The protein kinase A pathway-regulated transcriptome of endometrial stromal fibroblasts reveals compromised differentiation and persistent proliferative potential in endometriosis

Abstract

Intrinsic abnormalities in transplanted eutopic endometrium are believed to contribute to the pathogenesis of pelvic endometriosis. Herein we investigated transcriptomic differences in human endometrial stromal fibroblasts (hESFs) from women with (hESF(endo)) vs. without (hESF(nonendo)) endometriosis, in response to activation of the protein kinase A (PKA) pathway with 8-bromoadenosine-cAMP (8-Br-cAMP). hESF(nonendo) (n = 4) and hESF(endo) (n = 4) were isolated from eutopic endometrium and treated +/- 0.5 mm 8-Br-cAMP for 96 h. Purified total RNA was subjected to microarray analysis using the whole-genome Gene 1.0 ST Affymetrix platform. A total of 691 genes were regulated in cAMP-treated hESF(nonendo) vs. 158 genes in hESF(endo), suggesting a blunted response to cAMP/PKA pathway activation in women with disease. Real-time PCR and ELISA validated the decreased expression of decidualization markers in hESF(endo) compared with hESF(nonendo). In the absence of disease, 8-Br-cAMP down-regulated progression through the cell cycle via a decrease in cyclin D1, cyclin-dependent kinase 6, and cell division cycle 2 and an increase in cyclin-dependent kinase inhibitor 1A. However, cell cycle components in hESF(endo) were not responsive to 8-Br-cAMP, resulting in persistence of a proliferative phenotype. hESF(endo) treated with 8-Br-cAMP exhibited altered expression of immune response, extracellular matrix, cytoskeleton, and apoptosis genes. Changes in phosphodiesterase expression and activity were not different among experimental groups. These data support that eutopic hESF(endo) with increased proliferative potential can seed the pelvic cavity via retrograde menstruation and promote establishment, survival, and proliferation of endometriosis lesions, independent of hydrolysis of cAMP and likely due to an inherent abnormality in the PKA pathway.

Figure 1

A, IGFBP1 and PRL protein secretion in conditioned medium from hESFs treated with 0.5 mm cAMP for 96 h, normalized to total RNA, n = 4. *, Significance accepted at P ≤ 0.05. Error bars, ±sem. B, Hierarchical clustering analysis of hESF_nonendo (no endometriosis) and hESF_endo (endometriosis) samples treated with (+) or without (−) 0.5 mm cAMP for 96 h. C, Venn diagram of shared and unique genes between hESF_endo and hESF_nonendo response to cAMP treatment (gene lists in supplemental Tables S1 and S2).

Figure 2

QPCR validation of microarray data of gene expression in hESF_nonendo and hESF_endo decidualized in vitro with 0.5 mm with 8Br-cAMP for 96 h, expressed as fold change to the expression in 96 h vehicle controls. *, Significance accepted at P ≤ 0.05 (Mann-Whitney test). Error bars, ±sem. FC, Fold change; SST, somatostatin; CXCR4, C-X-C chemokine receptor type 4.

Figure 3

All hESFs were treated with or without 0.5 mm cAMP for 96 h. Validation by real-time QPCR expression of cell cycle genes’ mRNA. *, Significance accepted as P ≤ 0.05. Error bars, ±sem. FC, Fold change.

Figure 4

Analysis of PDE4 and PDE8 in hESF_nonendo and hESF_endo. A, Validation by real-time QPCR expression of PDE8B, PDE4B, and PDE4D mRNAs. All hESFs were treated with or without 0.5 mm cAMP for 96 h. B, Total and IBMX-insensitive cAMP PDE activity in hESFs was determined as described in Materials and Methods. Shown is the cAMP PDE activity of hESF lysates (n = 4 with and n = 3 without endometriosis) using 100 nm [³H] cAMP as substrate in the absence of added inhibitors (Total) or in the presence of 100 μm IBMX to measure the apparent PDE8 component (+IBMX). cAMP-PDE activity is expressed as picomoles of cAMP hydrolyzed per minute per milligram of total cell protein. Error bars, ±sem. C, PDE4 activity in hESF lysates. The PDE4 cAMP PDE activity in hESFs was determined as described in Materials and Methods by calculating the activity that was sensitive to 1 μm of the PDE4 selective inhibitor rolipram. The data represent n = 3 hESF_nonendo or hESF_endo assayed in duplicate. Error bars, ±sem. No endo, hESF_nonendo; endo, hESF_endo. *, Significant difference (P ≤ 0.05) compared with respective control. FC, Fold change.