Mammary Gland - Synergistic & Antagonistic Interactions of Transcription Factors of Milk Protein

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Synergistic-Antagonistic

Synergistic & Antagonistic Interactions of Transcription Factors of Milk Protein

1. INTRODUCTION

The activation of milk protein gene expression is an extensively studied example for the interaction of several signalling pathways. Long before the discovery of molecular details concerning the operation of the individual pathways, endocrinologists could demonstrate a synergism involving three hormones, namely the lactogenic hormones prolactin, glucocorticoids and insulin (reviewed). Later it became evident, that signals elicited by extracellular matrix components are also necessary for the activation of milk protein gene expression in mammary epithelial cells. Numerous other extracellular stimuli, most of them triggering the inhibition of milk protein gene expression, were discovered subsequently. Current knowledge indicates that many if not all of these extracellular signals influence milk protein gene expression at the level of transcription initiation. A summary of extracellular signals and transcription factors (TFs) implicated in the regulation of milk protein gene expression is given in Table 1. For the signals triggered by steroid hormones, prolactin, insulin, interleukin-4 (IL-4), and TNF-α, the transcription factors mediating the response to these stimuli have been identified. For other signals, such as insulin and TGF-β, the targeted transcription factor (TF) is either not known or the response to the signal is not dependent on the activation or repression of a TF. It should be noted that for several TFs with an established function in milk protein gene transcription such as NF-1, Oct- 1, CTF/NF-1 and Ets domain proteins, it has still to be established whether and how they are regulated by extracellular stimuli.

2. MODULAR ARRANGEMENT OF TRANSCRIPTION FACTOR BINDING SITES IN THE ß- CASEIN GENE PROMOTER

An important principle in directing the interaction between TFs is the modular arrangement of their binding sites in the DNA response regions of the regulated genes. Depending on the relative position of binding sites and the affinity of the TFs to these sites, cooperative binding or competition for binding sites can be observed, resulting in either synergy or antagonism. The structure of TF binding sites in the two lactogenic hormone response region (LHRR) of the β-casein gene has been studied in detail. Fig. 1 shows a schematic overview of the principle binding sites mapped in the rat, the bovine and the human gene and the conservation of these binding sites between species. A common feature of all LHRRs is the presence of multiple binding sites for CCAAT enhancer binding proteins and at least one binding site for STAT5. The human and ruminant proximal LHRR exhibits impaired functional activity in comparison to the LHRR of rodents.

Table 1. Extracellular signals and targeted TFs regulating milk protein gene transcription. So far, the activation of STAT5 by insulin has only been described in non-mammary tissues. Epidermal growth factor has a dual function on milk protein gene expression: it promotes the competence of mammary epithelial cells to express the β-casein gene, but represses the action of lactogenic hormones in terminal differentiated cells. A truncated version of the CCAAT enhancer protein C/EBP β is expressed in mammary epithelial cells (LIP) and is discussed to block the action of full length C/EBP β (LAP)

Effect on Transcription Ref.Extracellular Signal Targeted TF

Prolactin	STAT5	activation	6
interleukin-4	STAT6	activation	7
Glucocorticoids	Gl ucocorticoid receptor	activation	1
Progestins	Progesterone receptor	repression	1
Insulin	STAT5?	activation	3
Epidermal growth	STAT5	repression, (activation)	4,8,9
factor
Tumor necrosis factor	NF-κB	repression	10
aTransforming growth	?	repression	11
factor β
extracellular matrix	STATS, NF- 1 ?	activation	12,13
?	NF-1	activation	14-17
?	CCAAT enhancer	activation, repression	5,18
	binding proteins	(?)
?	Ets domain proteins	activation	19
?	octameric factor	activation	20
?	YY-1	repression	2 1,22

      This might relate to the lack of a second STAT5 binding site, which is required for the function of the LHRR in rodents (H. Weirich, unpublished results). In these species, activation of milk protein gene expression depends on a distal enhancer region with multiple C/EBP and STAT5 sites (Fig. 1).YY1 has been described as a factor participating in the repression of β-casein gene transcription by its binding to a site in the proximal LHRR. Due to the close proximity of binding sites, STAT5 inhibits binding of YY1. Such a STAT5 mediated relief of repression by YY1 has been postulated to be important for stimulation of β-casein gene expression. In the next sections two other examples for interactions of TFs with STAT5 are discussed. In both cases, a full understanding of their action on STAT5 signalling has to consider in addition mechanisms other than interactions mediated by the LHRR.



Fig. 1 Structural organisation of TF binding sites in the two defined regulatory regions of the β-casein gene. For rodents, the conserved transcription factor binding sites mapped in the sequence of the mouse and rat gene are shown. For ruminants, the depicted binding sites are identical for sheep, goat and cattle.

3. SYNERGY BETWEEN GLUCOCORTICOID RECEPTOR (GR) AND STAT5

     The proximal rat LHRR of the β-casein gene has been shown to direct the synergy between the GR and STAT5. Evidence obtained from immunoprecipitation experiments indicate protein-protein interactions between the GR and STAT5, which do not depend on the binding of the GR to DNA. These interactions appear to contribute to the functional synergy between GR and STAT5 However, mutational analysis of the GR binding sites in the β-casein gene promoter revealed that the structural integrity of GR binding sites is a prerequisite to achieve a synergy between these two TFs, indicating that binding of the GR to DNA is also important. A particularity of the binding of GR to the LHRR of milk protein genes is the recognition of half-palindromic sites, which do not function in the absence of activated STAT5 to mediate the action of GR on transcription. This mode of interaction provides an efficient means to restrict gene expression to conditions were both STAT5 and the GR are active.        In addition to the direct interactions between GR and STAT5 at the b-casein gene promoter, activated GR promotes STAT5 tyrosine pho~phorylation. Indirect actions of the GR, such as inhibition of the expression of cytokine inducible SH-2 domain proteins by glucocorticoids might be important for this effect and might explain the delayed effects and slow kinetics of glucocorticoid action on β-casein gene transcription. The left part of Fig. 2 illustrates the two levels of interaction between GR and STAT5.

Fig. 2 Cross-talk mechanisms between glucocorticoid,   prolactin and tumor necrosis-factor a induced signalling pathways in mammary epithelial cells.

4.  ANTAGONISM BETWEEN NF-kB AND STAT5

      Tumor necrosis factor a (TNF-a) has been shown to be a multifunctional regulator of mammary gland development with an inhibitory action on milk protein gene expression3536. It is an activator of NF-kB. We and others (CJ. Watson, personal communication) could demonstrate developmental regulated nuclear NF- kB p50/p65 in the mammary gland of mice, with peak levels between mid- and late-pregnancy. During the lactation period, DNA binding activity of NF- kB p50/p65 was low due to inhibited nuclear translocation (S.Geymayer et al., submitted). Co-transfection experiments performed with 293 cells revealed an inhibition of STAT5 dependent activation of(3-casein gene expression by NF- kB p50/p65. Similar as in the case of the above described synergism between STAT5 and GR, more than one mechanism is potentially involved in the antagonism between the STAT5 and NF-κB signalling pathways: (a) competition of STAT5 and NF-kB for binding in the β-casein gene promoter, mediated by binding sites for NF-kB in the proximal LHRR (Fig. 1); and (b) inhibition of STAT5 tyrosine phosphorylation, which was found to be inversely correlated to the NF-κB activation status in the mammary epithelial cell line HC11 (S.Geymayer et al., submitted).In conclusion, for the interaction of TFs involved in the regulation of milk-protein gene transcription, two different types of cross-talk mechanism are operative. The structural organization of the TF binding sites in the LHRR determines the gene specific response to simultaneous activation of signalling pathways. Thereby, species specific arrangement of sites are likely to contribute to the differences in strength and stage dependence of expression in different species. A second level of interaction, which does not necessarily require binding of interacting TF to DNA, is promoted by crosstalk between the signalling pathways leading to the activation of the TFs. A concise analysis of the co-operation or antagonism between TF has to consider both modes of interaction.

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