Abstract
HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with the lactogenic hormones prolactin, insulin, and glucocorticoids results in transcription of the beta-casein gene. Prolactin induces a signaling event which involves tyrosine phosphorylation of the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Here we show that HC11 cells express two Stat5 proteins, Stat5a and Stat5b. Phosphopeptide and phosphoamino acid analysis of Stat5a and Stat5b immunoprecipitated from phosphate-labeled HC11 cells revealed that both proteins were constitutively phosphorylated on serine. Lactogenic hormone treatment resulted in the appearance of a tyrosine-phosphorylated peptide in both Stat5 proteins. Consistent with this observation, a Western blot analysis of Stat5a and Stat5b showed that lactogenic hormones induced a rapid, transient increase in phosphotyrosine which paralleled the binding of Stat5 to its cognate recognition sequence in the beta-casein gene promoter. Lactogenic hormone treatment of the HC11 cells also led to a rapid activation of the mitogen-activated protein (MAP) kinase pathway. We examined the role of this pathway in beta-casein transcription using a specific MAP kinase kinase inhibitor, PD98059. Concentrations of PD98059 which completely abrogated lactogen-induced MAP kinase activation did not affect the phosphorylation state of Stat5, its DNA binding activity, or transcriptional activation of a beta-casein reporter construct. This indicates that the MAP kinase pathway does not contribute to lactogenic hormone induction of the beta-casein gene.
Highlights
HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression
The amplification capacity of the mitogen-activated protein (MAP) kinase cascade is well known [22], we cannot exclude that a MEK kinase distinct from Raf-1 might contribute to the hormone induction of MEK-1 and extracellular signalregulated kinase-2 (ERK2) in these cells [34]
In the present study we investigated the role of the MAP kinase pathway in lactogen-induced -casein promoter function and Stat5 phosphorylation
Summary
Signal transducer and activator of transcription; GAS, interferon-␥ activated site; MAP kinase, mitogenactivated protein kinase; MEK, MAP kinase kinase; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; ECM, extracellular matrix; IFN, interferon. Stat in Mammary Epithelial Cells data demonstrate that MAP kinase activation is not involved in the transcriptional induction of the -casein gene mediated by the lactogenic hormones
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