Differential regulation of members of the family of signal transducers and activators of transcription during mammary gland development.

Abstract

The JAK-STAT pathway is a direct signalling pathway from the cell surface to the nucleus which is utilised by the cytokine receptor superfamily [ 11. Upon ligand engagement, receptor chain(s) aggregate and this results in the activation of the catalytic activity of the associated Janus kinases (JAKs) by transphosphorylation. Subsequent phosphorylation of the receptor allows docking of specific members of the family of signal transducers and activators of transcription (STAT) factors which, upon phosphorylation on a single tyrosine residue, dimerise and translocate to the nucleus where the activated dimer binds to its recognition sequence. Seven members of the STAT family have been identified and of these, STAT5 is encoded by two closely related genes. We have previously shown that the promoter of the milk protein gene p-lactoglobulin (BLG) has three STAT factor binding sites within the proximal 400bp region and that these sites have different affinities for STAT5 [2]. We demonstrated, by site-directed mutagenesis of combinations of these motifs, that at least two functional sites are required for the transcriptional activation of BLG by STAT5 in transgenic mice and that mutation of a single site does not abolish the response to prolactin [3]. This suggests some synergism between the sites which may involve the formation of larger complexes or the interaction with additional factors such as nuclear factor 1 which has multiple binding sites in the BLG promoter [2]. We have also shown that STAT5 is a target for regulation by extracellular mamx [4] and that STAT factors are aberrantly activated in invasive breast cancers [5]. These results suggest that STATs may play a role in normal mammary gland development. STAT5 is expressed in most tissues and cell types. STATsl and 3, which are activated in response to a different subset of cytokines, are also widely expressed. In order to determine the specific requirement for individual STAT factors during mammary gland development, we investigated the expression and binding activity of STATs 1, 3, 5a and 5b throughout a complete cycle of mammary gland development. The developmental regulation of STAT5 binding activity during pregnancy, lactation and involution in the mouse mammary gland was investigated by electrophoretic mobility shift assay (EMSA). The three STAT binding sites in the BLG promoter were used as probes. The sequence of these motifs is shown in Figure 1 and compared with the STAT5 consensus.