Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine ␤-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of ␤-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with ␥-interferon activation site-related motifs within the ␤-lactoglobulin promoter, which we show are required for matrix dependence of ␤-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.Cell-matrix interactions are critical for regulating the phenotype of many cells. In mammary gland, basement membrane is necessary for the prolactin-mediated control of lactation (1-3). However, the mechanism by which extracellular matrix (ECM) 1 influences differentiation in mammary epithelial cells has not been elucidated. We have shown previously that functional ␤ 1 integrins are required for mammary differentiation (4) and that the basement membrane component laminin-1 directs milk protein gene transcription coordinately with prolactin (5).The prolactin pathway is driven through the protein tyrosine kinase Jak2 (6 -10) and one of its substrates, the transcription factor Stat5 (11,12). Stat factors associate with cytokine receptors following ligand binding and subsequently become phosphorylated by receptor-associated Jaks. They then dimerize and translocate to the nucleus where they bind specific DNA sequence motifs, thus contributing to transcriptional activation (13)(14)(15).In this paper we examine whether an element of the prolactin signaling pathway is modulated directly by cell-matrix interactions, thereby mediating the ECM control of transcription. Using primary and first passage cultures of mammary epithelial cells, we demonstrate that the activity of the promoter for the milk protein ␤-lactoglobulin (BLG) is dependent on basement membrane and that Stat5 recognition sites within this promoter are required for transcription. Moreover, we show that the ability of Stat5 to bind its cognate DNA sequence within the BLG promoter requires cell interactions with both basement membrane and prolactin. Thus, matrix and cytokine signals converge on a single pathway.Our data demonstrate, for the first time, that the activity of a Stat transcription factor is a target for regulation by ECM. This establishes a novel signaling...

Physiological apoptosis is induced by a switch from survival to death signalling. Dysregulation of this process is frequently associated with cancer. A powerful model for this apoptotic switch is mammary gland involution, during which redundant milk-producing epithelial cells undergo apoptosis. Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of this switch but the mechanism has not yet been defined. Stat3-dependent cell death during involution can be blocked by activation of Akt/protein kinase B (PKB), a downstream effector of the phosphoinositide-3-OH kinase (PI(3)K) pathway. Here we show that expression of the PI(3)K regulatory subunits p55alpha and p50alpha is induced by Stat3 during involution. In the absence of Stat3 in vivo, upregulation of p55alpha and p50alpha is abrogated, levels of activated Akt are sustained and apoptosis is prevented. Chromatin immunoprecipitation assays show that Stat3 binds directly to the p55alpha and p50alpha promoters in vivo. Overexpression of either p55alpha or p50alpha reduces levels of activated Akt. We propose a novel mechanism in which Stat3 regulates apoptosis by inducing expression of distinct PI(3)K regulatory subunits to downregulate PI(3)K-Akt-mediated survival signalling.

We have investigated the activity of STAT family members throughout a mammary developmental cycle. Transcripts for Stat 5 were upregulated during pregnancy whilst STAT1 and STAT3 mRNAs were expressed at constant levels. DNA binding complexes containing both STAT5a and 5b showed differing affinities for two naturally occurring STAT5 binding ~ites. In the involuting mammary gland STAT5 activity decreased whereas STAT3 was specifically activated. These observations reveal a complex pattern of activation of STAT tactors during mammary growth, differentiation and remodelling and provide the first evidence for the involvement of STAT3 in development of the mammary gland. ~. IntroductionSignal transducer and activator of transcription (STAT) !actors are latent cytoplasmic transcriptional regulators acti-'ated by cytokines and growth factors. Janus kinases (JAKs) tssociate with cytokine receptors and phosphorylate STAT actors on a particular tyrosine residue. Activated STATs hen form either homo of heterodimers and translocate to he nucleus where they bind to variations of a consensus paindrornic 9 bp recognition sequence [1]. To date six STAT amily members have been identified.The cytokine prolactin (PRL) promotes the growth and ,tifferentiation of the mammary gland. During lactation, i'RL stimulates the sustained activation of STAT5, a factor hat is essential for high level transcription of milk protein :genes [2,3]. STAT5 is encoded by two closely related genes, gTAT5a and 5b [4,5]. We have shown previously that the ~roximal 400 bp promoter of the milk protein gene ~-lactoglobulin (BLG) has three STAT factor binding sites and that hese sites have different affinities for STAT5 [6]. Additional "actors that modulate growth of the mammary gland such as growth hormone, epidermal growth factor (EGF) and fibro~last growth factor (FGF) have been shown to activate -;TATs 1, 3 and 5 in tissue culture cells. Therefore, the ";TAT factor binding sites in the promoters of milk protein genes, such as BLG, may not only act as PRL response elenents but also serve to integrate the array of signals that ~'ontrol differentiation of the mammary gland."Corresponding author. Fax: (44) (131) We investigated the expression and binding activity of STATs 1, 3, 5a and 5b throughout the mammary gland developmental cycle. STAT3 and STAT5 are activated in a reciprocal fashion, suggesting different roles for these factors during mammary development. Complexes containing STATs 5a and 5b are found from early pregnancy through lactation and have different relative affinities for the two highest affinity sites in the BLG promoter. Interestingly, STAT3 is most strongly activated during early involution when apoptosis is initiated. Materials and methods RNA extraction and Northern analysisRNA was isolated from HCll cells and mouse mammary gland tissue at various stages of development by the acid-phenol-guanidine thiocyanate single-step method [7]. Following electrophoresis on a 1.5% agarose gel containing 3.8% formaldehyde, RNA was transferre...

The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.