Hepatocyte growth factor (HGF)-induced activation of the AP-1 complex : a comparison between normal and transformed rat hepatocytes

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HEPATOCYTE GROWTH FACTOR (HGF)-INDUCED ACTIVATION OF THE AP-1 COMPLEX : A COMPARISON BETWEEN NORMAL AND TRANSFORMED RAT HEPATOCYTES.

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The response of normal and transformed hepatocytes to HGF has been shown to differ, suggesting that different signal transduction pathways are triggered by this growth factor in normal and transformed cells, Activation of the Fos/Jun AP-1 dimeric transcription factor is a key event of the cell response to growth factors. We have therefore compared HGF-induced activation of AP-1 in normal and transformed rat hepatocytes, the 7777 cell line. In both cell types, increased DNA-binding activity to an AP-1 probe was induced 2-6h after HGF stimulation and it was completely prevented in the presence of cycloheximide. Analysis of the type of Fos and Jun proteins recruited to the AP1 dimers after HGF activation indicated differences in the

composition of the HGF-induced DNA-binding complexes in the 2 cell types. While Fra-1 containing dimers were present in both cell types, c-Jun- and c-Fos-containing complexes were detected in hepatocytes and 7777: cells, respectively. Very close patterns of induction of the fos and jun genes at the mRNA level were found in the 2 cell types with a rapid (30 min) and transient accumulation of c-fos, fosB, c-jun and junB mRNAs. However, induction of the genes at the protein level differed: c-Jun, JunB and Fra-1 accumulated in both cell types, but c-Fos levels increased only in 7777 cells. These results show that the patterns of AP-1 activation by HGF differs between normal and transformed hepatocytes. The differences are mainly at the traductional and post-traductional level, leading to the recruitment of different Fos/Jun proteins into the AP-1 dimers.

1

ENHANCED AND STABLE INTEGRATION OF PLASMID DNA INTO THE GENOME OF LIVER CELLS BY NON-VIRAL GENE TRANSFER VIA ADENO-ASSOCIATED VIRUS INVERTED TERMINAL REPEATS R. Steiaemald. W.H. Caselmann*. M. Alt. MPI f. Biochemie, Dept. of Virus Research, Martinsried, *Dept. of Medicine, University of Bonn, Germany Since most gene therapy strategies require permanent expression of transgenes, integrating vectors like recombinant adeno-associated virus (AAV) have been developed. The aim of this study was to adapt the highly effective mechanism of integration of AAV to plasmid DNA, which can easily be combined with tissue-specific non-viral vector systems, e.g. for liver gene therapy. HepG2 cells were transfected with plasmids containing the neomycin resistance gene flanked by the inverted terminal repeats (ITRs) of AAV. the only &-active elements required for integration of AAV. ITR and control plasmids integrated randomly at low rates, as deduced from the number of resistant colonies. This result as well as the transient expression of different reporter genes indicate that the plasmids do not persist episomaly. To improve integration efficiency, plasmids containing the rep gene of AAV were constructed. Rep is the mediator of site-specific integration of wildtype AAV into human chromosome 19. In the presence of Rep, integration efficiency of ITR plasmids increased about eightfold. The site specificity of integration of these plasmids is evaluated by Southern-blot analysis. These data provide evidence that non-viral gene transfer using AAV ITRs can efficiently be used to integrate plasmid DNA into the hepatocyte genome.

REGULATION

OF HGF GmEEXPRIZZION

BY ‘IGF-01

Liver Unit and Department Molecular Medicine, King’s College School of medicine and Dentistry, London, UK Introduction Transforming growth factor-01 (TGF-l31) inhibits Hepatocyte growth factor (HGF) secretion by fibroblasts and reduces the expression of the full length 6.0 kb HGF mRNA transcript by an unknown mechanism. The effect on the alternatively spliced 1.5 kb HGF gene transcript, which encodes the HGF antagonist protein HGF-NK2, has not been reported. Methods and Results The effect of TGF-Dl on HGF gene expression was investigated in MRC-5 cells, using Northern blot analysis. TGF-I31 inhibited expression of the 6.0 kb HGF mRNA in a dose dependent manner and at 1 @ml for 24 hours reduced levels to only 7% of control values, whereas there was little effect on the 1.5 kb HGF-NK2 mRNA. The fall in the 6.0 kb mRNA species was apparent at 3 hours, was maximal by 9 hours, persisted up to 36 hours and recovered to basal levels at 48 hours; it required TGF-I31 to be present for only the first 2 hours. Using actinomycin D to block RNA synthesis, it was observed that TGF-I31 increased the rate of degradation of the 6.0 kb HGF mRNA transcript but had little effect on the stability of the 1.5 kb HGF-NK2 mRNA. TGF-81 had no effect on HGF promoter activity in MRC-5 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing HGF gene 5’-flanking sequences and ribonuclease protection mapping demonstrated that TGF-I31 had no effect on the relative use of the transcription initiation sites. Conclusion The differential effect of TGF-I31 on expression of the alternatively spliced HGF mRNA transcripts in MRC-5 cells is mediated at the post transcriptional level and favours production of the truncated HGF antagonist protein.