Hormonal induction of beta-casein gene expression: requirement of ongoing protein synthesis for transcription

Hormonal induction of beta-casein gene expression was studied using a two-step culture of mouse mammary explants and RNA blotting analysis. The explants prepared from pregnant mice were cultured first for 4 days under nonlactogenic conditions to reduce the initial level of beta-casein transcripts and then induced to express beta-casein gene in the presence of insulin, hydrocortisone, and PRL for up to 3 days. Among the six different combination of hormones tested in the first incubation period, the combinations of insulin and epidermal growth factor was found to reduce the residual level of beta-casein transcripts in cultured explants to a nearly undetectable level and allow the highest induction in the second incubation period. The increase in beta-casein transcripts was detected as early as 30 min of induction with insulin, hydrocortisone, and PRL, and its level increased more than 100-fold over the initial level at 24 h. The increase in beta-casein transcripts was blocked by concomitant addition of the protein synthesis inhibitors cycloheximide or puromycin with the three hormones. Cycloheximide inhibited the increase in beta-casein gene transcription that was elicited by insulin, hydrocortisone, and PRL, but did not alter the stability of beta-casein transcripts. These results suggested that protein synthesis was required for hormonal activation of beta-casein gene transcription.