ABSTRACT

We have explored the regulation of transforming growth factor beta (TGF-beta) activity in tissue repair by examining the interactions of Zf9/core promoter-binding protein, a Kruppel-like zinc finger transcription factor induced early in hepatic stellate cell (HSC) activation, with promoters for TGF-beta1 and TGF-beta receptors, types I and II. Nuclear extracts from culture-activated HSCs bound avidly by electrophoretic mobility shift assay to two tandem GC boxes within the TGF-beta1 promoter but minimally to a single GC box; these results correlated with transactivation by Zf9 of TGF-beta1 promoter-reporters. Zf9 transactivated the full-length TGF-beta1 promoter in either primary HSCs, HSC-T6 cells (an SV40-immortalized rat HSC line), Hep G2 cells, or Drosophila Schneider (S2) cells. Recombinant Zf9-GST also bound to GC box sequences within the promoters for the types I and II TGF-beta receptors. Both type I and type II TGF-beta receptor promoters were also transactivated by Zf9 in mammalian cells but not in S2 cells. In contrast, Sp1 significantly transactivated both receptor promoters in S2 cells. These results suggest that (a) Zf9/core promoter-binding protein may enhance TGF-beta activity through transactivation of both the TGF-beta1 gene and its key signaling receptors, and (b) transactivating potential of Zf9 and Sp1 toward promoters for TGF-beta1 and its receptors are not identical and depend on the cellular context.

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the aministration ofheparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 (6).Studies in vitro indicate that HL possesses both triglyceride hydrolase and phospholipase activities, and it has a high affinity for HDL particles (1). Administration of specific antibodies against HL in cats, rats, and monkeys indicated a role for this enzyme in the conversion of very low density lipoprotein (VLDL) remnants to low density lipoproteins (LDL) (7-9), in the metabolism of apolipoprotein (apo) B48-containing lipoproteins (10-12), and in the conversion of HDL2 to HDL3 (13,14). These findings are consistent with the phenotype of HL deficiency in human subjects. This disorder, in which the premature development of atherosclerosis is a cardinal feature, is characterized by elevated total plasma triglycerides and cholesterol corresponding to an increase in the amount of triglyceride-rich . However, the precise role of HL in lipoprotein metabolism remains unclear.To elucidate the physiological roles of HL, we generated transgenic rabbits that express human HL only in the liver, a major site of metabolic activity. The rabbit was selected as a model for studying HL functions because it has naturally decreased activity of this enzyme; it has about 1/10th as much activity as that of the rat (18, 19). Our results demonstrate that HL plays a key rate-limiting role in both IDL andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8724HDL metabolism; it has dramatic effects on the distribution and composition of these particles in plasma, with concomitant effects on plasma cholesterol levels. To our knowledge, this study is the first report of transgenic rabbits that overexpress an enzyme involved in lipid metabolism. MATERIALS AND METHODSCreation of the Human HL Constructs. Vectors designed for liver-specific expression contained sequences from the human apoE gene: 3 kb (plivhHL1) or 5 kb (plivhHL2) of 5'-flanking sequence, the first exon, first intron, the first six nucleotides (a nontranslated portion) of the second exon, a polylinker for cDNA insertion, the distal 92 nucleotides in the noncoding region of the fourth exon, the proximal 114 nucleotides of 3'-flanking sequence (20), and the hepatic control region (21, 22) of the apoE/C

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non‐permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C‐terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.