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17-β-estradiol inhibits hyperosmolarity-induced proinflammatory cytokine elevation via the p38 MAPK pathway in human corneal epithelial cells.

Wang C, Shi X, Chen X, Wu H, Zhang H, Xie J, Yang X, Gou Z, Ye J - Mol. Vis. (2012)

Bottom Line: Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared.The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol. 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs.The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Jiefang Road 88, Hangzhou, China.

ABSTRACT

Purpose: To evaluate the effects of 17-β-estradiol on hyperosmolar stress-induced proinflammatory cytokine production of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha (TNF-α) in SV40-immortalized human corneal epithelial cells (hCECs) and the regulatory effects of the mitogen-activated protein kinase (MAPK) signaling pathways in this process.

Methods: SV40 hCECs cultured in normal osmolar media were switched to a higher osmolarity (450 mOsM) by adding NaCl with or without pretreatment with 17-β-estradiol. Real-time polymerase chain reaction and ELISA were applied to characterize IL-6, IL-1, and TNF-α gene and protein expression. Cells were treated for 15-60 min, lysed in radioimmunoprecipitation assay (RIPA) buffer and subjected to a western blot with phospho (p)-specific antibodies against extracellular signal-regulated protein kinase 1/2 (ERK1/2), P38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2).

Results: The expression and production of IL-6, IL-1, and TNF-α in SV40 hCECs increased when the media osmolarity was switched to 450 mOsM. Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared. The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol.

Conclusions: 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs. The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs. These findings may contribute to an understanding of the etiologic roles and therapeutic implications of the hormone estrogen in dry eye disease.

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ELISA results regarding the amounts of IL-6, IL-1, and TNF-α present with inhibitors of the MAPK pathway in HCECs. Inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, but only SB203580 (an inhibitor of the p38 pathway) suppressed the elevation of cytokines caused by hyperosmolarity. 17-β-estradiol suppressed the hyperosmoticity-induced elevation of all of the three cytokines in conditions with or without JNK/ERK/p38 MAPK inhibitors. **p<0.01.
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f5: ELISA results regarding the amounts of IL-6, IL-1, and TNF-α present with inhibitors of the MAPK pathway in HCECs. Inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, but only SB203580 (an inhibitor of the p38 pathway) suppressed the elevation of cytokines caused by hyperosmolarity. 17-β-estradiol suppressed the hyperosmoticity-induced elevation of all of the three cytokines in conditions with or without JNK/ERK/p38 MAPK inhibitors. **p<0.01.

Mentions: Using phosphor-specific antibodies, western blot analysis revealed that the activated (phosphorylated) p38 was dramatically inhibited by pretreatment with 17-β-estradiol in SV40-immortalized hCECs within 15−60 min of exposure to 450 mOsM hyperosmolar media. In the control group, stimulated by the hyperosmolarity, p38 began to activate at the 15 min time point, and the maximum activation was observed after 30 min. A similar response was also observed in the case of p-ERK, with an increase by 15 min and the maximal activation being reached at 30 min. In the group of cells, which were pretreated with 17-β-estradiol, p38, and ERK were rarely activated by the hyperosmolarity (Figure 4). However, there was no difference in p-JNK between the pretreated cells and the controls. When inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, only the p38 inhibitor SB203580 could suppress the elevation of cytokines caused by hyperosmolarity (Figure 5). Figure 5 also showed that 17-β-estradiol pretreatment could suppress hyperosmotic-induced increases in any of the cytokines in all conditions with or without JNK/ERK/p38 MAPK inhibitors. Because all three inhibitors of the pathways were dissolved in DMSO, we used DMSO as a negative control.


17-β-estradiol inhibits hyperosmolarity-induced proinflammatory cytokine elevation via the p38 MAPK pathway in human corneal epithelial cells.

Wang C, Shi X, Chen X, Wu H, Zhang H, Xie J, Yang X, Gou Z, Ye J - Mol. Vis. (2012)

ELISA results regarding the amounts of IL-6, IL-1, and TNF-α present with inhibitors of the MAPK pathway in HCECs. Inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, but only SB203580 (an inhibitor of the p38 pathway) suppressed the elevation of cytokines caused by hyperosmolarity. 17-β-estradiol suppressed the hyperosmoticity-induced elevation of all of the three cytokines in conditions with or without JNK/ERK/p38 MAPK inhibitors. **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351403&req=5

f5: ELISA results regarding the amounts of IL-6, IL-1, and TNF-α present with inhibitors of the MAPK pathway in HCECs. Inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, but only SB203580 (an inhibitor of the p38 pathway) suppressed the elevation of cytokines caused by hyperosmolarity. 17-β-estradiol suppressed the hyperosmoticity-induced elevation of all of the three cytokines in conditions with or without JNK/ERK/p38 MAPK inhibitors. **p<0.01.
Mentions: Using phosphor-specific antibodies, western blot analysis revealed that the activated (phosphorylated) p38 was dramatically inhibited by pretreatment with 17-β-estradiol in SV40-immortalized hCECs within 15−60 min of exposure to 450 mOsM hyperosmolar media. In the control group, stimulated by the hyperosmolarity, p38 began to activate at the 15 min time point, and the maximum activation was observed after 30 min. A similar response was also observed in the case of p-ERK, with an increase by 15 min and the maximal activation being reached at 30 min. In the group of cells, which were pretreated with 17-β-estradiol, p38, and ERK were rarely activated by the hyperosmolarity (Figure 4). However, there was no difference in p-JNK between the pretreated cells and the controls. When inhibitors of the JNK pathway (SP600125 50 μM), the p38 pathway (SB203580 10 μM), and the ERK pathway (U0126 10 μM) were used, only the p38 inhibitor SB203580 could suppress the elevation of cytokines caused by hyperosmolarity (Figure 5). Figure 5 also showed that 17-β-estradiol pretreatment could suppress hyperosmotic-induced increases in any of the cytokines in all conditions with or without JNK/ERK/p38 MAPK inhibitors. Because all three inhibitors of the pathways were dissolved in DMSO, we used DMSO as a negative control.

Bottom Line: Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared.The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol. 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs.The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Jiefang Road 88, Hangzhou, China.

ABSTRACT

Purpose: To evaluate the effects of 17-β-estradiol on hyperosmolar stress-induced proinflammatory cytokine production of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha (TNF-α) in SV40-immortalized human corneal epithelial cells (hCECs) and the regulatory effects of the mitogen-activated protein kinase (MAPK) signaling pathways in this process.

Methods: SV40 hCECs cultured in normal osmolar media were switched to a higher osmolarity (450 mOsM) by adding NaCl with or without pretreatment with 17-β-estradiol. Real-time polymerase chain reaction and ELISA were applied to characterize IL-6, IL-1, and TNF-α gene and protein expression. Cells were treated for 15-60 min, lysed in radioimmunoprecipitation assay (RIPA) buffer and subjected to a western blot with phospho (p)-specific antibodies against extracellular signal-regulated protein kinase 1/2 (ERK1/2), P38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2).

Results: The expression and production of IL-6, IL-1, and TNF-α in SV40 hCECs increased when the media osmolarity was switched to 450 mOsM. Pretreatment with 10(-10) M 17-β-estradiol greatly inhibited the increased expression and production of IL-6, IL-1, and TNF-α induced by hyperosmolarity, whereas with the administration of SB203580 (10 μM), an inhibitor of the p38 pathway, the inhibiting effect of 17-β-estradiol disappeared. The western blot results showed that the increased phosphorylation level of p38 caused by hyperosmolarity was greatly inhibited by 17-β-estradiol.

Conclusions: 17-β-estradiol greatly inhibited the expression and production of proinflammatory cytokines IL-6, IL-1, and TNF-α, which were stimulated by hyperosmolarity in SV40-immortalized hCECs. The results also suggested that the p38 MAPK signaling pathway was involved in the regulatory effects of estrogen on hCECs. These findings may contribute to an understanding of the etiologic roles and therapeutic implications of the hormone estrogen in dry eye disease.

Show MeSH
Related in: MedlinePlus