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M Chojkier

Publications and source records attributed to M Chojkier.

At least 37 records · Page 2Linked to original sources
PubMed1994

LAP (NF-IL6) transactivates the collagen alpha 1(I) gene from a 5' regulatory region.

Although collagen is known to enhance hepatocyte differentiation and hepatocytes produce collagen in vivo, the transcriptional factors responsible for collagen type I gene expression in hepatic cells are not known. LAP (Liver Activator Protein) is a member of the C/EBP family, which in differentiated hepatocytes contributes to the high levels of liver-specific gene expression. In this study we show that LAP binds to the collagen alpha 1(I) promoter at both reverse CCAAT motifs and activates transcription. Furthermore, an upstream element, collagen element I (-370/-344), which shares homology with the LAP binding cis-element of the albumin promoter (9 of 13 bp) is described. This collagen element I stimulates transcription in both orientations and when placed in front of either a homologous or a heterologous chimeric report construct. These experiments suggest that LAP may be important in the expression of collagen in differentiated hepatocytes through both the promoter and a newly described upstream element.

Base Sequence↗
PubMed1994

Acetaldehyde-modified epitopes in liver biopsy specimens of alcoholic and nonalcoholic patients: localization and association with progression of liver fibrosis.

Acetaldehyde, the first product of ethanol oxidation, has been shown to stimulate collagen gene expression and to form protein-acetaldehyde adducts. Because little is known about these adducts in human liver tissue, we assessed, with an immunohistochemical procedure, the presence and location of acetaldehyde-protein adducts in liver biopsy specimens of alcoholic patients. In addition, we correlated the presence of adducts with the progression or subsequent occurrence of liver fibrosis. The group included 106 patients with high alcohol consumption (> 90 gm ethanol/day for the last 5 yr), 10 nonalcoholic patients with normal livers and 23 patients with other liver diseases. Sixty-four of the 106 alcoholic patients had a second liver biopsy, whose specimen was used to assess the progression of liver fibrosis. Polyclonal antibodies were produced against homologous low-density lipoprotein purified from rabbit serum and modified in vitro in the presence of acetaldehyde. Protein-acetaldehyde adducts could be detected by immunohistochemistry in biopsy specimens of 90 alcoholic patients (85%), in none of the 10 nonalcoholic patients with normal livers and in 65% of the patients with nonalcoholic liver disease. Acetaldehyde-modified epitopes were detected in the intracellular and extracellular compartment. Intracellular protein-acetaldehyde adducts were localized in the cytoplasm of hepatocytes with a more intense staining in zone 3. No correlation existed between the intensity of intracellular staining and the histologically assessed severity of liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1994

Stimulation of collagen alpha 1(I) gene expression is associated with lipid peroxidation in hepatocellular injury: a link to tissue fibrosis?

We have shown that lipid peroxidation stimulates collagen alpha 1(I) gene transcription in cultured cells. Because increased lipid peroxidation and collagen production coexist in many hepatic disorders, including experimental carbon tetrachloride intoxication, we investigated whether lipid peroxidation modulates collagen gene expression in rats treated with carbon tetrachloride. In this animal model, we show colocalization of increased collagen alpha 1(I) mRNA with lipid peroxidation by means of in situ hybridization and immunohistochemical study for malondialdehyde and 4-hydroxynonenal protein adducts, respectively. However, allyl alcohol treatment, which induced a similar degree of hepatocellular injury but without aldehyde-protein adducts, did not increase collagen alpha 1(I) gene expression, suggesting that hepatocyte necrosis is not sufficient to induce the expression of collagen type I. Furthermore, in the absence of an inflammatory response, coculture experiments of hepatocytes and Ito cells treated with carbon tetrachloride indicate that hepatocytes exert a "paracrine" stimulation of both lipid peroxidation and collagen gene expression in Ito cells. These experiments suggest that hepatocyte lipid peroxidation plays a major role in the regulation of collagen alpha 1(I) gene expression by Ito cells and that it may be a link between hepatocyte injury and hepatic fibrosis.

1-Propanol↗
PubMed1993

Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain.

One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2). NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators. Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide. The control of NF-IL6/LAP expression and activity is complex and poorly understood. Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines. Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway. We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy.

Animals↗
PubMed1992

The acute-phase response protects mice from D-galactosamine sensitization to endotoxin and tumor necrosis factor-alpha.

D-Galactosamine is an hepatocyte-specific inhibitor of RNA synthesis. It has been used to sensitize animals both to the lethal effects of bacterial endotoxin (lipopolysaccharide) and to a principal lipopolysaccharide-induced mediator of shock, tumor necrosis factor-alpha. The mechanism by which this sensitization occurs is unknown. Because lipopolysaccharide, acting through a network of cytokines, provokes the transcription of a number of hepatic acute-phase proteins, we postulated that the lipopolysaccharide-sensitizing effect of D-galactosamine could be caused by its inhibition of acute-phase product transcription. We confirmed that the acute-phase response to lipopolysaccharide was attenuated by simultaneous administration of D-galactosamine. However, when the acute-phase response was induced by subcutaneous turpentine 24 hr before D-galactosamine administration, the effect of D-galactosamine on circulating acute-phase reactants was negligible. Furthermore, induction of an a priori acute-phase response protected mice from both D-galactosamine/lipopolysaccharide and D-galactosamine/tumor necrosis factor-alpha-induced death. The turpentine-induced acute-phase response did not decrease endogenous tumor necrosis factor-alpha production after lipopolysaccharide, nor did it affect the clearance of larger doses of injected tumor necrosis factor-alpha. Thus we suggest that the acute-phase response protects against death in D-galactosamine-sensitized mice through an interaction with mediators of shock subsequent to tumor necrosis factor-alpha release.

Absorption↗
PubMed1991

Ascorbic acid stimulation of collagen biosynthesis independent of hydroxylation.

Ascorbic acid stimulates collagen gene expression in cultured fibroblasts but mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Because another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts, we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mmol/L) induced lipid peroxidation and stimulated collagen alpha 1(I) gene transcription in cultured human fibroblasts. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 mumol/L) or methylene blue (10 mumol/L) prevented the stimulation of collagen gene expression. Addition of malondialdehyde (200 mumol/L), a product of lipid peroxidation, to cultured human fibroblasts also increased two- to threefold collagen production and procollagen alpha 1(I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes, and this step may be necessary for stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.

Ascorbic Acid↗
PubMed1991

d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation.

Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1990

LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.

A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.

Albumins↗
PubMed1990

Tumor necrosis factor-alpha inhibits albumin gene expression in a murine model of cachexia.

The mechanisms responsible for decreased serum albumin levels in patients with cachexia-associated infection, inflammation, and cancer are unknown. Since tumor necrosis factor-alpha (TNF alpha) is elevated in cachexia-associated diseases, and chronic administration of TNF alpha induces cachexia in animal models, we assessed the regulation of albumin gene expression by TNF alpha in vivo. In this animal model of cachexia, Chinese hamster ovary cells transfected with the functional gene for human TNF alpha were inoculated into nude mice (TNF alpha mice). TNF alpha mice became cachectic and manifested decreased serum albumin levels, albumin synthesis, and albumin mRNA levels. However, even before the TNF alpha mice lost weight, their albumin mRNA steady-state levels were decreased approximately 90%, and in situ hybridization revealed a low level of albumin gene expression throughout the hepatic lobule. The mRNA levels of several other genes were unchanged. Hepatic nuclei from TNF alpha mice before the onset of weight loss were markedly less active in transcribing the albumin gene than hepatic nuclei from control mice. Therefore, TNF alpha selectively inhibits the genetic expression of albumin in this model before weight loss.

Animals↗
PubMed1990

Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload.

In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized.

Aldehydes↗
PubMed1990

Expression of collagen genes in the liver.

Hepatic production of type I collagen is markedly increased in liver cirrhosis. Previous studies using primary liver cell cultures have demonstrated that hepatocytes, lipocytes and endothelial cells are all capable of producing collagen. In this study in situ hybridization and hepatic cell sorting have been used to identify which cells are expressing the type I collagen gene, alpha 1(I), in normal rat liver. Northern blotting of mRNAs from purified hepatic cell populations demonstrated that both hepatocytes and several types of non-parenchymal cells express the collagen alpha 1(I) gene. Calculations based on cell numbers, yields of mRNA, and cellular mRNA concentration demonstrated that the majority of collagen alpha 1(I) mRNA originates from the hepatocytes in the normal liver. Localization of a collagen alpha 1(I) mRNA by in situ hybridization confirmed that both hepatocytes and non-parenchymal cells express this gene. Furthermore, collagen alpha 1(I) gene expression in hepatocytes was obtained by transfecting a reporter gene driven by the collagen alpha (I) 5' regulatory segment in primary liver cell cultures. Future experiments will further characterize the regulation of collagen alpha 1(I) gene expression in the liver.

Animals↗
PubMed1989

Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation?

Ascorbic acid stimulates collagen gene expression in cultural fibroblasts (Lyons, B. L., and Schwartz, R. L. (1984) Nucleic Acids Res. 12, 2569-2579), but the mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Since another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts (Brenner, D. A., and Chojkier, M. (1987) J. Biol. Chem 262, 17690-17696), we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mM) induced lipid peroxidation in cultured human fibroblasts judging by the production of thiobarbituric acid-reactive substances and carbonyl groups, and by the presence of malondialdehyde- and 4-hydroxynonenal-protein adducts. Ascorbic acid stimulated (2-3-fold) the net production of collagen relative to total proteins, the levels of procollagen alpha 1 (I) mRNA and the transcription of this gene. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 microM) or methylene blue (10 microM) prevented the stimulation of collagen gene expression. The addition of malondialdehyde (200 microM), a product of lipid peroxidation, to cultured human fibroblasts also increased 2-3-fold collagen production and procollagen alpha 1 (I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes and this step may be necessary for the stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.

Ascorbic Acid↗
PubMed1989

Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes.

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.

Aging↗
PubMed1989

Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is secreted by macrophages in response to inflammation, infection and cancer. Sublethal doses of recombinant TNF-alpha to rats causes cachexia, anaemia and inflammation. TNF-alpha plays a major part in tissue inflammation and remodelling by stimulating production of collagenase. Cellular responses to TNF-alpha are initiated by binding to high-affinity cell surface receptors. TNF-alpha then profoundly affects gene regulation, stimulating the fos, myc, interleukin-1 and interleukin-6 genes and inhibiting the type I collagen gene. Here we demonstrate that TNF-alpha also stimulates collagenase gene transcription; this stimulation is mediated by an element of the gene that is responsive to the transcription factor AP-1, the major component of which (jun/AP-1) is encoded by the jun gene; and that TNF-alpha stimulates prolonged activation of jun gene expression. This prolonged induction of jun contrasts with its transient activation by the phorbol ester TPA and provides a physiological example of the ability of jun/AP-1 to stimulate its own transcription. This may be a key mechanism for mediating at least some of the biological effects of TNF-alpha.

Blotting, Northern↗
PubMed1988

Tumor necrosis factor alpha inhibits collagen gene transcription and collagen synthesis in cultured human fibroblasts.

The effects of recombinant human tumor necrosis factor alpha (TNF alpha) on collagen production and gene expression in cultured fibroblasts were studied. Cells were labeled with [3H]proline, and the radioactivity of collagenase-sensitive and -resistant proteins were used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by TNF alpha (0-1.2 nM) in a dose- and time-related manner. The specific activities of the free [3H]proline pool, which were similar in control and TNF alpha-treated cells, were used to calculate the absolute rates of protein production. The absolute rate of collagen production was decreased by 50% in the presence of 1.2 nM TNF alpha during 24-h incubations (851 +/- 104 versus 426 +/- 39 pmol/micrograms of DNA/h; p less than 0.01), whereas noncollagen protein production and the rate of procollagen secretion were unchanged. We found no evidence of cellular toxicity in cultured cells treated with TNF alpha. In addition, TNF alpha did not affect cell proliferation as determined by [6-3H]thymidine incorporation into DNA. Most of the collagen produced by the cultured fibroblasts was type I. Using hybridization with specific DNA probes there was an approximately 50% decrease in the quantity of procollagen alpha 1(I) mRNA, without changes in the quantity of alpha tubulin mRNA or the size of the transcripts, in cells incubated with TNF alpha. Interleukin-1 (2.5 ng/ml) also decreased the levels of procollagen alpha 1(I) mRNA by approximately 50%. Cycloheximide (0.1 mM), an inhibitor of protein synthesis, blocked the inhibitory effect of both TNF alpha and interleukin-1 on procollagen alpha 1(I) mRNA. Nuclear run-off assays demonstrated that TNF alpha decreased procollagen alpha 1(I) transcriptional activity by 50% and had no effects on alpha tubulin gene transcription. Thus, TNF alpha decreases collagen gene transcription, collagen mRNA levels, and collagen production in cultured fibroblasts.

Cell Division↗
PubMed1987

Acetaldehyde increases collagen gene transcription in cultured human fibroblasts.

Acetaldehyde, the first metabolite of ethanol, mediates many of the biological effects of ethanol. We have previously shown that acetaldehyde, but not ethanol, stimulates collagen production in cultured human fibroblasts (Holt, K., Bennett, M., and Chojkier, M. (1984) Hepatology 4, 843-848). Here, we examined the effects of acetaldehyde on collagen gene expression. Confluent human fetal fibroblasts were incubated for up to 4 h in the presence of ascorbate (0.2 mM) alone or with the addition of either ethanol (12 mM) or acetaldehyde (200 microM). Acetaldehyde induced the production of collagen (up to 2.5-fold) and had a small inhibitory effect on procollagen secretion (-20%). The steady-state levels of mRNAs were measured by hybridizing total cellular RNA to specific cDNA probes at high stringency. Acetaldehyde increased the steady-state level of collagen alpha 1(I) and collagen alpha 2(I) mRNAs about 3-fold and had small effects on beta-actin mRNA (+50%) and collagenase mRNA (-50%). Northern blots revealed that the RNAs were intact and that acetaldehyde preferentially increased the abundance of the longer of the two collagen alpha 1(I) transcripts. Acetaldehyde increased both collagen alpha 1(I) and collagen alpha 1(III) transcriptional activity by 2.5-fold and had small effects on beta-actin and collagenase gene transcription. The increase in both collagen production and collagen mRNA levels induced by acetaldehyde was blocked by methylene blue, a scavenger of reducing equivalents. These data indicate that reducing equivalents, which enhance the formation and stability of acetaldehyde-protein adducts, may be required for acetaldehyde-stimulated collagen production. Thus, this study suggests that acetaldehyde increases collagen production by increasing collagen gene transcription in cultured human fibroblasts.

Acetaldehyde↗