Constitutive expression and hormonal regulation of male sexually differentiated cytochromes P450 in primary cultured rat hepatocytes

Experiments, predominantly performed in vivo, have shown that the pattern of growth hormone (GH) release from the pituitary gland is a major regulator of sex-specific cytochromes P450 (P450) in rats and other rodents. However, difficulty in constitutively expressing male-specific forms of P450 using in vitro models, such as primary cell culture, has impeded efforts to examine the direct actions of hormones on these enzyme forms. In the present study mRNA species for the male-specific P450 2C11 and 2C13, but not 3A2, were successfully expressed in primary hepatocytes cultured on a laminin-rich extracellular matrix (matrigel) in a serum-free, chemically defined medium containing insulin as the only hormone. When cells were exposed to GH (100 ng/ml), 2C11 mRNA expression was virtually abolished and 2C13 expression decreased to approximately 50% of control values, demonstrating that the negative regulation of these P450 forms by GH is a direct action on hepatocytes. Dexamethasone (DEX, 10^-8 m) increased the expression of 2C11 to 195% while decreasing 2C13 expression to 25% of control values. When GH and DEX were administered concurrently 2C11 was downregulated, indicating that GH is the dominant regulatory hormone for this form. l-Triiodothyronine (T₃) (10^-9 m) suppressed 2C11 (46% of control) but had no effect on 2C13 mRNA expression. The positive regulatory effect of glucocorticoids on 2C11 was also found to occur in vivo and demonstrated to operate predominantly at the transcriptional level. This study demonstrates that primary cultures of hepatocytes are a suitable in vitro model for studies on regulation of some male-specific P450 forms and that GH, DEX, and T₃ act directly on hepatocytes at a pretranslational level to regulate these forms.