Transcriptional Hyperactivity of Human Progesterone Receptors Is Coupled to Their Ligand-Dependent Down-Regulation by Mitogen-Activated Protein Kinase-Dependent Phosphorylation of Serine 294

Abstract

Breast cancers often exhibit elevated expression of tyrosine kinase growth factor receptors; these pathways influence breast cancer cell growth in part by targeting steroid hormone receptors, including progesterone receptors (PR). To mimic activation of molecules downstream of growth factor-initiated signaling pathways, we overexpressed mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase) kinase kinase 1 (MEKK1) in T47D human breast cancer cells expressing the B isoform of PR. MEKK1 is a strong activator of p42 and p44 MAPKs. MEKK1 expression increased progestin-mediated transcription 8-to 10-fold above normal PR-driven transcription levels. This was dependent on the presence of a progesterone response element and functional PR. PR protein levels were unchanged by MEKK1 alone but were extensively down-regulated by MEKK1 plus the progestin R5020. MEKK1 expression resulted in phosphorylation of PR on Ser294, a MAPK consensus site known to mediate ligand-dependent PR degradation. MEK inhibitors blocked phosphorylation of Ser294 and attenuated PR transcriptional hyperactivity in response to MEKK1 plus R5020; stabilization of PR by inhibition of the 26S proteasome produced similar results. T47D cells stably expressing mutant S294A PR, in which serine 294 is replaced by alanine, fail to undergo ligand-dependent down-regulation and are resistant to MEKK1-plus-R5020-induced transcriptional synergy but respond to progestins alone. Similarly, c-myc protein levels are synergistically increased by epidermal growth factor and R5020 in cells expressing wild-type PR, but not S294A PR. Thus, highly stable mutant PR are functional in response to progestins but are incapable of cross talk with MAPK-driven pathways. These studies demonstrate a paradoxical coupling between steroid receptor down-regulation and transcriptional hyperactivity. They also suggest a link between phosphorylation of PR by MAPKs in response to peptide growth factor signaling and steroid hormone control of breast cancer cell growth.Many solid tumors, including breast cancers, exhibit elevated mitogen-activated protein kinase (MAPK) expression and/or activities (13, 40), presumably due to increased expression of growth factor receptors that couple to MAPK activation. Overexpression of type I tyrosine kinase growth factor receptors in the epidermal growth factor (EGF) receptor/cErbB family is believed to contribute to proliferative signaling in breast cancer and to be indicative of a poor prognosis. Analogous to other members of the steroid receptor superfamily, human estrogen receptors (ER) and progesterone receptors (PR) are highly phosphorylated and therefore sensitive to growth factor-initiated signaling pathways. Indeed, the same phosphorylation sites on ER and/or PR can be regulated in response to steroid hormone or growth factor treatment of cells (reviewed in references 18 and 49). Although the role of direct phosphorylation of steroid hormone receptors and the exact kinase-signaling pathways involved remain large...

Linkage of progestin and epidermal growth factor signaling: Phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth

“…In these cell line models, progestins (10nM R5020) typically stimulated PRE-luciferase expression 10-20-fold above vehicle-treated controls, while EGF alone (30ng/ml) was without significant effect. In contrast to our previous reports using transiently expressed constitutively activated protein kinases (16,17), the combination of EGF and R5020 (simultaneously delivered) produced a modest increase in PR transcriptional activity relative to R5020 alone, but this did not reach statistical significance. The same treatments were without effect in control experiments using T47D-Y PR-null cells (not shown).…”

“…Western blotting using phospho-specific antibodies indicated that EGF stimulation of T47D cells induces phosphorylation of PR Ser294 at 5-10 min, similar to that induced by R5020 at early time points (15,16). Both agents are known to activate MAPKs within 5-15 minutes (25,26).…”

“…We previously reported transcriptional synergy in progestin-treated cells transiently expressing activated MEKK1, a kinase pathway that mediates robust p42/p44 MAPK activation via the action of the classical Ras/Raf/MEK1-2 module in breast epithelial cells (16). Additionally, EGF and progestins synergistically activated transcription from the p21 and c-fos promoters that lack classical PRE sites, but presumably respond to liganded PR via the action of activated STATs (5,30) and/or PR interaction with SP-1 molecules (31).…”

“…PR Ser294 is a hormone-inducible MAPK consensus site in the PR N-terminus that may serve as a direct "sensor" for MAPK-dependent input to PR transcriptional activity (6,(13)(14)(15)(16)19,32). Therefore, we included our well-characterized S294A PR mutant receptor in the above experiments (Fig.…”

“…kinase cascades) by peptide growth factors or liganded steroid hormone receptors stationed at the cell membrane is the direct phosphorylation of nuclear steroid receptors. We have previously defined functional roles for phosphorylation of human progesterone receptor (PR) Ser294 by mitogen-activated protein kinase (14)(15)(16) and PR Ser400 by cell-cycle-dependent protein kinase two (17). Phosphorylation of these sites is regulated in response to progestins or growth factor mitogens, including EGF and heregulin, and can potentiate ligand-dependent PR transcriptional activity, as well as induce ligand-independent PR actions (17,18).…”