The expression of casein gene in the mammary gland is stimulated by the synergistic actions of insulin, glucocorticoid and prolactin in vitro through enhancement of both transcription of the gene and stability of the transcripts. Our recent study suggests that protein synthesis is required for hormonal stimulation of casein gene transcription, but not for the increased stability of casein transcripts. To study the molecular mechanism of hormone action on casein gene expression, we have isolated the genomic clones of mouse beta-casein and the complete nucleotide sequence of the 6.8-kb casein gene and its immediate 6-kb (5') and 10-kb (3') flanking region has been determined. To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kb of the 51 and 1.6 kb of the 31 flanking sequences of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The chimeric gene was transfected into primary mouse mammary epithelial cells prepared from pregnant mice. Expression of the beta-casein-CAT chimeric gene required the syngeristic actions of insulin, hydrocortisone and prolactin. Expression of the chimeric gene was also dependent on the appropriate substratum, since the degree of hormonal induction of the chimeric gene was much higher in cells cultured on a reconstituted basement membrane (Matrigel) than in cells cultured on either type I collagen gel or plastic. Additional transfection experiments using a series of beta-casein-CAT constructs suggested the existence of positive and negative regulatory elements responsible for.hormonal induction. We have extended our studies to investigate the possible role of EGF in other organs and also the control mechanisms involved in EGF gene expression and to identify diseases associated with EGF deficiency. Our studies indicate that in diabetic mice, the level of EGF and its MRNA in the submandibular gland as well as circulating EGF are greatly reduced and that insulin treatment corrects the defect in EGF production. The possibility that EGF deficiency is involved in manifestation of pathological complications in diabetes mellitus is currently investigated.
