Studies on growth hormone regulation of the CYP2C12 gene in rat liver

Growth hormone (GH) is secreted from the pituitary gland in a sexually dimorphic manner in the rat. This leads to a sexual dimorphism in the expression of a number of hepatic GH-target genes including members of the cytochrome P450 (CYP) family. Transcription of the female specific CYP2C12 gene is dependent on continuous presence of GH in serum, which characterizes the female rat. To understand which component(s) of the GH secretion pattern that is recognized as male or female by the hepatocyte, it is essential to understand how GH transduces its signal into the cell. This thesis focuses on elucidating signaling events in response to continuous GH exposure and the expression of the female specific CYP2C12 gene. GH elicits its effects by dimerizing membrane bound receptors (GHR). When primary rat hepatocytes were exposed to increasing doses of rat GH (rGH), the expression of P450 2C12 mRNA reached a plateau. The induction of IGF-I, a classical GH response gene, appeared biphasic. This is in line with a sequential binding mechanism, as described for hGH. When hepatocytes were treated with a site 2 mutant, G118RrGH, an unexpected agonistic effect was observed. The lack of antagonism together with the difficulty in achieving bell-shaped dose-response curves with rGH, indicate that the binding sites of rGH have similar affinity. Moreover, the agonistic effect of G118RrGH was bell-shaped, indicating an interaction with two receptors. This could imply that G118RrGH, via its site 2, can interact with another receptor than GHR. GH regulates expression of CYP2C12 at the transcriptional level. In gel shift experiments, using an element in the proximal promoter of CYP2C12, binding of HNF-3 proteins was observed with liver nuclear extracts from rats devoid of GH, and a complex distinct from the HNF-3 complexes was detected with liver nuclear extracts from rats exposed to GH. The bound protein was identified as HNF- 6, a liver enriched transcription factor. Transient cotransfection experiments showed that both HNF-3beta and HNF-6 could transactivate CYP2C12-luciferase constructs, indicating a physiological function of these transcription factors in the regulation of the gene. The CYP2C12 gene harbors Stat5 binding elements both in the 5flanking region and in the 3 untranslated region (UTR). Interestingly, several different STAT5 complexes were formed on the 3UTR element. In addition to full-length STAT5 also carboxy-truncated STAT5 (STAT5beta)- proteins, lacking their transactivating domain, bound to this element. Moreover, in transient transfection studies, we demonstrated that the presence of the 3UTR element reduced GH activation of a CYP2C12-luciferase construct conveyed by the 5-STAT5 elements. Thus, it is possible that binding of STAT50 to the 3UTR element could be of relevance for the GH-dependent and sex-specific expression of CYP2C12. The family of SOCS/CIS proteins represents negative regulators of cytokine signal transduction. We demonstrated that SOCS-2 and CIS expression in rat liver is dependent on the presence of GH. Furthermore, in cotransfection studies, overexpression of CIS, but not SOCS-2, inhibited GH-induced transactivation of a STAT5-responsive reporter gene construct. This suggests that GH induction of CIS could be one mechanism behind the desensitization of GH-dependent JAK/STAT5 signaling in liver cells continuously exposed to GH