A-Kinase Anchoring Protein 13 (AKAP13) Augments Progesterone Signaling in Uterine Fibroid Cells

Abstract

Context: Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood.

Objective: Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids.

Methods and results: Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus.

Conclusion: These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.

Trial registration: ClinicalTrials.gov NCT00290251.

Figures

Figure 1.

Immunohistochemical (IHC) staining showed altered expression of AKAP13 and PR in ulipristal acetate‒treated fibroids and myometrium. Fibroid tissues were collected from patients who received placebo or ulipristal acetate treatment (20 mg per dose). Tissues were stained with (A) antihuman AKAP13 antibody, (B) antihuman PR antibody (PR-A and PR-B), or (C) antihuman PR-B‒specific antibody to show the abundance of these proteins in these tissues. (A) Black arrowheads show the intense staining of AKAP13 in placebo samples but its disappearance in ulipristal acetate‒treated samples. Images of two representative patients from each group are shown. Six patients from each group were evaluated with similar results. Magnification = 20×. Scale bar represents 1 μm.

Figure 2.

Progesterone (P4)-dependent gene activation by PR-B is augmented by expression of AKAP13. (A) Human fibroid cells derived from immortalized human fibroid tissues (P51F) and (B) cells from the monkey kidney cell line COS-7 were cotransfected with plasmids of PRE-luc and human PR-B. The cotransfection of AKAP13 and the addition of P4 (40 nM) show augmented PRE-luc activity. (C) COS-7 cells were cotransfected with PRE-luc and either PR-A or PR-B. (D) COS-7 cells were cotransfected with MMTV-luc, PR-B, and AKAP13 as indicated by plus (+) or absence (−). Luciferase activity was determined by luminometer and normalized by protein concentration in each well. The fold-changes were calculated by normalization with luciferase value in wells without AKAP13 transfection and no P4 treatment. Each condition was repeated in triplicate. Each graph represents the experimental data with SEM of five to seven experiments with similar results. Statistical analysis was done using the Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. ns, no statistical significance.

Figure 3.

AKAP13 knockdown by siRNA reduced expression of a progesterone (P4)-responsive gene. The human breast cancer cell line T47D was transfected with siRNA targeting AKAP13 or nontargeting siRNA negative control. Two d after transfection, cells were treated with the indicated dose of P4. Twelve h after P4 treatment, the expression of the P4-responsive gene alkaline phosphatase was measured by absorbance at 405 nm. Representative data with SEM of triplicate are shown. The experiment was repeated twice with similar results. Statistical analysis was done using the Student t test. **P < 0.01; ***P < 0.001.

Figure 4.

Augmentation of PR-B activity by AKAP13 was affected by the inhibition of the ERK pathway and not by the p38 MAPK pathway. (A) COS-7 cells were treated with p38 inhibitor SB203580 (10 μM) or SB202190 (2 μM) for 1 h. Cell lysate was harvested, and the quantification of phospho-p38 MAPK (active) and total p38 MAPK was done by western analysis using specific antibodies. (B) COS-7 cells were cotransfected with MMTV-luc reporter plasmid, human PR-B, and human AKAP13. Twenty-four h after transfection, the p38 inhibitors SB203850 and SB202190 were added to the cell culture for 1 h before progesterone (P4) treatment. (C) COS-7 cells were treated with the ERK inhibitor U0126 (0.1, 1, and 10 μM) for 1 h before P4 treatment. Cell lysate was collected 24 h after P4 treatment. Luciferase activity was determined by luminometer and normalized by protein concentration in each well. MMTV-luc activity in cells transfected with AKAP13 without any inhibitors was designated to be 100%, and the ERK inhibitor, U0126, was added in increasing concentrations as noted by the black triangles. Each condition was repeated in triplicate. Presence (+) or absence (−) of factor, as noted. Each graph represents data with the SEM of five to seven experiments with similar results. Statistical analysis was done using the Student t test. **P < 0.01.

Figure 5.

Interaction between AKAP13 and PR. (A) Upper panel shows the schematic diagram of two human AKAP13-GST fusion proteins used for the pull-down assay: the 1.8-kb version (GST-FL) and the C-terminal version (GST-R5). Both versions contain the NRID domain that was previously shown to bind ER. GST-0 is the GST-his tag alone without any fusion. Lower panel shows that the C-terminal version of human PR protein (PR687C) was 35S-labeled and used in this study, as shown. (B) Labeled PR-B C-terminus (PR687C) protein was incubated with various fusion proteins. Lane 1, 35S-labeled PR-B input. Lane 2, GST-0 did not precipitate labeled PR-B. Lane 3, GST-FL AKAP13 did precipitate labeled PR-B (black arrow). Lane 4, the NRID region of AKAP13 in GST-R5 precipitated labeled PR-B. (C) Luciferase was 35S-labeled (Lane 1, input) and incubated with GST-FL (Lane 2) or GST-0 (Lane 3) to serve as a negative control for AKAP13 binding. No binding was observed (open arrow).

Figure 6.

Proposed model of AKAP13 and PR-B signaling in fibroids. Our working model suggests that AKAP13 augments ligand-dependent activity of PR-B through ERK, possibly through close interaction of PR-B with AKAP13. Because AKAP13 is an essential regulator of the transcription factor YAP in mechanotransduction, it may potentially regulate PR activity through crosstalk with the Hippo pathway. Red = actin filament; blue box = integrin complex. VEGF, vascular endothelial growth factor. AKAP13 activates Rho (+) and is capable of augmenting ligand-dependent PR activity (+). PR then may affect gene transcription. The involvement of AKAP13 in both processes suggests integration of mechanical and progesterone signaling. Arrows suggest possible relationships of the factors in the pathway.