tagged-RXRa did localize to the speckle. These results indicate that the secondary structure of both N and C terminus is critical for speckle localization. Reporter assay revealed that basal level of transcription of DR1 mediated by RXR homodimer was not promoted by additional ligand, suggesting reduction of accessibility and lesser availability of RXRa to the response element of the target gene as the responsible agent. It was also found that overexpressed RXRa restored RA sensitivity in MB-231 cells by inducing cell growth inhibition in the presence of ligand.
| Andreola, Fausto; Calvisi, Diego F; Elizondo, Guillermo et al. (2004) Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion. Hepatology 39:157-66 |
| Andreola, Fausto; Hayhurst, Graham P; Luo, Gang et al. (2004) Mouse liver CYP2C39 is a novel retinoic acid 4-hydroxylase. Its down-regulation offers a molecular basis for liver retinoid accumulation and fibrosis in aryl hydrocarbon receptor-null mice. J Biol Chem 279:3434-8 |
| Tanaka, T; Dancheck, B L; Trifiletti, L C et al. (2004) Altered localization of retinoid X receptor alpha coincides with loss of retinoid responsiveness in human breast cancer MDA-MB-231 cells. Mol Cell Biol 24:3972-82 |
