LAP/C/EBP␤ is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP␤ mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP␤ gene transcription. Deletion analysis of the 5-flanking region, located upstream of the start site of transcription in the LAP/C/EBP␤ gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP␤ promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP␤ inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP␤ gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP␤ mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP␤ transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP␤ via CREB under different physiological conditions.

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LAP/C/EBP␤ is a member of the C/EBP family of transcription factors and contributes to the regulation of the acute phase response in hepatocytes. Here we show that IL-6 controls LAP/C/EBP␤ gene transcription and identify an IL-6 responsive element in the LAP/C/EBP␤ promoter, which contains no STAT3 DNA binding motif. However, luciferase reporter gene assays showed that STAT3 activation through the gp130 signal transducer molecule is involved in mediating IL-6-dependent LAP/ C/EBP␤ transcription. Southwestern analysis indicated that IL-6 induces binding of a 68-kDa protein to the recently characterized CRE-like elements in the LAP/C/ EBP␤ promoter. Transfection experiments using promoter constructs with mutated CRE-like elements revealed that these sites confer IL-6 responsiveness. Further analysis using STAT1/STAT3 chimeras identified specific domains of the protein that are required for the IL-6-dependent increase in LAP/C/EBP␤ gene transcription. Overexpression of the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in IL-6-mediated transcription of the LAP/C/ EBP␤ gene. These data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBP␤ gene transcription. Our analysis describes a new mechanism by which STAT3 controls gene transcription and which has direct implication for the acute phase response in liver cells.

OBJECTIVE-The nuclear receptor hepatic nuclear factor 4␣ (HNF4␣) is a master regulatory protein and an essential player in the control of a wide range of metabolic processes. Dysfunction of HNF4␣ is associated with metabolic disorders including diabetes. We were particularly interested in investigating molecular causes associated with diabetic nephropathy.RESEARCH DESIGN AND METHODS-Novel disease candidate genes were identified by the chromatin immunoprecipitation-cloning assay and by sequencing of immunoprecipitated DNA. Expression of candidate genes was analyzed in kidney and liver of Zucker diabetic fatty (ZDF) and of streptozotocin (STZ)-administered rats and after siRNA-mediated silencing of HNF4␣.RESULTS-We identified the calcium-permeable nonselective transient receptor potential cation channel, subfamily C, member 1 (TRPC1) as a novel HNF4␣ gene target. Strikingly, TRPC1 is localized on human chromosome 3q22-24, i.e., a region considered to be a hotspot for diabetic nephropathy. We observed a significant reduction of TRPC1 gene expression in kidney and liver of diabetic ZDF and of STZ-administered rats as a result of HNF4␣ dysfunction. We found HNF4␣ and TRPC1 protein expression to be repressed in kidneys of diabetic patients diagnosed with nodular glomerulosceloris as evidenced by immunohistochemistry. Finally, siRNA-mediated functional knock down of HNF4␣ repressed TRPC1 gene expression in cell culture experiments.CONCLUSIONS-Taken collectively, results obtained from animal studies could be translated to human diabetic nephropathy; there is evidence for a common regulation of HNF4␣ and TRPC1 in human and rat kidney pathologies. We propose dysregulation of HNF4␣ and TRPC1 as a possible molecular rationale in diabetic nephropathy.

Interleukin-6 (IL-6) triggers pivotal pathways in vivo.The designer protein hyper-IL-6 (H-IL-6) fuses the soluble IL-6 receptor (sIL-6R) through an intermediate linker with IL-6. The intracellular pathways that are triggered by H-IL-6 are not defined yet. Therefore, we studied the molecular mechanisms leading to H-IL-6-dependent gene activation. H-IL-6 stimulates haptoglobin mRNA expression in HepG2 cells, which is transcriptionally mediated as assessed by run-off experiments. The increase in haptoglobin gene transcription correlates with higher nuclear translocation of tyrosinephosphorylated STAT3 and its DNA binding. As H-IL-6 stimulates STAT3-dependent gene transcription, we compared the molecular mechanism between IL-6 and H-IL-6. Transfection experiments were performed with a STAT3-dependent luciferase construct. The same amount of H-IL-6 stimulated luciferase activity faster, stronger, and for a longer period of time. Dose response experiments showed that a 10-fold lower dose of H-IL-6 stimulated STAT3-dependent gene transcription comparable with the higher amount of IL-6. Cotransfection with the gp80 and/or gp130 receptor revealed that the effect of H-IL-6 on STAT3-dependent gene transcription is restricted to the gp80/gp130 receptor ratio. High amounts of gp130 increased and high amounts of gp80 decreased the effect on H-IL-6-dependent gene transcription. To investigate the in vivo effect of H-IL-6 on gene transcription in the liver, H-IL-6 and IL-6 were injected into C3H mice. H-IL-6 was at least 10-fold more effective in stimulating the DNA binding and nuclear translocation of STAT3, which enhances haptoglobin mRNA and protein expression. Thus H-IL-6 stimulates STAT3-dependent gene transcription in liver cells in vitro and in vivo at least 10-fold more effectively than IL-6. Our results provide evidence that H-IL-6 is a promising designer protein for therapeutic intervention during different pathophysiological conditions also in humans.

The purpose of this study was to develop a protocol suitable for membrane protein extraction from limited starting material and to identify appropriate conditions for two-dimensional (2-D) gel electrophoresis. We used A549 cells, a human alveolar type II cell line, and evaluated three protein extraction methods based on different separation principles, namely protein solubility, detergent-based and density-based organelle separation. Detergent-based extraction achieved the highest yield with 14.64% +/- 2.35 membrane proteins but sequential extraction with 7.35% +/- 0.78 yield and centrifugal extraction with 4.1% +/- 0.54 yield produced the purest fractionation of membrane proteins. Only the sequential and the detergent-based extraction proved suitable for small volumes of starting material. We identified annexin I + II, electron transfer flavoprotein beta-chain, H(+)-transporting ATP synthase, mitofilin and protein disulfide isomerase A3 as membrane and cytokeratin 8 + 18, actin and others as soluble proteins using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis and started to map the A549 cell proteome. Our data suggest that membrane proteins can be extracted efficiently from small samples using a simple sequential protein extraction method. They can be separated and identified successfully using optimized conditions in 2-D gel electrophoresis. The presented methods will be useful for further investigations of membrane proteins of alveolar and bronchial carcinomas.

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