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Biomedical subjects

D A Brenner

Publications and source records attributed to D A Brenner.

At least 109 records · Page 6Linked to original sources
PubMed1995

Activation of Jun kinase is an early event in hepatic regeneration.

Compensatory hepatic regeneration after partial hepatectomy (PH) is dependent upon the extent of resection. This study analyzes the regulation of the AP-1 transcription factor c-Jun during hepatic regeneration. There is a progressive increase in c-jun mRNA levels after sham operation, one-third PH, and two-thirds PH. A concomitant increase in AP-1 binding activity is also observed. The c-Jun protein is a major constituent of the AP-1 complex in quiescent and early regenerating liver. The activity of c-Jun nuclear kinase (JNK), which phosphorylates the activation domain of the c-Jun protein, is markedly stimulated after one-third PH. JNK1 or an immunologically related kinase is a constituent of this stimulated JNK activity after PH. When primary cultures of adult rat hepatocytes are incubated with epidermal growth factor or transforming growth factor-alpha, AP-1 transcriptional activity is increased and the activation domain of the c-Jun protein is further potentiated. Phosphopeptide mapping of the endogenous c-Jun protein in proliferating cultured hepatocytes demonstrates phosphorylation of the c-Jun activation domain. Combining the results of these in vivo and culture studies, we conclude that the minimal stimulation of one-third PH activates JNK, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes.

Animals↗
PubMed1995

Insulin-like growth factor-I and epidermal growth factor interact to regulate growth and gene expression in IEC-6 intestinal epithelial cells.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) exert trophic effects on bowel mucosa. Each growth factor uses a distinct tyrosine kinase receptor but the receptors share some common signal transduction pathways. In other systems, regulation of cell growth involves interactions among multiple growth factors. We used IEC-6 cells, an epithelial cell line established from rat small intestine, to test whether EGF and IGF-I interact to regulate intestinal epithelial cell growth. EGF and IGF-I alone each stimulated DNA synthesis in IEC-6 cells. EGF was more potent than IGF-I, and effects of the two growth factors in combination were synergistic. Characterization of the IGF system [IGF-I, IGF-II, type 1 IGF receptor, and six IGF binding proteins (IGFBPs) 1-6] revealed that IEC-6 cells express high levels of type 1 IGF receptor mRNA, low or undetectable levels of IGF-I and IGF-II mRNAs, and mRNA for only one of the six IGFBPs, IGFBP2. IGF-I decreases expression of type 1 IGF receptor mRNA in IEC-6 cells and EGF attenuates this effect. EGF and IGF-I both reduce IGFBP2 mRNA expression, and inhibitory effects of EGF and IGF-I in combination are additive. EGF reduces IGFBP2 accumulated in conditioned medium relative to levels observed with IGF-I alone. These effects of EGF on type 1 IGF receptor expression and on levels of IGFBP2 mRNA and IGFBP2 in medium may contribute to synergistic mitogenic effects with IGF-I by promoting IGF-I responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Sp1 binding activity increases in activated Ito cells.

Ito cells are the primary cell type in the liver responsible for increased type I collagen production observed during fibrogenesis. After a fibrogenic stimulus, Ito cells change from their normal quiescent state to an activated state. In this study, we examined the expression of the alpha 1(I) collagen gene in Ito cells that were freshly isolated from normal rat liver (quiescent) and Ito cells that were activated by culture on plastic. Northern blots showed that alpha 1(I) collagen messenger RNA levels were low in freshly isolated Ito cells and Ito cells cultured on plastic for up to 4 days, but were induced after 7 days in culture. To determine the location of important transcriptional regulatory elements within the 5'-flanking region of the alpha 1(I) collagen gene, transient transfections using various portions of the alpha 1(I) collagen gene promoter linked to the luciferase gene were performed in cultured Ito cells. As in fibroblast cells, only 220 base pairs (bp) of immediate 5'-flanking sequence of the collagen alpha 1(I) gene was necessary for efficient expression of the reporter gene. Deoxyribonuclease I footprinting analysis and mobility shift assays showed dramatic differences in the DNA binding proteins that interact with the 220 base pair promoter region of the alpha 1(I) collagen gene between freshly isolated and activated Ito cells. Sp1 binding activity was greatly increased in activated Ito cells, as was binding activity for CCAAT binding factor (CBF), a transcription factor previously shown to interact with the alpha 2(I) collagen gene promoter. The increased Sp1 binding activity to the alpha 1(I) collagen gene promoter involves a posttranscriptional event, as shown by Northern and Western blots. Therefore, we show that activated Ito cells have a different pattern of transcription factor binding activity interacting with the alpha 1(I) collagen gene promoter compared with quiescent Ito cells, including increased Sp1 binding activity, and that this may contribute to the increase in alpha 1(I) collagen gene expression during fibrosis.

Animals↗
PubMed1995

Current and prospective therapies for hepatic fibrosis.

The prospects for new, effective treatments for hepatic fibrosis are real. Currently there are several drugs being evaluated in clinical trials, including polyunsaturated lecithin and prostaglandins. In addition, the authors' (J.F.T. and D.A.B.) knowledge of the pathophysiology of hepatic fibrosis has increased dramatically during the past few years. These advances are being incorporated into novel therapies for hepatic fibrogenesis such as TGF-beta antagonists. Hopefully, definitive treatments for hepatic fibrosis will become available in the near future.

Collagen↗
PubMed1995

Post-transcriptional regulation of collagen alpha 1(I) mRNA in hepatic stellate cells.

Posttranscriptional events may play an important role in regulating collagen alpha 1(I) mRNA in hepatic stellate cells. Quiescent stellate cells contain a very low level of this mRNA, but treatment of the cells with Actinomycin D or cycloheximide increases the mRNA accumulation 5 fold in 3 hours. The same treatment has no effect on activated stellate cells, which produce a large amount of the collagen alpha 1(I) mRNA. This suggests that there is a labile degrading activity which normally prevents accumulation of the mRNA in quiescent cells. Half life of the mRNA in activated cells is about 48 hours. We detected a sequence specific RNA binding activity present in these cells which binds to the C-rich sequence in the 3' UTR of collagen mRNA. Such binding can not be detected in quiescent cells and may be responsible for the stabilization of the mRNA in activated stellate cells. Thus, collagen alpha 1(I) mRNA levels in stellate cells may be modulated by negative regulation in quiescent cells and by positive regulation in activated cells.

Animals↗
PubMed1994

A single promoter directs both housekeeping and erythroid preferential expression of the human ferrochelatase gene.

We have isolated and characterized the 5'-flanking region of the gene for human ferrochelatase (HFC), the last enzyme of the heme biosynthetic pathway. The proximal promoter of the gene is contained within a region that structurally resembles a CpG island and is devoid of general cis elements such as TATA and CAAT boxes. Recognition sites for the ubiquitous Sp1 family of transcription factors, as well as for the erythroid-specific trans-acting factors NF-E2 and GATA-1 were found, and binding of regulatory proteins to these elements was analyzed by in vitro DNase I protection assays. The contribution of the various cis elements to both ubiquitous and erythroid preferential expression of the HFC gene was assessed by using transient transfection assays. These showed that a minimal Sp1-driven promoter devoid of the upstream erythroid-specific elements was sufficient for erythroid preferential expression of the HFC gene. However, elimination of a repressor sequence lying between the minimal promoter and the erythroid-specific elements resulted in high levels of expression in human erythroleukemic K562 cells only when the cis elements recognized by GATA-1 and NF-E2 were present, suggesting that the activity of these factors is regulated by a downstream repressor in erythroid cells.

Animals↗
PubMed1994

Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase.

Tumor necrosis factor alpha (TNF alpha) has multiple biological functions including the prolonged activation of the collagenase and c-jun genes, which are regulated via their AP-1 binding sites. We show that incubating human fibroblasts with TNF alpha induces prolonged activation of JNK, the c-Jun kinase, which phosphorylates the transactivation domain of c-Jun. Furthermore, an immune complex kinase assay specifically demonstrates that TNF alpha stimulates the activity of JNK1, the recently described predominant form of JNK. TNF alpha also produces a small and transient increase in extracellular signal-regulated kinase (ERK) activity and no measured increase in Raf-1 kinase activity. On the other hand, epidermal growth factor causes a prolonged activation of Raf-1 kinase and ERK activity and a smaller, more transient activation of JNK, whereas the phorbol ester phorbol 12-myristate 13-acetate causes a small stimulation of Raf-1 kinase and a pronounced stimulation of ERK activity. The activation of JNK by TNF alpha does not correlate with Raf-1 or ERK activity. The kinetics of Raf-1, ERK, and JNK induction by epidermal growth factor, phorbol 12-myristate 13-acetate, or TNF alpha indicate distinct mechanisms of activation in human fibroblasts.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1994

The CAAX peptidomimetic compound B581 specifically blocks farnesylated, but not geranylgeranylated or myristylated, oncogenic ras signaling and transformation.

Recently developed CAAX peptidomimetic compounds have been shown to be potent and specific inhibitors of farnesyl protein transferase activity and to block the growth of Ras-transformed cells. However, whether this growth inhibitory action is specifically a consequence of blocking oncogenic Ras signaling has not been determined. To address this question, we have utilized mutants of the normally farnesylated oncogenic Ras protein (Ras-F) that are modified by alternative lipids, a geranylgeranyl isoprenoid (Ras-GG) or the fatty acid myristate (Myr-Ras), to determine the specificity of the CAAX peptidomimetic compound, B581. Like Ras-F, both Ras-GG and Myr-Ras are membrane-associated and transforming. Unexpectedly, NIH 3T3 cells transformed by each of the three Ras mutants underwent morphological alteration to a less transformed, but not normal, morphology. However, B581 inhibited the ability of only Ras-F-transformed cells, but not Ras-GG- or Myr-Ras- (or Raf-) transformed cells, to grow in soft agar. Furthermore, although all three lipid-modified versions of Ras stimulated mitogen-activated protein kinase activation, and both Jun and Elk-1 transcriptional activity, B581 inhibited only farnesylated Ras activation of these three downstream components of Ras signaling. Therefore, B581 prevents the growth of Ras-transformed cells by specifically antagonizing Ras-mediated signaling.

3T3 Cells↗
PubMed1994

Oncogenic Ras activates c-Jun via a separate pathway from the activation of extracellular signal-regulated kinases.

c-Jun transcriptional activity is augmented by expression of oncogenic Ras and Raf proteins. This study demonstrates a direct correlation between Ras transforming activity and c-Jun activation, supporting an important role for c-Jun in transformation by Ras. Since we observed that Ras activated c-Jun transcriptional activity by increasing phosphorylation of the c-Jun activation domain at residues Ser-63/Ser-73 and that oncogenic Ras proteins activated extracellular signal-regulated protein kinases (ERK1 and ERK2) (also known as mitogen-activated protein kinases), we evaluated the possibility that ERKs were directly responsible for c-Jun activation. Coexpression of wild-type ERKs with oncogenic Ras proteins potentiated, while kinase-defective ERKs inhibited, Ras-induced transcriptional activation from the Ras-responsive element (Ets-1/AP-1) present in the NVL-3 enhancer and the serum-response element in the c-fos promoter. In contrast, coexpression of either wild-type or kinase-defective ERKs inhibited Ras and Raf activation of c-Jun transcriptional activity. Thus, although activation of both ERK and c-Jun are downstream consequences of activation of the Ras signal transduction pathway, our results suggest that Ras-induced c-Jun phosphorylation and transcriptional activation are not a direct consequence of ERK1 and ERK2 activation.

3T3 Cells↗
PubMed1994

DNA methylation represses the murine alpha 1(I) collagen promoter by an indirect mechanism.

Several lines of evidence indicate that DNA methylation plays a role in the transcriptional regulation of the murine alpha 1(I) collagen gene. To study the molecular mechanisms involved, a reporter gene construct containing the alpha 1(I) promoter and part of the first exon linked to the luciferase gene (Col3luc) was methylated in vitro and transfected into murine fibroblasts and embryonal carcinoma cells. Methylation resulted in repression of the alpha 1(I) promoter in both cell types, although it was less pronounced in embryonal carcinoma cells than in fibroblasts. The extent of repression depended on the density of methylation. DNase footprint and mobility shift assays indicated that the trans-acting factors binding to the alpha 1(I) promoter and first exon are ubiquitous factors and that their DNA binding is not inhibited by methylation. Transfection of Col3luc into Drosophila SL2 cells together with expression vectors for the transcription factors Sp1 and NF-1 showed that DNA methylation also inhibits the alpha 1(I) promoter in nonvertebrate cells, although to a much lesser extent than in murine cells. However, Sp1 and NF-1 transactivated the unmethylated and methylated reporter gene in SL2 cells equally well, confirming that these factors can bind and transactivate methylated DNA and indicating that DNA methylation represses the alpha 1(I) promoter by an indirect mechanism. This was further confirmed by cotransfection experiments with unspecific methylated competitor DNA which partially restored the activity of the methylated alpha 1(I) promoter. Our results suggest that DNA methylation can inhibit promoter activity by an indirect mechanism independent of methyl-C-binding proteins and that in vertebrate cells, chromatin structure and methyl-C-binding proteins cooperatively mediate the transcriptional inhibitory effect of DNA methylation.

Animals↗
PubMed1994

Tumor necrosis factor-alpha induces c-jun during the regenerative response to liver injury.

After liver injury, remaining hepatocytes proliferate to regenerate the liver. Although the precise mechanisms that initiate and localize regeneration are unknown, local induction of c-jun is a critical, early step in the response. Treatment of rats with antibodies to tumor necrosis factor-alpha (TNF-alpha), a mediator of liver injury, inhibits regenerative induction of jun nuclear kinase activity and nuclear c-jun expression and alters the DNA binding activity of the c-jun transcription factor, AP-1, in liver. Pretreatment with anti-TNF antibodies does not affect pulmonary or renal c-jun expression or AP-1 binding activity post-partial hepatectomy. In primary hepatocyte cultures, TNF-alpha directly promotes the proliferative actions of mitogens, supporting in vivo evidence that it sensitizes hepatocytes to mitogens. Thus local release of TNF may act in a paracrine fashion to initiate regeneration in the injured liver by promoting induction of critical growth-related genes, such as c-jun.

Animals↗
PubMed1993

Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Activation of activating protein 1 during hepatic acute phase response.

During an acute phase response following inflammatory stimuli, specific changes occur in the synthesis and secretion of many hepatic proteins. Because the expression of differentiated function requires the coordinated regulation of many genes, we investigated the activity of general and tissue-specific transcription factors using a rat liver model of the acute phase response induced by Freund's adjuvant. Nuclear extracts and RNAs were prepared throughout a 48-h posttreatment period. Mobility shift assays revealed increased binding activity by nuclear factor-kappa B, interleukin-6 (IL-6) responsive element binding protein, and activating protein 1 (AP-1). Two AP-1 complexes were induced during the acute phase response, and correlation between their presence and transcription activity was demonstrated by transfection studies. Elevated binding activity of AP-1 also correlated with elevated levels of c-jun, junD, junB, and c-fos mRNAs. Western blots showed elevated hepatic levels of c-Jun but not c-Fos proteins during the acute phase response. In addition, IL-6, tumor necrosis factor-alpha, and IL-1 beta, cytokine regulators of the acute phase response, stimulated expression of an AP-1 responsive reporter gene introduced by DNA-mediated transfection into adult rat hepatocytes in primary culture. These findings demonstrate the complexity of AP-1 hepatic transcription factor responses to humoral regulators with direct hepatocellular effects.

Acute-Phase Reaction↗
PubMed1993

Type I collagen gene regulation and the molecular pathogenesis of cirrhosis.

Cirrhosis is characterized by an increased deposition of extracellular matrix proteins, including type I collagen. Type I collagen is a product of two genes, alpha 1(I) and alpha 2(I), which are generally coordinately regulated. Since expression of type I collagen genes is increased during cirrhosis, understanding the structure and function of the regulatory components of the type I collagen genes should provide insight into the molecular pathogenesis of cirrhosis. This review will analyze the collagen alpha 1(I) gene with respect to chromatin structure, DNA methylation, regulation by agonists, and DNA-protein interactions.

Animals↗