The importance of PRL in physiological proliferation and differentiation of the mammary gland, together with the high level of PRLR in breast tumors, the association of circulating PRL with incidence of breast cancer, and the recognition of locally produced PRL, call for greater understanding of its actions in mammary disease. Little is known about the breadth of pathways whereby this cytokine/hormone may directly modulate or synergize with other oncogenes in the disease process. Here we have demonstrated that PRL is a potent inducer of AP-1 activity in breast cancer cells, which may alter expression of many target genes that are critical for neoplasia, including cell survival, proliferation, differentiation, angiogenesis, and invasion. As depicted in , PRL activates this transcription factor by complex signaling cascades, involving multiple proximal pathways, including Jak2, c-Src, PI3K, and PKC, although only Jak2 is required for a PRL response. ERK1/2 are the primary downstream activators of c-Jun, JunB, and c-Fos in this system, resulting in increased DNA binding of these components. Although JNK1/2 activity is a major determinant of the magnitude of AP-1 activity in this system, it is not necessary for the response to PRL, in contrast to other reports. ERK5 and p38 are minor effectors of the PRL response.
Model for PRL Activation of AP-1
Most studies have focused on Jak2 as a proximal mediator of PRL action. However, PRL also activates other pathways in various target cells, including c-Src, PI3K, and PKC (reviewed in Refs. 1
), although the hierarchical relationships between Jak2 and these other kinases are not well understood. Because these proteins also activate MAPKs, it is not surprising that all these pathways play roles in PRL signaling to AP-1. We have previously shown the importance of Jak2, PI3K, and c-Src in PRL induction of the critical cell cycle regulator, cyclin D1, and in PRL-induced proliferation of this MCF-7 derived cell line (27
). PRL induction of c-Src has been also reported to be a critical mediator of proliferation and activation of PI3K and ERK1/2 in MCF-7 parent cells as well as T47D breast cancer cells (39
). In contrast, although PRL has been shown to activate PKC-linked signaling cascades (40
), the role of these kinases in PRL action has received relatively little attention in any cell type, especially in breast cancer cells. Whereas Jak2/Stat5 mediates most PRL actions in alveolar development (reviewed in Ref. 7
), and Jak2 is required for PRL activation of AP-1 in PRL-deficient MCF-7 cells, Stat5 is not required for the AP-1 response, and indeed inhibits this pathway (Gutzman, J. H., L. M. Arendt, D. E. Rugowski, S. E. Nikolai, H. Rai, and L. A. Schuler, manuscript in preparation). Our findings underscore the wide range of signaling pathways activated by PRL and the need for further investigation into this kinase network.
PRL also activated many MAPK family members in these breast cancer cells. Whereas ERK1/2 and JNK1/2 have been reported to be downstream of the PRLR in several in vitro
models, our data indicate that PRL also is able to initiate phosphorylation of ERK5 and p38, suggesting other unexplored distal targets. Whereas various p38 isoforms have been reported to differentially affect AP-1 activity (41
), p38α accounted for the modest effect observed with a p38-selective inhibitor in the current study. All of these MAPKs have been shown to modulate AP-1 activity in response to other stimuli (reviewed in Refs. 9
); the ability of DN constructs specific for each family member to partially inhibit PRL-induced activation of AP-1 here suggests that they play specific roles in mediating PRL action and that they are not redundant pathways.
AP-1 enhancer activation requires increased Jun/ Fos expression, DNA binding affinity, and/or transactivational potential of one or more of these proteins. Our studies showed that PRL employed all of these mechanisms to increase the activity of dimers composed of c-Jun, JunB, and/or c-Fos. Phosphorylation of c-Jun at Ser-63 was the earliest detectable modification after exposure to PRL; this modification by JNK1/2 is a well-characterized event that potentiates its transactivation properties by enhancing recruitment of coactivators (reviewed in Ref. 22
). Activated c-Jun, in combination with other transcription factors stimulated by a complex network of other MAPKs, increases c-jun
). Indeed, in our studies, increased c-Jun protein was detectable after 1 h exposure to PRL. U0126 also partially blocked the PRL-induced rise in c-Jun levels; this is consistent with roles for both JNK1/2 as well as the U0126-sensitive ERKs in this event. In addition, PRL elevated levels of JunB, apparent after 1 h. Like the other AP-1 components, amounts of this protein can be regulated at many levels. However, two Serum Response Elements in the promoter can be activated by MAPK-activated Ternary Complex Factor, providing at least one potential mechanism. In contrast, net levels of c-Fos were not altered over the time examined here. Although MAPKs can also activate multiple cis-
inducible elements in the c-fos
promoter (reviewed in Ref. 22
), it is possible that increased protein turnover may have stabilized steady-state levels in our studies (38
). However, PRL-induced, U0126-sensitive phosphorylation of c-Fos was clearly evident. This is consistent with the well-characterized phosphorylation of c-Fos at multiple sites in the C-terminal transactivation domain primarily by ERK1/2 and downstream kinases, which enhances its transactivation potential (38
). Together, our data support a model whereby PRL directs activation of a complex web of MAPKs to alter AP-1 activity at multiple levels. Interestingly, the effects observed here on AP-1 proteins were transient; the dynamic mechanism(s) sustaining the PRL-induced activity are under investigation.
These studies document the ability of PRL to augment signaling through AP-1 components but do not address the likely plethora of target genes. The clear evidence that DNA binding of AP-1 proteins is dependent on flanking sequences and promoter context (reviewed in Refs. 3
) suggest that the interactions detected with our reporter construct and EMSAs may be a subset of the total activity. Analysis of gene expression in diverse cell types in response to overexpression of individual components and tethered AP-1 dimers has confirmed the multiplicity of factors determining these targets and is beginning to reveal the diverse AP-1 repertoire (12
). Cell context, dictating levels of potential dimerization partners and activity of cooperating pathways, is paramount in determining the response. JunB, for example, is generally considered antiproliferative; however, it increases cyclin A transcription permitting entry into S phase (50
), up-regulates angiogenic factors during progression of fibrosarcomas (51
), transforms Rat1a fibroblasts (48
), and can replace c-Jun during development (reviewed in Refs. 8
). In addition to the direct DNA binding examined here, PRL-activated AP-1 components may also modify gene expression via interaction with a multitude of other transcription factors, such as nuclear factor-κ B, NFAT, Smad, Ets, Stats, and various nuclear receptors (reviewed in Refs. 13
). This well-documented underlying complexity suggests that targets of PRL-activated AP-1 may differ in breast tumors of various etiologies or as tumorigenesis progresses.
Our studies demonstrated the ability of PRL to direct a complex signaling network resulting in activation of AP-1 components in breast cancer cells, which may modify numerous aspects of tumor cell behavior. Many other growth factors and hormones that play central roles in the normal mammary gland or carcinogenesis can also activate AP-1, providing opportunities for cross-talk. Investigation of PRL interactions with these factors in different cell contexts and responsive AP-1 target genes will increase our understanding of the role of PRL in mammary neoplasia and aid in the development of new therapeutic approaches for breast cancer.