<i>In vivo</i> studies of altered expression patterns of <i>p53</i> and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis

Borrás, Elisa; Zaragozá, Rosa; Morante, María; García, Carlos J. Gómez; Gimeno, Amparo; López-Rodas, Gerardo; Barber, Teresa; Miralles, Vicente J.; Viña, Juan R.; Torres, Luís

doi:10.1046/j.1432-1033.2003.03511.x

Cited by 17 publications

(13 citation statements)

References 42 publications

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“…Chromatin from mammary tissue was fixed and immunoprecipitated according to Borrás et al (5). Briefly, mammary tissue samples were excised and immersed in PBS, pH 7.4, and 1% formaldehyde for 8 min to crosslink the transcription factors to DNA.…”

Section: Methodsmentioning

confidence: 99%

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

Zaragozá

Gimeno²,

Miralles³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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Retinoic acid (RA) is a signaling molecule in the morphogenesis of the mammary gland, modulating the expression of matrix metalloproteinases (MMPs). The aim of this paper was to study the role of RA during weaning, which consists of three events: apoptosis of the secretory cells, degradation of the extracellular matrix, and adipogenesis. CRABP II and CRBP-1 carrier proteins increased significantly during weaning compared with lactating glands but reverted to control values after the litter resuckled. The effects of RA are mediated by the nuclear receptors RAR␣, RAR␤, RAR␥, and RXR␣, which underwent an increase in protein levels during weaning. In an attempt to elucidate the RAR␣-dependent signaling pathway, ChIP assays were performed. The results showed the binding of RAR␣ to the MMP-9 promoter after 24-and 72-h weaning together with its coactivator p300; this fact could be responsible for the increase found in MMP-9 mRNA and protein levels in these conditions. Expression of related MMPs (MMP-2 and MMP-3) was also increased during weaning. Using gelatine zymography, we observed a time-dependent increase in active forms of MMP-9 and MMP-2. On the other hand, the inhibitor of MMPs, TIMP-1, was almost undetectable at 24-and 72-h weaning by Western blot. The role of retinoids in matrix remodeling is reinforced by the fact that administration of an acute dose of retinol palmitate to control lactating rats also induces MMP-9 expression. This emphasizes the importance of retinoids in vivo to regulate mammary gland involution. mammary gland involution; matrix metalloproteinase-9; stromelysin-1; retinoic acid; retinoid acid receptor-␣; TO FEMALE MAMMALS, THE COST OF LACTATION is exorbitant compared with other physiological processes. Thus, to assure a good milk production, the lactating animal has developed several physiological changes that include hyperphagia, liver and mammary gland hypertrophy, increased cardiac output, and increased blood flow to the gland, which, together with widespread changes in the metabolism of different tissues, assure a sufficient supply of substrates to the gland for milk production.Few adult tissues exhibit extensive apoptosis under physiological conditions. These include the small intestine, adipose, uterus, ovary, and mammary gland (1, 9). After being weaned, the mammary gland is remodeled in preparation for the next pregnancy/lactation cycle through a complex cellular program.This weaning process has two phases. The first phase is reversible; it depends on p53 and is characterized by the disappearance of the physiological adaptations and by an increase in the number of apoptotic events in the epithelia of the lobulo-alveolar compartment (15, 33). The second phase is p53 independent; it is irreversible and includes a proteolytic degradation of the basement membrane and remodeling of the mammary gland. To achieve the latter events, two main families of extracellular matrix (ECM)-degrading proteinases are activated, matrix metalloproteinases (MMPs) and serine proteinases, that are involved...

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Section: Methodsmentioning

confidence: 99%

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

Zaragozá

Gimeno²,

Miralles³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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“…Cells were treated with 1% formaldehyde in phosphate-buffered saline buffer under gentle agitation for 10 min at room temperature to cross-link transcription factors to DNA. Thereafter, cells were collected by centrifugation, washed, resuspended in lysis buffer as described previously (Borras et al, 2003), and sonicated on ice for 10 steps of 10 s at 30% output in a Branson sonicator. Samples were centrifuged to clear the supernatants.…”

Section: Methodsmentioning

confidence: 99%

“…The inmunocomplexes were affinity-absorbed with 10 mg of protein A-Sepharose (prewashed with lysis buffer for 4 h at 4°C under gentle rotation) and collected by centrifugation (6500g, 1 min). The antibody-bound and background DNA fractions were washed as described previously (Borras et al, 2003). The cross-links were reversed by heating the samples at 65°C overnight.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Regulation of the Human HepaticCYP3A4: Identification of a New Distal Enhancer Region Responsive to CCAAT/Enhancer-Binding Protein β Isoforms (Liver Activating Protein and Liver Inhibitory Protein)

Martínez-Jiménez¹,

Gómez-Lechón²,

Castell³

et al. 2005

Mol Pharmacol

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CCAAT/enhancer-binding proteins (C/EBPs) are key transcription factors involved in the constitutive expression of several cytochrome P450 genes in the liver. Their concentration and activity change in several pathophysiological conditions. For instance, during inflammation, released cytokines induce repressive C/EBP␤-liver inhibitory protein (LIP), which antagonizes constitutive C/EBP transactivators [C/EBP␣ and C/EBP␤-liver activating protein (LAP)], down-regulating genes such as CYP3A4. However, the mechanism by which hepatic C/EBP factors modulate transcription of the CYP3A4 gene is not known. To elucidate the mechanism of action, we cotransfected luciferase reporter vectors, containing 5Ј-flanking deletions of the CYP3A4 gene, along with expression vectors for C/EBP␤-LAP, C/EBP␤-LIP, and C/EBP␣, in hepatic (HepG2) and nonhepatic (HeLa) cells. Analysis of the Ϫ3557 to Ϫ6954 base pair (bp) region demonstrated the existence of a 288-bp sequence at Ϫ5.95 kilobases (kb), which showed maximal response to C/EBP␤-LAP (ϳ30-fold increase in HepG2 cells). Coexpression of LAP with increasing amounts of LIP reduced the activating effect by ϳ70%. Site-directed mutagenesis of predicted C/EBP␤ binding sites demonstrated the presence of four functional C/EBP␤-responsive motifs within this distal flanking region. Further experiments using chromatin immunoprecipitation proved the binding of endogenous C/EBP␤ to the Ϫ5.95-kilobase enhancer of the CYP3A4 gene in human hepatocytes. Expression of recombinant LAP and LIP by means of adenoviral vectors resulted in their binding to this region, which was followed by activation/repression of CYP3A4. Together, our results uncover a new distal enhancer site in the CYP3A4 gene where C/EBP␤-LAP binds and activates transcription, whereas the truncated form, C/EBP␤-LIP, antagonizes LAP activity and causes gene repression.

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“…Chromatin of cultured cells was fixed and immunoprecipitated according to Borrás et al 34 Crosslinking between transcription factors and chromatin was achieved via the addition of formaldehyde (1% final concentration) for 10 min at 37 1C, and then halted via the addition of 125 mM glycine for 5 min at room temperature (25 1C). Chromatin solutions were sonicated and incubated with anti-LXRa, anti-SREBP-1c (both from Abcam), and an antibody against RNA polymerase II (Santa Cruz Biotechnology), 35 and then rotated overnight at 4 1C.…”

Section: Chip (Chromatin Immunoprecipitation) Assaysmentioning

confidence: 99%

Liver X receptor α-mediated regulation of lipogenesis by core and NS5A proteins contributes to HCV-induced liver steatosis and HCV replication

García‐Mediavilla

Pisonero-Vaquero

Lima-Cabello

et al. 2012

Laboratory Investigation

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Molecular mechanisms contributing to hepatitis C virus (HCV)-associated steatosis are not well established, although HCV gene expression has been shown to alter host cell cholesterol/lipid metabolism. As liver X receptors (LXRs) play a role as key modulators of metabolism signaling in the development of steatosis, we aimed to investigate in an HCV in vitro model the effect of HCV NS5A protein, core protein, and viral replication on the intracellular lipid accumulation and the LXRa-regulated expression of lipogenic genes. The effects of LXRa siRNA or agonist GW3965 treatment on lipogenesis and HCV replication capacity in our HCV replicon system were also examined. NS5A-and core-expressing cells and replicon-containing cells exhibited an increase of lipid accumulation by inducing the gene expression and the transcriptional activity of LXRa, and leading to an increased expression of its lipogenic target genes sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor-g, and fatty acid synthase. Transcriptional induction by NS5A protein, core protein, and viral replication occurred via LXR response element activation in the lipogenic gene promoter. No physical association between HCV proteins and LXRa was observed, whereas NS5A and core proteins indirectly upregulated LXRa through the phosphatidylinositol 3-kinase pathway. Finally, it was found that LXRa knockdown or agonist-mediated LXRa induction directly regulated HCV-induced lipogenesis and HCV replication efficiency in replicon-containing cells. Combined, our data suggest that LXRa-mediated regulation of lipogenesis by core and NS5A proteins may contribute to HCV-induced liver steatosis and to the efficient replication of HCV.

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In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis

Abstract: Several clinical trials have revealed that individuals who were given b)carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo 2,3 ; 2,3 rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G

Cited by 17 publications

References 42 publications

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

Transcriptional Regulation of the Human HepaticCYP3A4: Identification of a New Distal Enhancer Region Responsive to CCAAT/Enhancer-Binding Protein β Isoforms (Liver Activating Protein and Liver Inhibitory Protein)

Liver X receptor α-mediated regulation of lipogenesis by core and NS5A proteins contributes to HCV-induced liver steatosis and HCV replication

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