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The Classical Progesterone Receptor Associates with p42 MAPK and Is Involved in Phosphatidylinositol 3-Kinase Signaling inXenopus Oocytes*

  • Christoph P. Bagowski
    Affiliations
    From the Division of Chemical Biology, Stanford University, Stanford, California 94305-5174
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  • Jason W. Myers
    Affiliations
    From the Division of Chemical Biology, Stanford University, Stanford, California 94305-5174
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  • James E. Ferrell Jr.
    Footnotes
    Affiliations
    From the Division of Chemical Biology, Stanford University, Stanford, California 94305-5174
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant GM46383 and postdoctoral fellowships from the Lalor Foundation and the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ To whom all correspondence should be addressed: Division of Chemical Biology, Stanford University, 269 W. Campus Dr., Stanford, CA 94305-5174. Tel.: 650-725-0765; Fax: 650-723-2253; E-mail: [email protected]
Open AccessPublished:October 05, 2001DOI:https://doi.org/10.1074/jbc.M104582200
      The induction of Xenopus laevis oocyte maturation by progesterone is a striking example of a steroid hormone-mediated event that does not require transcription. Here we have investigated the role of the classical progesterone receptor in this nongenomic signaling. TheXenopus progesterone receptor (XPR) was predominantly cytoplasmic; however, a significant fraction (∼5%) of one form of the receptor (p82 XPR) was associated with the plasma membrane-containing P-10,000 fraction, compatible with the observation that membrane-impermeant derivatives of progesterone can induce maturation. XPR co-precipitated with active phosphatidylinositol 3-kinase. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin delayed progesterone-induced maturation and completely blocked the insulin-dependent maturation, indicating that the association of XPR with PI3-K could be functionally important. We also examined whether the nongenomic signaling properties of XPR can account for the ability of glucocorticoids and the progesterone antagonist RU486 to induce maturation. We found that none of these steroids cause XPR to become associated with active PI3-K; thus, association of XPR with active PI3-K is progesterone-specific. Finally, we showed that p42 mitogen-activated protein kinase (MAPK) associates with XPR after progesterone-induced germinal vesicle breakdown and that active recombinant MAPK is able to phosphorylate p110 XPR in vitro. These findings demonstrate that the classical progesterone receptor is involved in progesterone-induced nongenomic signaling inXenopus oocytes and provide evidence that p42 MAPK and PI3-K activity are directly associated with the classical progesterone receptor.
      ERK
      extracellular signal-regulated kinase
      GVBD
      germinal vesicle breakdown
      MAPK
      mitogen-activated protein kinase
      PI3-K
      phosphatidylinositol 3-kinase
      PR
      progesterone receptor
      PVDF
      polyvinylidene difluoride
      XPR
      Xenopus progesterone receptor
      PMSF
      phenylmethylsulfonyl fluoride
      Many effects of steroid hormones result from changes in transcription; the hormone binds to a classical steroid hormone receptor in the cytoplasm or nucleus and changes the receptor's transcriptional regulatory properties (
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schutz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      ,
      • Beato M.
      • Herrlich P.
      • Schutz G.
      , ). However, steroids can also exert fast, nontranscriptional effects (
      • Moore F.L.
      • Evans S.J.
      ,
      • Levin E.R.
      ,
      • Ruehlmann D.O.
      • Mann G.E.
      ,
      • Schmidt B.M.
      • Gerdes D.
      • Feuring M.
      • Falkenstein E.
      • Christ M.
      • Wehling M.
      ). One physiologically important process that clearly depends upon nontranscriptional effects of steroid hormones is the maturation of fish and amphibian oocytes. Maturation is the series of events through which a fully grown oocyte becomes ready for ovulation and fertilization (
      • Maller J.L.
      ,
      • Ferrell Jr., J.E.
      ,
      • Ferrell Jr., J.E.
      ). Immature oocytes are arrested in a G2-like state with an intact nuclear envelope or germinal vesicle. The steroid hormones progesterone (in frogs) or 17α,20β-dihydroxy-4-pregnen-3-one (in fish) cause the oocyte to undergo germinal vesicle breakdown (GVBD),1 condense its chromosomes and organize a meiotic spindle, complete the first meiotic division, enter meiosis II, and then arrest in metaphase of meiosis II. Steroids may be important in the regulation of mammalian oocyte maturation as well (
      • Byskov A.G.
      • Andersen C.Y.
      • Leonardsen L.
      • Baltsen M.
      ,
      • Hegele-Hartung C.
      • Kuhnke J.
      • Lessl M.
      • Grondahl C.
      • Ottesen J.
      • Beier H.M.
      • Eisner S.
      • Eichenlaub-Ritter U.
      ,
      • Grondahl C.
      • Ottesen J.L.
      • Lessl M.
      • Faarup P.
      • Murray A.
      • Gronvald F.C.
      • Hegele-Hartung C.
      • Ahnfelt-Ronne I.
      ).
      Many of the biochemical and cell biological events of progesterone-induced frog oocyte maturation can occur in the absence of transcription. Progesterone can induce germinal vesicle breakdown in the presence of the transcriptional inhibitor actinomycin D (
      • Schuetz A.W.
      ) and can trigger Cdc2 activation in oocytes whose nuclei have been microsurgically removed (
      • Masui Y.
      • Markert C.L.
      ,
      • Smith L.D.
      • Ecker R.E.
      ). Cdc2 activation is weaker and less sustained in enucleate Xenopus oocytes than it is in intact oocytes (
      • Iwashita J.
      • Hayano Y.
      • Sagata N.
      ); nevertheless, it does occur. These findings demonstrate that nontranscriptional effects are of central importance in progesterone-induced oocyte maturation.
      The nongenomic effects of progesterone on oocytes could be mediated either by a classical steroid hormone receptor or by some novel type of steroid hormone receptor. Until recently, it was generally believed that the latter was the case, based on several observations. First, progesterone is more effective in inducing maturation when applied to the outside of an oocyte than it is when microinjected into the cytoplasm or nucleus, consistent with a plasma membrane localization for the maturation-inducing receptor (
      • Masui Y.
      • Markert C.L.
      ,
      • Smith L.D.
      • Ecker R.E.
      ). In addition, steroids immobilized on agarose beads (
      • Ishikawa K.
      • Hanaoka Y.
      • Kondo Y.
      • Imai K.
      ) or covalently coupled to a synthetic polymer (
      • Godeau J.F.
      • Schorderet-Slatkine S.
      • Hubert P.
      • Baulieu E.E.
      ) can still induce maturation. Since classical steroid hormone receptors are generally found in the cytoplasm or nucleus, the receptor responsible for oocyte maturation was thought not to be a classical steroid hormone receptor. In addition, progesterone can cause rapid, GTP-dependent inhibition of adenylyl cyclase in washed oocyte membranes (
      • Sadler S.E.
      • Maller J.L.
      ,
      • Sadler S.E.
      • Maller J.L.
      ,
      • Finidori-Lepicard J.
      • Schorderet-Slatkine S.
      • Hanoune J.
      • Baulieu E.E.
      ). This supports the idea that the progesterone receptor may be localized to the membrane and suggests that the plasma membrane progesterone receptor is a seven-pass, G-protein-coupled receptor.
      Two recent papers have prompted reexamination of the idea that the oocyte receptor is something other than a classical steroid hormone receptor (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ,
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ). Both papers reported the cloning and characterization of cDNAs for Xenopus homologs of the classical progesterone receptor, one of which appears to represent a complete cDNA, designated XPR-1 (
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ). The XPR-1 sequence shows high similarity to the mammalian progesterone receptor proteins in the C-terminal hormone binding domain and central DNA binding domain and more limited similarity in the N-terminal region that is present in mammalian PR-B proteins and absent from the smaller PR-A proteins (
      • Kastner P.
      • Krust A.
      • Turcotte B.
      • Stropp U.
      • Tora L.
      • Gronemeyer H.
      • Chambon P.
      ). Both groups presented evidence that this classical progesterone receptor plays a role in progesterone-induced maturation. Injection of a truncated XPR mRNA was found to accelerate progesterone-induced Mos synthesis, p42 MAPK activation, and oocyte maturation, showing that a classical steroid hormone receptor can promote maturation (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ,
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ). The transcription inhibitor actinomycin D did not affect the ability of XPR to promote maturation (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ,
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ). In addition, XPR-1 antisense oligonucleotides were found to inhibit progesterone-induced maturation, and co-injection of sense XPR or human PR-B mRNAs restored progesterone-induced maturation (
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ). These findings support the hypothesis that oocyte maturation is mediated by nontranscriptional effects of a classical progesterone receptor.
      However, there are a number of aspects of steroid-induced oocyte maturation that are difficult to reconcile with the hypothesis that it is mediated by XPR-1 (
      • Maller J.L.
      ). The first is the apparent localization of the maturation-inducing progesterone receptor to the plasma membrane. Although some classical steroid hormone receptors have been found to associate with the plasma membrane when overexpressed (
      • Razandi M.
      • Pedram A.
      • Greene G.L.
      • Levin E.R.
      ), Bayaaet al. reported that endogenous XPR-1 is exclusively cytosolic (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ). If so, it would seem unlikely that XPR-1 mediates the progesterone-induced inhibition of adenylyl cyclase in washed membrane preparations or the induction of maturation by immobilized steroids. In addition, a diverse group of nonprogesterone-like steroids, including the glucocorticoids hydrocortisone and deoxycorticosterone, are potent inducers of oocyte maturation (
      • Baulieu E.E.
      • Godeau F.
      • Schorderet M.
      • Schorderet-Slatkine S.
      ). Unless XPR-1 differs markedly from other progesterone receptors in its ability to be activated by nonprogestins, it seems unlikely that it could mediate the effects of these steroids.
      Here we have examined whether the location of XPR is consistent with its hypothesized role as a mediator of maturation and whether the steroid specificity of XPR-associated effects is consistent with the steroid specificity of maturation. In addition, we looked for a mechanistic connection between XPR and the signal transduction machinery of maturation. We found that mammalian progesterone receptor antibodies recognize two main protein bands in blots and immunoprecipitates of Xenopus oocyte lysates, a prominent 82-kDa band and a less prominent 110-kDa band. We found that about 5% of the oocyte's 82-kDa XPR protein can be recovered from washed oocyte membranes, indicating that XPR could initiate signals at the plasma membrane. Moreover, progesterone caused XPR to become associated with phosphatidylinositol 3-kinase (PI3-K) activity. This association is detectable within 30 min of progesterone treatment and continues to increase until the time of germinal vesicle breakdown. The physical association of XPR with PI3-K provides one possible functional link between XPR and maturation. We also found that XPR becomes associated with p42 MAPK after germinal vesicle breakdown and that p42 MAPK is able to phosphorylate p110 XPR in vitro. This raises the possibility that XPR is regulated by the p42 MAPK pathway. Finally, we showed that RU486, hydrocortisone, and deoxycorticosterone, three steroids that can induce maturation but are not agonists at classical progesterone receptors, do not cause XPR to become associated with PI3-K activity. This finding suggests that although XPR may contribute to progesterone-induced maturation, it probably does not account for the maturation-inducing effects of nonprogestins.

      DISCUSSION

      The recent reports that overexpression of XPR accelerates progesterone-induced maturation (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ) and antisense XPR oligonucleotides inhibit progesterone-induced maturation (
      • Tian J.
      • Kim S.
      • Heilig E.
      • Ruderman J.V.
      ) support the idea that a classical progesterone receptor mediates progesterone-induced oocyte maturation, a physiological process long thought to be mediated by some other type of receptor. Here we have examined three questions that emerge out of these studies: (i) Can the localization of XPR account for the ability of bead-linked steroids to induce maturation? (ii) Is XPR able to directly engage signal transduction pathways that are involved in maturation? (iii) Can the steroid specificity of XPR-mediated signaling responses account for the steroid specificity of maturation?

      Can the Localization of XPR Account for the Ability of Bead-linked Steroids to Induce Maturation?

      Bayaa et al. (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ) found no evidence for association of XPR with the plasma membrane. They proposed instead that the evidence that the maturation-inducing receptor was located in the plasma membrane needed to be reexamined. They pointed out that the induction of maturation by bead-linked steroids could result from the leaching of steroids off the beads and into the cytoplasm. They also noted that not all laboratories agree that microinjected progesterone is ineffective at inducing maturation (
      • Bayaa M.
      • Booth R.A.
      • Sheng Y.
      • Liu X.J.
      ). Even so, it would be difficult to reconcile the observation that washed plasma membranes respond to progesterone with the hypothesis that the progesterone receptor is exclusively cytoplasmic.
      The present studies provide a resolution to this problem. We found that about 5% of the oocytes' p82 XPR is present in washed membranes. This amount of membrane-associated p82 XPR cannot be accounted for by cytoplasmic or nuclear contamination. Thus, p82 XPR could be responsible for the maturation-inducing effects of bead-linked steroids and the effects of progesterone on adenylyl cyclase activity in washed plasma membrane preparations (
      • Masui Y.
      • Markert C.L.
      ,
      • Smith L.D.
      • Ecker R.E.
      ,
      • Ishikawa K.
      • Hanaoka Y.
      • Kondo Y.
      • Imai K.
      ,
      • Godeau J.F.
      • Schorderet-Slatkine S.
      • Hubert P.
      • Baulieu E.E.
      ,
      • Sadler S.E.
      • Maller J.L.
      ,
      • Sadler S.E.
      • Maller J.L.
      ,
      • Finidori-Lepicard J.
      • Schorderet-Slatkine S.
      • Hanoune J.
      • Baulieu E.E.
      ).

      Is XPR Able to Directly Engage Signal Transduction Pathways That Are Involved in Maturation?

      We found two links between XPR and the biochemistry of oocyte maturation. First, we found that progesterone causes XPR to become associated with PI3-K activity. No significant amount of PI3-K activity was found in control immunoprecipitates, indicating that the association is specific to XPR, and no XPR-associated PI3-K activity was found in oocytes induced to mature with RU486, hydrocortisone, or deoxycorticosterone, indicating that the association depends upon the interaction of XPR with a progestin agonist.
      In agreement with previous studies (
      • Liu X.J.
      • Sorisky A.
      • Zhu L.
      • Pawson T.
      ), we found that PI3-K activation plays an important role in insulin-induced maturation. In addition, PI3-K may facilitate progesterone-induced maturation. Oocytes treated with low concentrations of wortmannin (10 nm) exhibited an ∼2-h delay in progesterone-induced maturation when submaximal concentrations of progesterone (0.6 μm) were used. Consistent with these findings, Andersen et al.
      C. B. Andersen, H. Sakaue, M. Wu, R. A. Roth, and M. Conti, manuscript in preparation.
      have recently shown that inhibition of Akt, an important downstream mediator of PI3-K activity, blocks insulin-induced maturation and delays progesterone-induced maturation, but only when submaximal concentrations of progesterone are used. Taken together, these findings support the hypothesis that PI3-K and Akt are essential mediators of insulin-induced maturation and contributors to progesterone-induced maturation. However, we found that increasing concentrations of wortmannin (>10 nm) cause increasing degrees of inhibition of maturation, although PI3-K is maximally inhibited by 10 nm wortmannin. This finding raises the possibility that the effects of wortmannin on progesterone-induced maturation are unrelated to inhibition of PI3-K.
      We also found that XPR interacts with p42 MAPK. This interaction was only detectable after GVBD, suggesting that the activation of p42 MAPK (which occurs prior to GVBD) does not depend upon this association and is not sufficient to bring about this association. In many cell types, active p42 MAPK concentrates in the nucleus. Since XPR is cytoplasmic, perhaps active p42 MAPK is sequestered away from XPR until GVBD occurs. p42 MAPK was able to phosphorylate p110 XPR in XPR immunoprecipitates, raising the possibility that p42 MAPK triggers the degradation of p110 XPR, as has been seen in breast cancer cells (
      • Lange C.A.
      • Shen T.
      • Horwitz K.B.
      ). However, although some experiments showed an apparent decrease in p110 XPR abundance in progesterone-treated oocytes (see, for example, Fig. 5A), this was not a consistent finding (cf. Fig. 1, Aand B).

      Can the Steroid Specificity of XPR-mediated Signaling Responses Account for the Steroid Specificity of Maturation?

      Finally, we examined whether the steroid specificity of XPR-linked signaling can account for the ability of oocytes to mature in response to RU486, hydrocortisone, and deoxycorticosterone (as well as progesterone). We found no association of XPR with active PI3-K in oocytes that had been induced to mature with RU486, hydrocortisone, or deoxycorticosterone. Thus, some receptor other than XPR is probably responsible for the maturation-inducing effects of these hormones. One attractive hypothesis is that other classical steroid hormone receptors, such as the glucocorticoid receptor, mediate these effects, although the potent glucocorticoid dexamethasone, which would be expected to interact with a classical glucocorticoid receptor, fails to induce maturation (
      • Baulieu E.E.
      • Godeau F.
      • Schorderet M.
      • Schorderet-Slatkine S.
      ). Recently, Morrison et al.(
      • Morrison T.
      • Waggoner L.
      • Whitworth-Langley L.
      • Stith B.J.
      ) have shown that progesterone can bring about the activation of a tyrosine-specific protein kinase in oocyte membrane/cortex preparations. It will be of interest to see whether this tyrosine kinase is also stimulated by RU486, hydrocortisone, and deoxycorticosterone.
      In summary, our studies provide new evidence for the involvement of a classical progesterone receptor in meiotic maturation. The identity of the receptor responsible for maturation is becoming less elusive, although perhaps not yet conclusive.

      Acknowledgments

      We thank C. Dreyer for the generous gift of the anti-N1/N2 antibodies. We are grateful to Kristina Kovacina for help with the PI3-K studies and to Carsten Andersen, Marco Conti, and Richard Roth for helpful discussions and for sharing unpublished information. We thank John Yoon for help; Mike Sohaskey for ideas; and Jaya Besser, Joe Pomerening, and Jianbo Yue for critical reading of the manuscript.

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