Identification and Characterization of the Protein-associated Splicing Factor as a Negative Co-regulator of the Progesterone Receptor

Progesterone is essential in all species for the maintenance of pregnancy, and its withdrawal is required to activate the myometrium and to initiate labor. However, unlike most other species, progesterone levels do not fall at term in humans, raising the paradox as to how labor can occur under the continued influence of progesterone. We hypothesized that an endogenous (myometrial) repressor of the progesterone receptor (PR) could induce a functional withdrawal of progesterone and hence lead to the initiation of labor. We used the human PR as bait in a protein pull-down assay and identified polypyrimidine tract-binding protein-associated splicing factor (PSF) as a PR-interacting protein. PSF functions as a potent inhibitor of PR (but not estrogen receptor) transcriptional activity in mammalian cells. It acts through two novel mechanisms, inducing degradation of the PR through the proteasomal pathway and also interfering with binding of PR to its DNA response element. Importantly, in vivo studies in rats demonstrated a dramatic increase in myometrial PSF expression at term that was temporally associated with reduced levels of the myometrial PR. Accordingly, we propose that PSF acts as a PR corepressor and contributes to the functional withdrawal of progesterone and the initiation of human labor. Progesterone is essential in all species for the maintenance of pregnancy, and its withdrawal is required to activate the myometrium and to initiate labor. However, unlike most other species, progesterone levels do not fall at term in humans, raising the paradox as to how labor can occur under the continued influence of progesterone. We hypothesized that an endogenous (myometrial) repressor of the progesterone receptor (PR) could induce a functional withdrawal of progesterone and hence lead to the initiation of labor. We used the human PR as bait in a protein pull-down assay and identified polypyrimidine tract-binding protein-associated splicing factor (PSF) as a PR-interacting protein. PSF functions as a potent inhibitor of PR (but not estrogen receptor) transcriptional activity in mammalian cells. It acts through two novel mechanisms, inducing degradation of the PR through the proteasomal pathway and also interfering with binding of PR to its DNA response element. Importantly, in vivo studies in rats demonstrated a dramatic increase in myometrial PSF expression at term that was temporally associated with reduced levels of the myometrial PR. Accordingly, we propose that PSF acts as a PR corepressor and contributes to the functional withdrawal of progesterone and the initiation of human labor. Progesterone is an essential regulator of the reproductive events associated with the establishment and maintenance of pregnancy through its ligand-activated progesterone receptor (PR) 1The abbreviations used are: PR, progesterone receptor; PRA, progesterone receptor A; PRB, progesterone receptor B; PRE, progesterone response element; DBD, DNA-binding domain; AF, activation function; CBP, cAMP response element-binding protein-binding protein; GST, glutathione S-transferase; PSF, polypyrimidine tract-binding protein-associated splicing factor; ER, estrogen receptor; SHMs, Syrian hamster myocytes; WCL, whole cell lysate; DMEM, Dulbecco's modified Eagle's medium; MALDI, matrix-assisted laser desorption ionization time-of-flight; RRM, RNA recognition motif. (1.Challis J.R.G. Matthews S.G. Gibb W. Lye S.J. Endocr. Rev. 2000; 5: 514-550Google Scholar). Progesterone actions include the suppression of genes encoding contraction-associated proteins (e.g. oxytocin and prostaglandin receptors and connexin-43) that are required for myometrial activation and the onset of labor. In humans, progesterone levels remain elevated throughout labor, raising a paradox as to how labor can be initiated. Even in species in which progesterone levels fall at term, concentrations are likely sufficiently high to inhibit contraction-associated protein gene expression. This suggests there must be an active mechanism for inducing a functional withdrawal of progesterone at term. We have previously suggested that a blockade of PR signaling in the myometrium could induce a “functional withdrawal” of progesterone that would result in the initiation of labor (2.Challis J.R.G. Lye S.J. Creasy R.K. Resnik R. Maternal-Fetal Medicine Principles and Practice. 5th Ed. W. B. Saunders Co., Philadelphia2004: 79-87Google Scholar). A number of mechanisms have been proposed to effect such a functional withdrawal, including changes in the expression of the PR or PR isoforms (3.Mesiano S. Chan E.C. Fitter J.T. Kwek K. Yeo G. Smith R. J. Clin. Endocrinol. Metab. 2002; 6: 2924-2930Crossref Scopus (350) Google Scholar) as well as altered transcriptional activity of the PR as a result of changes in the expression of essential co-regulators (both coactivators and corepressors) (4.Condon J.C. Jeyasuria P. Faust J.M. Wilson J.W. Mendelson C.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9518-9523Crossref PubMed Scopus (243) Google Scholar). The PR is a member of the steroid receptor superfamily of ligand-dependent transcriptional factors. In the human myometrium, the PR is transcribed as full-length PRB and an N-terminally truncated (164 amino acids) PRA isoform (5.Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. EMBO J. 1990; 5: 1603-1614Crossref Scopus (1339) Google Scholar). PRA is generally a weaker transcriptional activator than PRB (6.Tora L. Gronemeyer H. Turcotte B. Gaub M.P. Chambon P. Nature. 1988; 333: 185-188Crossref PubMed Scopus (319) Google Scholar, 7.Meyer M.E. Quirin-Stricker C. Lerouge T. Bocquel M.T. Gronemeyer H. J. Biol. Chem. 1992; 267: 10882-10887Abstract Full Text PDF PubMed Google Scholar, 8.Giangrande P.H. McDonnell D.P. Recent Prog. Horm. Res. 1999; 54 (Discussion 313–314): 291-313PubMed Google Scholar) and can also act as a repressor of PRB as well as of other steroid receptors (9.Vegeto E. Shahbaz M.M. Wen D.X. Goldman M.E. O'Malley B.W. McDonnell D.P. Mol. Endocrinol. 1993; 10: 1244-1255Google Scholar, 10.Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 4: 1847-1857Crossref Scopus (175) Google Scholar). Upon ligand binding through the hormone-binding domain, the activated PR undergoes a conformational change enabling it to bind to specific progesterone response elements (PREs) through its DNA-binding domain (DBD). This in turn facilitates recruitment of the general transcriptional machinery, either directly (11.Ing N.H. Beekman J.M. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar) or indirectly via co-regulators (12.Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2063) Google Scholar, 13.Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1086) Google Scholar), which act to positively or negatively modulate the transcription rate of target genes. Two common transcriptional activation domains exist within PRs, a hormone-dependent activation function domain (AF2) in the C-terminal hormone-binding domain and a ligand-independent domain (AF1) in the N-terminal region (14.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2702) Google Scholar). In addition, PRB possesses a third activation function domain (AF3) within its unique N-terminal region (15.Sartorius C.A. Melville M.Y. Hovland A.R. Tung L. Takimoto G.S. Horwitz K.B. Mol. Endocrinol. 1994; 8: 1347-1360Crossref PubMed Scopus (244) Google Scholar). Interactions between the N-terminal AF domains (AF1 and AF3) and the AF2 domain (either direct or indirect via co-regulators) elicit maximal hormone-dependent activity (16.Tetel M.J. Giangrande P.H. Leonhardt S.A. McDonnell D.P. Edwards D.P. Mol. Endocrinol. 1999; 6: 910-924Crossref Scopus (144) Google Scholar). Our knowledge of nuclear receptor co-regulators has increased markedly over recent years, revealing their multifaceted roles in regulating gene transcription. Besides the autonomous activation domains in steroid receptor coactivators and repression domains in the nuclear receptor corepressor and SMRT, many co-regulators possess acetylase (such as steroid receptor coactivators, p300/CBP, and p300/CBP-associated factor) or (such as C.K. Rosenfeld M.G. 2000; PubMed Google Scholar). in the of and nuclear receptor activation co-regulators can be through their which is their functions Smith E. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, J. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Biochem. Mol. Biol. 1999; PubMed Scopus Google Scholar). In an to co-regulators within the myometrium that with the PR and modulate PR we used glutathione proteins to pull-down protein myometrium cell of the associated proteins identified tract-binding protein-associated splicing factor was to inhibit the transcriptional activity of the PR mechanisms that with PR binding to the and degradation of the PR protein through the proteasomal the that the expression of PSF increased in the myometrium at term pregnancy in with reduced levels of the myometrial PR to that novel PR corepressor to the functional withdrawal of progesterone and the initiation of labor. and and and the proteasomal inhibitor and and was inhibitor was was PR expression and P. used as the a of amino and with a an and an and a and a high DNA expression and and also of the of at the and was as the PR and at the and PSF was a J. G. PSF and its and also with a an and an and a an and a and at the and and at the and The of all was DNA proteins also specific receptor and expression of the and of the estrogen response was previously J.R.G. Lye S.J. J. Mol. Endocrinol. PubMed Scopus Google Scholar). of proteins as previously J.R.G. Lye S.J. J. Mol. Endocrinol. PubMed Scopus Google Scholar). proteins in with to a of and in and inhibitor and with of a of and nuclear of Syrian hamster an nuclear and through and with proteins to for at The with and with or The associated proteins the of glutathione and with and with as K. S. T. Biol. 2003; 4: Scopus Google Scholar). the in with and in a of and was a ionization to a high the was The and of the was used to the and We the the and of the PR and was in cells. in and to A of of with and in inhibitor concentrations of the whole cell the M.M. Biochem. PubMed Scopus Google Scholar) and was to in A of was at in with either or the of of protein for with with and The was and of the endogenous PR and PSF, in in of inhibitor with of or or at the of of protein for at with and with The proteins with the whole cell or pull-down assay was as previously J.R.G. Lye S.J. J. Mol. Endocrinol. PubMed Scopus Google Scholar). and its proteins The was at with the PR or transcribed and in the of The was with the of to associated The proteins with and and and in Dulbecco's modified Eagle's as A. J. Lye S.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). steroid the was with was to the at a of to the The DNA and and to the at in of of which used for the and activity activity was the to the was to cell in inhibitor of protein and with the of PR was in the in with PR protein was of with the with and nuclear the nuclear and The nuclear was with and used in a a with and through in a of in of of and of and receptor protein. In or was as The binding was to for at was of for an the of in at the and and rats under and and rats with of was as the a was The of under was the of Our for labor of at and myometrial and or and was at all with the the labor was the at myometrial and and The was the myometrial We have previously that the and the of the M. Lye S.J. 1995; PubMed Google Scholar). The myometrial was in other and and at the and in at RNA was the and as A. Lye S.J. Biol. PubMed Scopus Google Scholar). The used to PSF was a number The T. was used as a A of of to of to between with the of for at The expression of the PR protein was was under a and The was for in assay and with and inhibitor at for at and the was to a to a protein concentrations protein assay in and at for at with to and The was and with as a of and the to of to between with the of for at of PSF as a PR-interacting PR-interacting proteins to and with either or nuclear through proteins and Two protein in nuclear of SHMs, identified that at the of and to and was to bind at a of concentrations and could bind at This suggests that a with two and for within the proteins that two proteins are The within the to a protein PSF, previously identified as an Two PSF protein isoforms and have been two isoforms are through amino with amino and amino J. P. B. 1993; PubMed Scopus Google Scholar). PSF two RNA recognition and within amino and an N-terminal region in and and to as an protein Our not specific to the of PSF with the PR in to the between the PR and PSF in was of the PSF the of the with the PR expression as with and to that two proteins in the We with either or with the to that target proteins could be the protein whole cell PRB PSF with and with protein The associated proteins and for the of was in the of the PR was used to WCL, we that the of the PR could be in the of PSF was also with the endogenous PR the cell of the PR or PSF was or was with in vivo the between the PR and PSF in the pull-down to an in vivo between PSF and the PR was the mammalian PSF was to the of the in the and PRB or PRA was of the activation domain in the with in a in activity with the However, of activation and with a dramatic increase in activity as a result of the between PSF and This was ligand-independent the of progesterone not a in that the PR and PSF in within the PR and the between PSF and the PR is direct to the of the an in pull-down assay was PSF was the in and its binding to a of proteins was PSF to proteins the to a to the domain of PRB, not bind to other of the PR. also to the direct binding of the full-length PR to proteins the domain, and the binding of PRB was with proteins in PRB binding to proteins not to of proteins of the of proteins of protein that the between the PR and PSF is direct and through the domain and of the PR and of of the between PSF and the of the between PSF and the PR the transcriptional of was in We two the and the of the of PSF effect either of However, PSF PRA and PRB of in a also in the in not that effect is not the cell or The PSF expression was also with and in a We not of PSF either or that PSF than PSF the of the PR through the the that the of PSF the PR not to the reduced expression of PR of was of the PSF and PR expression as for we that the of the PSF expression the of a of the in a in the levels of PRA and PRB proteins This to the that PSF the degradation of the PR through the proteasomal of PSF or truncated of PSF the C-terminal and in a of PR proteins However, the of the proteasomal inhibitor at a of for an the of the PR protein. also the degradation of PR that region of PSF is to the PR and that the isoform would also have PSF PR through Two the PSF repression of activation domains of the PR, we used a in which of the activation domains of the PR was to the of the in the and with the a increase in activity with the of of PSF in a in activity with a of protein. PSF also and the degradation of region the domain and However, PSF effect and domain of the a binding for PSF, or the AF2 Importantly, PSF inhibit the of the AF2 domain and in the protein of domain that the of PSF to inhibit and to induce the degradation of the and domains is the of PSF with the domain and the other PSF also directly with of the function of domain a mechanism mechanisms include domains within PSF or the recruitment of other transcriptional protein a was in which of PSF the and with the and genes. 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PubMed Scopus Google Scholar) have that PSF can with and transcriptional through the recruitment of to the receptor Our mechanisms which PSF PR of the degradation of the PR protein through the proteasomal pathway as well as with PR binding to the of PSF and the PR in PR and effect could be the of the proteasomal inhibitor that PSF can PR protein degradation through the proteasomal It is to that PSF possess activity or can the PR with protein the The that a truncated of PSF can the PR suggests that and possesses PR degradation The of and to the and nuclear that could target the PR for degradation as as protein in the and would target the nuclear This in function of PSF in the PR for degradation is increased functional in vivo a between the increased expression of PSF in the myometrium at term and reduced levels of the PR protein. A mechanism which PSF PR signaling is through with PR binding to the as Our at for the AF2 domain of the PR, with DNA binding is of the effect of PR It to be in the of the whole receptor blockade of PR binding to the is a for the PR for Our also that two within the of PSF possess transcriptional This is with recent that a truncated of PSF gene transcription of the binding to a DNA within its J. 2002; Google Scholar). has been that PSF can other transcriptional protein through M. Mol. Cell. Biol. PubMed Scopus Google Scholar), the that the of PSF possess functions be Our that PSF the function of the PR well as the and J. R. G. and S. J. not the or suggests that is that the of nuclear receptors has a high of to the it be that PSF binding to the of PR a of This is the PSF can with the PR in the domain and DBD, the of is with binding between PSF and the of PR. This to the domain a and that PRB to an domain, it is that PSF a with PRB and PRB with as well as of mechanisms which PSF steroid of receptor with and recruitment of The to which of mechanisms is in vivo and occur or remain to be However, in the an in vivo between increased PSF expression and PR Recent have suggested other mechanisms which a functional progesterone withdrawal occur in human have proposed that PRA can act as a repressor of PRB within the myometrium and that an increase in the expression of PRA PRB induce a functional withdrawal of progesterone (3.Mesiano S. Chan E.C. Fitter J.T. Kwek K. Yeo G. Smith R. J. Clin. Endocrinol. Metab. 2002; 6: 2924-2930Crossref Scopus (350) Google Scholar, M. Mol. Scopus Google Scholar). (4.Condon J.C. Jeyasuria P. Faust J.M. Wilson J.W. Mendelson C.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9518-9523Crossref PubMed Scopus (243) Google Scholar) have expression of steroid receptor coactivators in human myometrium at term that PR are not the for progesterone withdrawal at term, it is that mechanisms exist to a functional progesterone withdrawal and the initiation of labor. In has identified a novel function for PSF as a PR PSF to act the of the PR to bind to its DNA response and to target the PR for degradation through the proteasomal as we do not have direct of an of PSF in the initiation of human labor, it has been that there is a in the binding of the PR term myometrial protein for the PRE, that in term myometrium to of the between the PR and Wilson T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Our that PSF to PSF be a of the which a functional withdrawal of progesterone occur in human pregnancy and a target for at the of labor at term and We for and We also for

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