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AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts

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Science Signaling  21 Nov 2017:
Vol. 10, Issue 506, eaac6160
DOI: 10.1126/scisignal.aac6160
  • Fig. 1 The distribution of AKAP95 and AKAP79 in human fetal membranes and amnion fibroblasts.

    (A to D) Immunohistochemical staining of AKAP95 (A and B) and AKAP79 (C and D) in human fetal membranes. ae, amnion epithelial cells; af, amnion fibroblasts; ct, chorion trophoblasts. (E) Detection of AKAP95 and AKAP79 proteins in different subcellular fractions of human amnion fibroblasts by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calnexin, lamin A/C, and vimentin are markers for the cytoplasm, membrane and organelles, nucleus, and cytoskeleton, respectively. n = 2 experiments with samples from different patients. Scale bars, 50 μm.

  • Fig. 2 Involvement of AKAP79 and AKAP95 in the induction of COX-2 expression by cortisol (F) and activation of the cAMP pathway in human amnion fibroblasts.

    (A) Quantitative Western blots and quantitative polymerase chain reaction (qPCR) analysis showing the abundance of COX-2 mRNA and protein in human amnion fibroblasts treated with cortisol (“F,” for Kendall’s compound F) in the presence of PKI or control peptide m-PKI. (B) Quantification of COX-2 mRNA and protein in cortisol-treated human amnion fibroblasts in the presence of Ht31 or Ht31C. (C and D) Quantification of COX-2 mRNA and protein in cortisol-treated (C) or FSK-treated (D) human amnion fibroblasts transfected with scrambled (−) or AKAP79-targeted (+) siRNA. (E to G) Quantification of COX-2 mRNA and protein in cortisol-treated (E), FSK-treated (F), and dbcAMP-treated (G) human amnion fibroblasts transfected with scrambled (−) or AKAP95-targeted (+) siRNA. (H) Quantification of PGE2 in cortisol-treated human amnion fibroblasts in the presence of absence of siRNA-targeting AKAP95. *P < 0.05, **P < 0.01, ***P < 0.001 against control with m-PKI, Ht31C, or scrambled siRNA; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to cells treated with cortisol, FSK, or dbcAMP [by one-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test]. Data are means ± SEM of three to five experiments, with representative blots.

  • Fig. 3 The distribution of AKAP95, PKA RIIα, total and phosphorylated CREB, and STAT3 in human amnion fibroblasts.

    (A) Fluorescence microscopy images showing immunofluorescence staining of AKAP95 (green) in human amnion fibroblasts [marked by vimentin staining (red) and nuclear stain DAPI (4′,6-diamidino-2-phenylindole) (blue)]. (B) Confocal microscopy images showing immunofluorescence colocalization of PKA RIIα (green) and the Golgi apparatus marker Golgin-97 (red). Nuclei were stained with DAPI (blue). (C and D) Effects of siRNA-mediated knockdown of AKAP95 on the abundance of PKA RIIα and Cα in the cytoplasm and nucleus of human amnion fibroblasts. GAPDH and lamin A/C served as markers for the cytoplasm and nucleus, respectively. *P < 0.05 compared to cells transfected with scrambled siRNA (by paired Student’s t test). Data are means ± SEM of four experiments, with representative blots. (E) Total and phosphorylated (p) CREB and STAT3 abundance in the cytoplasmic and nuclear fractions in human amnion fibroblasts from two patients. (F to I) Fluorescence microscopy images showing immunofluorescence staining of total or phosphorylated CREB [red; (F) and (G), respectively] and STAT3 [green; (H) and (I), respectively] in human amnion fibroblasts. Nuclei were stained with DAPI (blue). Images are representative of two experiments. Scale bars, 25 μm.

  • Fig. 4 Involvement of AKAP95 in the phosphorylation of CREB but not STAT3 in response to cortisol (F) and activation of the cAMP pathway in human amnion fibroblasts.

    (A to D) Effects of Ht31 and siRNA-mediated knockdown of AKAP95 on the phosphorylation of CREB at Ser133 and STAT3 at Tyr705 in response to cortisol. (E to H) Effects of siRNA-mediated knockdown of AKAP95 on the phosphorylation of CREB at Ser133 and STAT3 at Tyr705 in response to FSK or dbcAMP. *P < 0.05, **P < 0.01, ***P < 0.001 against control with Ht31C or scrambled siRNA; #P < 0.05, ##P < 0.01, ###P < 0.001 against cortisol-, FSK-, or dbcAMP-treated cells (by one-way ANOVA followed by the Newman-Keuls multiple comparison test). Data are means ± SEM from three to five experiments, with representative blots.

  • Fig. 5 The effects of cortisol (F) on AKAP95, PKA RIIα, and Cα protein abundance in human amnion fibroblasts.

    (A and B) Concentration-dependent effects of cortisol on cellular AKAP95, PKA RIIα mRNA, and protein abundance in human amnion fibroblasts (by one-way ANOVA followed by the Newman-Keuls multiple comparison test). (C and D) Effects of cortisol on the abundance of PKA RIIα and Cα in the cytoplasm and nucleus of human amnion fibroblasts. GAPDH and lamin A/C are markers for cytoplasm and nucleus, respectively. *P < 0.05, **P < 0.01 against control cells without cortisol (by paired Student’s t test). Data are means ± SEM from four to five experiments, with representative blots.

  • Fig. 6 Changes in the abundance of AKAP95, COX-2, and phosphorylated and total CREB in human amnion tissue at parturition.

    (A) Western blots showing the abundance of AKAP95, COX-2, and phosphorylated and total CREB protein in human amnion tissue collected upon cesarean section without labor at term [term non-labor (TNL); n = 6] and upon delivery after spontaneous labor [term labor (TL); n = 6]. (B to E) Quantification (means ± SEM) of the Western blotting assays represented in (A). *P < 0.05, **P < 0.01 against TNL (by unpaired Student’s t test).

  • Fig. 7 A working model illustrating the role of AKAP95 in cortisol-induced PTGS2 expression in human amnion fibroblasts.

    By inducing AKAP95 expression, cortisol increases the abundance of PKA anchored in the nucleus, thereby enhancing the activation of nuclear PKA by the cAMP pathway that is coupled with the PGE2 receptor. Consequently, the phosphorylation of nuclear CREB is increased. In contrast, the phosphorylation of STAT3 may occur in the cytoplasm. Phosphorylated CREB, STAT3, and activated glucocorticoid receptor (GR) interact at the PTGS2 promoter, thereby leading to the increased PTGS2 expression and subsequent PGE2 production in human amnion fibroblasts. Gs, stimulatory G protein; AC, adenylate cyclase; ATP, adenosine 5′-triphosphate.

Supplementary Materials

  • Supplementary Materials for:

    AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts

    Jiangwen Lu, Wangsheng Wang, Yabing Mi, Chuyue Zhang, Hao Ying, Luyao Wang, Yawei Wang, Leslie Myatt, Kang Sun*

    *Corresponding author. Email: sungangrenji{at}hotmail.com

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    • Fig. S1. The efficiency of siRNA-mediated knockdown of AKAP79 and AKAP95 in human amnion fibroblasts.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Confocal z-stack imaging of the colocalization of PKA RIIα and Golgin-97 in human amnion fibroblasts.

    Citation: J. Lu, W. Wang, Y. Mi, C. Zhang, H. Ying, L. Wang, Y. Wang, L. Myatt, K. Sun, AKAP95- mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts. Sci. Signal. 10, eaac6160 (2017).

    © 2017 American Association for the Advancement of Science

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