Abstract 488 Cytokines: Intracellular Signalling Platform, Tuesday, 5/4

The rat liver serpin family contains three highly homologous serine protease inhibitor (Spi) genes. Two (Spi 2.1 and Spi 2.3) are growth hormone (GH) responsive, while a third (Spi 2.2) is acute-phase-responsive. During induction of an acute-phase response (APR) by lipopolysaccharide (LPS) treatment, Spi 2.2 expression is activated while the expression of Spi 2.1 and Spi 2.3 is decreased. Several cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and interleukin-1β (IL-1β), are produced during induction of such an inflammatory response. We hypothesized that IL-6 would be the primary cytokine mediating the changes in Spi 2.1 and 2.2 expression after an APR induction. To study the the effects of these cytokines on the APR responses of Spi 2.1 and Spi 2.2, functional assays were carried out in primary rat hepatocyte cultures transfected with reporter constructs linked to the promoters of the Spi 2.1 and Spi 2.2 genes. Treatment of hepatocytes with both IL-6 (5-20 ng/ml) and GH (500 ng/ml) did not decrease the activity of Spi-(-275/+85)-CAT when compared to treatment with GH alone. In addition, treatment with IL-6 alone unexpectedly led to an induction of Spi 2.1-CAT activity similar to that of GH. However, simultaneous addition of IL-6 (0.5 ng/ml), TNFα (0.5 ng/ml) and IL-1β (0.5 ng/ml) in the presence of GH (50 ng/ml) led to a three fold reduction of the Spi 2.1-(-275/+85)-CAT activity. Control experiments performed with Spi 2.2-(-319/+85)-CAT showed that Spi 2.2 was responsive only to IL-6 and not to GH. Simultaneous treatment with GH, IL-6, IL-1β and TNFα led to only a modest reduction of the IL-6 response. Northern blots of hepatocytes that were treated with either IL-6 or GH showed that Spi 2.1 mRNA was induced by GH only, but not by IL-6. Simultaneous treatment with both GH and IL-6 led to a 3 fold reduction of the GH response. In contrast, Spi 2.2 mRNA was only induced by IL-6. Simultaneous treatment with IL-6 and GH did not alter its response to IL-6. To correlate these findings to the intact animal and to elucidate the mechanism by which the APR down-regulates Spi 2.1's GH response, hypophysectomized rats were treated with LPS for varying times, with each receiving GH (30 µg/100 g bw) 1 h before sacrifice. The results of electrophoretic mobility assays (EMSA) using radiolabeled GH-response element (GHRE) from the Spi 2.1 promoter and the hepatic nuclear proteins extracted from these animals showed that LPS treatment diminishes the binding of signal transducer and activator of transcription (Stat)5 to the GHRE in a time-dependent manner. We conclude that the modulation of Spi 2.1's GH response during induction of an APR is not mediated exclusively by IL-6, but involves several cytokines, and that both transcriptional and post-transcriptional modulation of expression occurs.

(Support from NIH DK32817)