In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin‐responsive cell clone, HC11, from the COMMA‐1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co‐cultivation with other cell types for the prolactin‐dependent in vitro induction of the endogenous beta‐casein gene by lactogenic hormones. Induction of beta‐casein mRNA is rapid and was detected 3 h after hormone stimulation. A prolactin‐dependent increase in the rate of transcription of the beta‐casein gene was shown in an in vitro nuclear transcription assay. beta‐Casein protein was detected in an immunoblot assay after 24 h, and further accumulated during 5 days of hormone treatment. To identify low‐abundance proteins induced directly after prolactin stimulation, mRNA was accumulated during 5 h of stimulation of HC11 cells with prolactin in the presence of cycloheximide. Following cycloheximide removal, the mRNA was translated into protein during a 60‐min [35S]methionine pulse and the proteins were resolved by DEAE ion exchange HPLC and SDS‐PAGE. A strong induction of a 120‐kd cytosolic protein was detected which was maximally expressed within 6 h of hormone stimulation.

Nitric oxide (NO) produced from L‐arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post‐transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non‐macrophage cell lines activates RNA binding by iron regulatory factor (IRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO‐induced binding of IRF to iron‐responsive elements (IRE) specifically represses the translation of transfected IRE‐containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Transcription from the ,i-casein milk protein gene promoter is induced by the synergistic action of glucocorticoid and prolactin hormones in the murine mammary epithelial cell line, HC11. We analyzed the binding of nuclear proteins to the promoter and determined their binding sites. Site-directed mutagenesis was used to determine the function of nuclear factor binding. During lactogenic hormone induction of HCll cells, the binding of two nuclear factors increased. The binding of two other nuclear factors, present in uninduced cells, decreased. The basal activity of the promoter could be increased to and above the level of the induced wild-type promoter when the recognition sequences of the negatively regulated factors were mutated. This suggests that the li-casein promoter is regulated by the relief of the repression of transcription. An essential tissue-specific factor was also found in nuclear extracts from the mammary glands of mice. Mutation of its recognition sequence in the j-casein promoter led to the abolition of the induction of transcription by lactogenic hormones. The DNA sequences recognized by all five of these nuclear factors are conserved in the promoters of different casein genes from several species, confirming their importance in the regulation of milk protein gene transcription.The tissue-and stage-specific expression of milk protein genes is the result of complex hormonal regulation exerted at both the transcriptional and posttranscriptional levels (35,39,42). 1-Casein is one of the most abundant proteins of murine milk. Studies on mammary explant cultures and on mammary epithelial cell lines revealed the preponderant role of prolactin and glucocorticoids in the regulation of P-casein gene transcription (3,19). cis-acting DNA sequences required for the regulation of the rat ,B-casein gene promoter were determined by using the HCll cell line (12), derived from the mammary epithelial cell line COMMA-1D (10). HC11 cells retain hormone responsiveness under simple culture conditions (3). Introduction of P-casein gene promoter-CAT (chloramphenicol acetyltransferase) constructs demonstrated that 338 bp 5' of the P-casein transcription start site was sufficient to confer hormone responsiveness to the CAT reporter gene (12). Prolactin and glucocorticoids individually have only a weak effect on P-casein-CAT transcription. Synergy of both hormones, which leads to the strong induction of ,-casein gene transcription, was observed. These effects were seen in confluent HC11 cells but not in growing cells.Regulation of gene transcription is mediated by the complex interplay of nuclear proteins binding to promoter or enhancer elements of eukaryotic genes (38). The concentration or the functional modifications of these positively or negatively acting factors determine the rate of transcription (24, 33). We are investigating the mechanism of synergy between members of the steroid and peptide hormone families which regulates ,-casein gene transcription. Little is known about how prolactin regulates transcription...

We have detected hormone response elements in the promoter region of the rat f3-casein gene that confer the synergistic action of prolactin and glucocorticoid hormones upon transcription of chimeric gene constructs. A 2800-base-pair (bp) rat (3-casein gene fragment containing 2300 bp of 5' flanking sequence was placed in front of a chloramphenicol acetyltransferase (CAT) reporter gene and stably transfected into the mouse mammary epithelial cell line HC11. Addition of prolactin or dexamethasone alone was sufficient for a modest induction of the fusion gene. The simultaneous presence of both hormones produced a strongly synergistic effect, which did not require the presence of insulin. Induction of the fi-casein-CAT gene was only observed in stably transfected confluent cell cultures. Analysis of a 5' deletion series of the j3-casein-CAT gene construct revealed a stepwise loss of hormone inducibility; 285 bp of 5' flanking sequence was sufficient to mediate the synergistic action of lactogenic hormones on expression. The response was reduced by half when compared with the construct containing 2300 bp of the 5' flanking region. Synergistic inducibility further decreased in deletion mutants between -285 and -265 and was completely abolished between -180 and -170. Thus, the 5' flanking region between -285 and -170 contains cis-acting sequences, which are required for mediating the effect of prolactin and dexamethasone.