Identification of new sites of binding and activation of the murine alpha1(I) collagen promoter by CCAAT/enhancer binding protein beta.

The CCAAT/enhancer binding protein beta (C/EBPbeta) was previously shown to bind to the alpha(1)(I) collagen promoter at -365 to -335 (site 1) and to activate it. Acetaldehyde also activates the promoter, and this effect is mediated by an increase in stellate-cell C/EBPbeta protein and C/EBPbeta binding. The present study identified two additional distal sites (sites 2 and 3) of binding of C/EBPbeta, in the nuclear extracts of stellate cells, at -399 to -370 and -623 to -592 in the alpha(1)(I) collagen promoter. The C/EBPbeta protein activates the promoter at all three sites. Acetaldehyde increases C/EBPbeta binding to all three sites. Activation by acetaldehyde is abrogated in the transfected promoter mutated at either site 1 or site 3 but is not affected by mutation at site 2. Binding of the 20-kDa C/EBPbeta isoform (p20C/EBPbeta), which is eliminated by mutation at the distal site 3 of C/EBP binding, is necessary for the activation by acetaldehyde of the alpha(1)(I) collagen promoter.

Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.

CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde.

Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.

A transient increase in c-myc precedes the transdifferentiation of hepatic stellate cells to myofibroblast-like cells.

AIMS/BACKGROUND: Liver stellate cells are transdifferentiated to collagen-producing myofibroblast-like cells in vivo during liver injury or when placed in culture. The purpose of this study was to determine the presence of retinoids and the expression of the immediate early genes as they relate to the transdifferentiation of liver stellate cells in culture. METHODS: Rat liver stellate cells were studied immediately after isolation or sequentially after culture for varying periods of time. RNA was isolated and specific messages were determined by RT-PCR. Cells were also isolated for determination of retinoid autofluorescence and immunofluorescent staining with specific antibodies by laser confocal microscopy. RESULTS: c-fos message and immunoprotein were high in the freshly isolated cells prior to culture, while c-myc expression increased markedly after one day of culture. Both c-fos and c-myc gene expression decreased prior to the transdifferentiation of the cells to myofibroblast-like cells and to the increase in alpha 1(I) and alpha 2(I) collagen messages and collagen production. The presence of retinoid autofluorescence and retinoic acid receptor (RAR-alpha and RAR-beta) messages and RAR-beta immunoprotein persisted during initial transdifferentiation of the stellate cells. CONCLUSIONS: This study shows a high initial level of c-fos expression and a transient increase in c-myc expression followed by a decrease to lower levels prior to transdifferentiation and collagen production by stellate cells. A total loss of retinoid autofluorescence or a decrease in RAR-alpha or RAR-beta are not required for initial transdifferentiation of stellate cells or collagen production.

Identification of two repressor elements in the mouse alpha 2(I) collagen promoter.

We recently identified three areas of Sp1 binding located between -568 and -453 of the 5' flanking region of the murine alpha2(I) collagen promoter which are necessary for optimal activity. We now identify two additional regions of Sp1 binding located at -371 to -351 (region 4) and at -690 to -613 (region 5), which when mutated increased promoter activity in transfected rat hepatic stellate cells indicating they contain negative regulatory elements. AP-2 bound to region 4 while YY1 bound most strongly to region 5. AP-2 decreased Sp1 binding to region 4 and had a dual effect on Sp1 binding to region 5 decreasing and increasing Sp1 binding at low and high concentrations of AP-2, respectively. YY1 enhanced Sp1 binding to both regions. AP-2 inhibited or enhanced the stimulatory effect of a transfected Sp1 expression vector on the alpha2(I) collagen promoter in Drosophila cells at low or high AP-2 expression, respectively. YY1 enhanced or inhibited the activation of the promoter by low or high Sp1 expression, respectively. This study identifies two negative regulatory elements in the murine alpha2(I) collagen promoter and shows that AP-2 and YY1 interact with Sp1 at these sites and can inhibit the activating action of Sp1.

Effect of dihydrotestosterone on turnover of alcohol dehydrogenase in rat hepatocyte culture.

Dihydrotestosterone decreased alcohol dehydrogenase (ADH) activity and enzyme-protein in rat hepatocytes in culture. This effect was observed after the hepatocytes had been exposed to dihydrotestosterone for 3 days at concentrations of 0.5 micromol/L or higher. Dihydrotestosterone did not decrease alcohol dehydrogenase messenger RNA (mRNA) but, rather, resulted in small increases in ADH mRNA after 3 days of exposure. To further determine the mechanism for the effects of dihydrotestosterone in decreasing the enzyme, the turnover of ADH was determined after incorporation of [3H]-leucine into the enzyme protein. Dihydrotestosterone did not alter the initial 2-hour incorporation of [3H]-leucine into the enzyme protein. Dihydrotestosterone, however, resulted in an increase in the fractional rate of degradation (Kd) of the enzyme from 0.12 +/- 0.013 to 0.23 +/- 0.004 per hour (P < .001) accompanied by a much smaller increase in the fractional rate of synthesis (Ks) from 0.12 +/- 0.028 to 0.17 +/- 0.031 per hour (P > .05). Hence, the mechanism for the fall in ADH in the presence of dihydrotestosterone is an increase in enzyme degradation which is not accompanied by a sufficient increase in enzyme synthesis.

Effect of acetaldehyde on Sp1 binding and activation of the mouse alpha 2(I) collagen promoter.

Acetaldehyde activates the mouse alpha 2(I) collagen promoter and this effect is mediated in part by increased binding of nuclear factor I (NF-I). Additional mechanisms may exist since deletions in the promoter upstream to the NF-I binding site prevented enhancement by acetaldehyde. Three adjacent areas of binding by nuclear proteins from activated hepatic stellate cells were identified at -568 to -554 (region 1), -542 to -518 (region 2), and -473 to -453 (region 3) of the promoter using DNase I protection analyses. Multiple DNA-protein complexes were formed in electrophoretic mobility shift assays with oligonucleotide probes specifying the three regions. Sp1 and NF-1 bound to all three regions, while Sp3 bound to region 2. Acetaldehyde decreased nuclear protein binding to all three regions. Mutations of regions 1, 2, and 3 reduced basal activity of the promoter and inhibited acetaldehyde stimulation in transfected stellate cells. Acetaldehyde inhibited the stimulatory effect of the Sp1 vector pPacSp1 on the promoter in transfected Drosophila cells. In conclusion, three regions of Sp1 binding were identified and are required for optimal activity of the alpha 2(I) collagen promoter. Sp1 is required for basal activity of the alpha 2(I) collagen promoter; however, the enhancing effect of acetaldehyde on the promoter is not mediated by Sp1.

Activation by acetaldehyde of the promoter of the mouse alpha2(I) collagen gene when transfected into rat activated stellate cells.

The effect of acetaldehyde in activating the mouse alpha2(I) collagen promoter in transiently transfected rat activated stellate cells and the possible mediating effect of transforming growth factor beta1 (TGFbeta1) on type I collagen gene expression were determined. Acetaldehyde and TGFbeta1, each had a similar effect in activating the wild-type promoter, but failed to activate the promoter with a -352 to -104 deletion, or the promoter containing a 3-bp substitution between -305 and -303 in the putative nuclear factor I (NF-I) binding site. The effects of acetaldehyde and TGFbeta1 are therefore mediated by a similar factor or factors that bind to the NF-I consensus sequence within the region -352 to -104. Additional factors may also play a role in the effects of acetaldehyde and TGFbeta1, which have similar effects on the wild-type promoter, but become additive in activating the promoter with a more distal deletion containing a cis-repressor element. Pretreatment of activated stellate cells with antibodies to TGFbeta1 suppressed the effect of acetaldehyde in increasing the alpha1(I) collagen message, indicating that TGFbeta1 mediates the effect of the acetaldehyde-induced increase in the expression of the alpha1(I) collagen gene which also contains NF-I binding sites.

Effects of acetaldehyde on nuclear protein binding to the nuclear factor I consensus sequence in the alpha 2(I) collagen promoter.

Acetaldehyde has been shown to increase collagen production in cultured rat myofibroblastlike cells and to activate the mouse alpha 2(I) collagen promoter in transfected NIH 3T3 cells. Nuclear factor I (NF-I), a CCAAT binding transcription factor, is known to bind and activate the alpha 1(I) and alpha 2(I) collagen genes. Activation of the alpha 2(I) collagen promoter was not observed when the NF-I binding site of the promoter was deleted. In this study, we determined if acetaldehyde influences the binding of NF-I to the alpha 2(I) collagen promoter. Nuclear proteins extracted from NIH 3T3 cells, or myofibroblastlike cells, 36 hours after the addition of acetaldehyde (200 mumol/L) in serum-free media showed increased binding to the consensus sequence of the NF-I binding site by DNase I protection analysis and by electrophoretic mobility shift assay (EMSA) as compared with control nuclear extracts that were not exposed to acetaldehyde. Furthermore, nuclear proteins extracted from myofibroblastlike cells that had been previously exposed to acetaldehyde had a marked increase in NF-I protein, as shown by Western blot with NF-I antibodies. Antisera to NF-I resulted in a slow migrating DNA-protein-antibody complex (supershift) on EMSA. However, the NF-I antibody did not supershift all the DNA-protein complexes, and the supershift band was not increased with nuclear proteins from acetaldehyde-treated cells despite the increased binding of these nuclear protein preparations to the NF-I oligo. Therefore, nuclear proteins, in addition to NF-I, bind to the NF-I consensus sequence and may have their binding altered by acetaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)