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. 2023 Jun 29;24(13):10881.
doi: 10.3390/ijms241310881.

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition

Fan Zhang  1   2 , Jiang-Wen Lu  1   2 , Wen-Jia Lei  1   2 , Meng-Die Li  1   2 , Fan Pan  1   2 , Yi-Kai Lin  1   2 , Wang-Sheng Wang  1   2 , Kang Sun  1   2
Affiliations

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition

Fan Zhang et al. Int J Mol Sci. .

Abstract

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

Keywords: 15(S)-HETE; ALOX15; PGE2; glucocorticoids; inflammation; parturition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of cortisol on ALOX15 and ALOX15B expression in human amnion fibroblasts. (A,B) Abundance of cortisol (A) and 15(S)-HETE (B) in the human amnion obtained from deliveries at term with labor (TL; n = 7) and without labor (TNL; n = 7). (C) Pearson analysis showing the positive correlation between 15(S)-HETE and cortisol levels in the human amnion (n = 14). (D) Scatter plot of the transcriptomic sequencing data displaying the fold change (FC) in ALOX family members in human amnion fibroblasts treated with or without cortisol (1 µM; 24 h). Y-axis represents −log (p-value) and X-axis represents log (FC). n = 3. (E) Heatmap of the transcriptomic sequencing data displaying the mRNA abundance of ALOX family members in human amnion fibroblasts with and without cortisol (1 µM; 24 h) treatment. Blue to red represents expression levels from low to high. n = 3. (F–I) Concentration-dependent induction of ALOX15 and ALOX15B mRNA (F, n = 6; G, n = 4) and protein (H, n = 3; I, n = 4) expression by cortisol (0.01, 0.1 and 1 µM; 24 h) in human amnion fibroblasts. (J) Concentration-dependent induction of 15(S)-HETE production by cortisol (0.01, 0.1, and 1 µM; 24 h) in human amnion fibroblasts. n = 3. (K) Representative images of immunofluorescence staining of ALOX15 (red) and ALOX15B (green) in human amnion fibroblasts with or without cortisol (1 µM; 24 h) treatment. Nuclei were stained with DAPI (blue). Scale bars, 50 μm. n = 3. Data are represented as means + SEM. Statistical analysis was performed with unpaired Student’s t-test (A,B) or one-way ANOVA test followed by Newman–Keuls multiple comparisons test (F–I,J). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNL or control group.
Figure 2
Figure 2
Involvement of p300 in the induction of ALOX15 and ALOX15B expression by cortisol in human amnion fibroblasts. (A) Induction of p300 mRNA (n = 3) and protein (n = 3) expression by cortisol (1 µM; 24 h). (B,C) Blockade of cortisol (1 µM; 24 h)-induced ALOX15 and ALOX15B mRNA (n = 4) and protein (n = 4) expression by p300 inhibitor C646 (10 µM). (D,E) Diagrams showing the putative binding sites of p300 in ALOX15 (D) and ALOX15B (E) gene promoters. Brown box indicates p300 binding sites. Arrows indicate primer aligning positions in ChIP assay. P1, forward primer; P2, reverse primer; TSS, transcription start site. (F,G) ChIP assay showing enrichments of p300 and H3K27ac at ALOX15 (F) and ALOX15B (G) gene promoters in human amnion fibroblasts with cortisol treatment (1 μM; 12 h). n = 4. IgG served as negative control. Statistical analysis was performed with paired Student’s t-test (A,F,G) or one-way ANOVA test followed by Newman–Keuls multiple comparisons test (B,C). Top panels of A−C are the representative immunoblots. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cortisol-treated group.
Figure 3
Figure 3
Involvement of GR, CREB, and STAT3 in the induction of ALOX15 and ALOX15B expression by cortisol in human amnion fibroblasts. (A–D) Blockade of cortisol (1 µM; 24 h)-induced ALOX15 and ALOX15B mRNA (A,C, n = 4) and protein (B,D, n = 4) expression by GR antagonist RU486 (1 µM). (E–H) Blockade of cortisol (1 µM; 24 h)-induced ALOX15 and ALOX15B mRNA (E,G, n = 4) and protein (F,H, n = 4) expression by STAT3 inhibitor S3I-201 (10 μM). (I–L) Blockade of cortisol (1 µM; 24 h)-induced ALOX15 and ALOX15B mRNA (I,K, n = 4) and protein (J,L, n = 4) expression by CREB inhibitor 666-15 (10 μM). Data are represented as means + SEM. Statistical analysis was performed with one-way ANOVA test followed by Newman–Keuls multiple comparisons test. Top panels of B, D, F, H, J, and L are the representative immunoblots. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cortisol-treated group.
Figure 4
Figure 4
Increased enrichments of GR, CREB, and STAT3 at ALOX15 and ALOX15B gene promoters in human amnion fibroblasts by cortisol treatment. (A,B) Diagrams showing the putative binding sites for GR, STAT3, and CREB in ALOX15 (A) and ALOX15B (B) gene promoters. Red box indicates GR binding sites, blue box indicates STAT3 binding sites, and gray box indicates CREB binding sites. Arrows indicate primer aligning positions in ChIP assay. P1, forward primer; P2, reverse primer; TSS, transcription start site. (C,D) ChIP assay showing the enrichments of GR, STAT3, and CREB at ALOX15 (C) and ALOX15B (D) gene promoters in human amnion fibroblasts upon cortisol treatment (1 μM; 12 h). n = 4. IgG served as negative control. (E) CoIP assay showed the interaction of p300 with STAT3, CREB, and GR in human amnion fibroblasts with cortisol (1 µM; 12 h) treatment. Input and IgG served as positive and negative control, respectively. n = 3. Data are represented as means + SEM. Statistical analysis was performed with paired Student’s t-test. * p < 0.05 vs. control group.
Figure 5
Figure 5
Role of PGE2-EP2-PKA pathway in the induction of CREB and STAT3 phosphorylation, and ALOX15/15B expression by cortisol in human amnion fibroblasts. (A–F) Blockade of cortisol (1 µM)-induced phosphorylation of CREB (A, n = 3) and STAT3 (B, n = 3) and increases in ALOX15 and ALOX15B mRNA (C,D, n = 4) and protein (E,F, n = 3) abundance by EP2 receptor antagonist PF-04418948 (PF; 10 μM). (G–L) Blockade of cortisol (1 µM)-induced phosphorylation of CREB (G, n = 3) and STAT3 (H, n = 3) and increases in ALOX15 and ALOX15B mRNA (I,J, n = 4) and protein abundance (K,L, n = 4) by PKA inhibitor PKI 14-22 amide (PKI; 5 μM). Data are represented as means + SEM. Statistical analysis was performed with one-way ANOVA test followed by Newman–Keuls multiple comparisons test. Top panels of (A,B,E,F,G,H,K,L) are the representative immunoblots. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cortisol-treated group.
Figure 6
Figure 6
Involvement of CREB but not STAT3 in the regulation of p300 expression by cortisol in human amnion fibroblasts. (A) Blockade of cortisol (1 µM; 24 h)-induced EP300 (encoding p300) mRNA expression by CREB inhibitor 666-15 (10 μM). n = 4. (B) Ineffectiveness of STAT3 inhibitor S3I-201 (10 μM) (B, n = 4) on the induction of EP300 mRNA expression by cortisol (1 µM; 24 h). (C) Diagrams illustrating the putative binding sites of CREB in EP300 gene promoter. Brown box indicates CREB binding sites. Arrows indicate primer aligning positions in ChIP assay. P1, forward primer; P2, reverse primer; TSS, transcription start site. (D) ChIP assay showing increased CREB enrichment at EP300 gene promoter in human amnion fibroblasts with cortisol treatment (1 μM; 12 h). n = 4. IgG served as negative control. (E,F) Blockade of cortisol (1 µM; 24 h)-induced EP300 mRNA expression by EP2 receptor antagonist PF-04418948 (PF; 10 μM; E) and PKA inhibitor PKI 14-22 amide (PKI; 5 μM; F). n = 4. (G) Induction of EP300 mRNA expression by PGE2 (1 µM; 6 h). n = 3. (H,I) Blockade of PGE2 (1 µM; 6 h)-induced EP300 mRNA expression by EP2 receptor antagonist PF-04418948 (PF; 10 μM; H) and PKA inhibitor PKI 14-22 amide (PKI; 5 μM; I). n = 4. Data are represented as means + SEM. Statistical analysis was performed with paired Student’s t-test (D,G) or one-way ANOVA test (A, B, E, F, H, and I) followed by Newman–Keuls multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cortisol- or PGE2-treated group.
Figure 7
Figure 7
Increased abundance of ALOX15, ALOX15B, p300, p-CREB, and p-STAT3 in the human amnion at parturition. (A–F) Abundance of ALOX15, ALOX15B, p300, p-CREB, and p-STAT3 in the human amnion collected from term delivery with labor (TL; n = 7) and without labor (TNL; n = 7) as measured with Western blotting. (G–I) Pearson analysis showing positive correlation between ALOX15 and p300 (G), p-CREB (H), or p-STAT3 (I) abundance in the human amnion (n = 14). (J–L) Pearson analysis showing positive correlation between ALOX15B and p300 (J), p-CREB (K), or p-STAT3 (L) abundance in the human amnion (n = 14). (M) Pearson analysis showing positive correlation between p300 and p-CREB in the human amnion (n = 14). Statistical analysis was performed with unpaired Student’s t-test (C–F) or Mann–Whitney U test (B). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNL.
Figure 8
Figure 8
Diagram illustrating the pathway underlying the induction of ALOX15/15B expression by cortisol in human amnion fibroblasts. By binding to EP2 receptors, PGE2 activates the cAMP/PKA pathway, followed by CREB and STAT3 phosphorylation. Phosphorylated CREB and STAT3 not only induce PTGS2 (encoding COX-2) but also ALOX15/15B expression. PGE2 produced by COX-2 further reinforced this feed-forward loop. In addition, phosphorylated CREB also induces p300 expression. Increased p300 not only leads to H3K27 acetylation at ALOX15/15B gene promoters but also interacts with phosphorylated CREB and STAT3 at ALOX15/15B gene promoters to induce ALOX15/15B expression and 15(S)-HETE production. When cortisol is present, GR activated by cortisol interacts with phosphorylated CREB and STAT3 to further stimulate PTGS2 and ALOX15/15B expression. Gαs, stimulatory G protein; AC, adenylate cyclase; ATP, adenosine 5′-triphosphate; cAMP, cyclic adenosine monophosphate.
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