The role of MAPK11/12/13/14 (p38 MAPK) protein in dopamine agonist-resistant prolactinomas

Abstract

Background: Prolactinoma is a functional pituitary adenoma that secretes excessive prolactin. Dopamine agonists (DAs) such as bromocriptine (BRC) are the first-line treatment for prolactinomas, but the resistance rate is increasing year by year, creating a clinical challenge. Therefore, it is urgent to explore the molecular mechanism of bromocriptine resistance in prolactinomas. Activation of the P38 MAPK pathway affects multidrug resistance in tumours. Our previous studies have demonstrated that inhibiting MAPK14 can suppress the occurrence of prolactinoma, but the role of MAPK11/12/13/14 (p38 MAPK) signalling in dopamine agonist-resistant prolactinomas is still unclear.

Methods: A prolactinoma rat model was established to determine the effect of bromocriptine on MAPK11/12/13/14 signalling. DA-resistant GH3 cells and DA-sensitive MMQ cells were used, and the role of MAPK11/12/13/14 in bromocriptine-resistant prolactinomas was preliminarily verified by western blot, RT-qPCR, ELISA, flow cytometry and CCK-8 experiments. The effects of MAPK11 or MAPK14 on bromocriptine-resistant prolactinomas were further verified by siRNA transfection experiments.

Results: Bromocriptine was used to treat rat prolactinoma by upregulating DRD2 expression and downregulating the expression level of MAPK11/12/13/14 in vivo experiments. The in vitro experiments showed that GH3 cells are resistant to bromocriptine and that MMQ cells are sensitive to bromocriptine. Bromocriptine could significantly reduce the expression of MAPK12 and MAPK13 in GH3 cells and MMQ cells. Bromocriptine could significantly reduce the expression of MAPK11, MAPK14, NF-κB p65 and Bcl2 in MMQ but had no effect on MAPK11, MAPK14, NF-κB p65 and Bcl2 in GH3 cells. In addition, knockdown of MAPK11 and MAPK14 in GH3 cells by siRNA transfection reversed the resistance of GH3 cells to bromocriptine, and haloperidol (HAL) blocked the inhibitory effect of bromocriptine on MAPK14, MAPK11, and PRL in MMQ cells. Our findings show that MAPK11 and MAPK14 proteins are involved in bromocriptine resistance in prolactinomas.

Conclusion: Bromocriptine reduces the expression of MAPK11/12/13/14 in prolactinomas, and MAPK11 and MAPK14 are involved in bromocriptine resistance in prolactinomas by regulating apoptosis. Reducing the expression of MAPK11 or MAPK14 can reverse bromocriptine resistance in prolactinomas.

Keywords: Bromocriptine; Dopamine agonist; Drug resistance; MAPK11/12/13/14; Prolactinomas.

Fig. 1

Bromocriptine upregulates DRD2 levels and reduces MAPK11/12/13/14 and PRL levels in rat prolactinomas. a Analysis of the appearance of the pituitary in the Control, Prolactinoma, and BRC groups. b Ratio of pituitary weight to body weight in rats,^****P < 0.0001 vs. Control;^####P < 0.0001 vs. Prolactinoma, (n = 10). c Detection of serum PRL level in rats by ELISA.^***P < 0.001 vs. Control; ^###P < 0.001 vs. Prolactinoma,(n = 6). d The protein expression levels of DRD2, MAPK11, MAPK12, MAPK13, MAPK14 and PRL in rat pituitary were detected by western blot. e The expression levels of DRD2, MAPK11, MAPK12, MAPK13, MAPK14 and PRL mRNA in rat pituitary were detected by RT-qPCR.^*p < 0.05 vs. control, ^***p < 0.001 vs. control, ^#p < 0.05 vs. prolactinoma,^###p < 0.001 vs. Prolactinoma,(n = 3)

Fig. 2

GH3 cells are resistant to bromocriptine and MMQ cells are sensitive to bromocriptine. a, b Western Blot analysis of the DRD2 protein expression level of different concentrations of bromocriptine (12.5 μM, 25 μM, 50 μM, 100 μM) treated MMQ cells for 48 h.^**P < 0.01 vs. 0 μM,^***P < 0.001 vs. 0 μM, (n = 3). c RT-qPCR analysis of DRD2 mRNA expression levels in MMQ cells treated with different concentrations of bromocriptine(12.5 μM, 25 μM, 50 μM, 100 μM) for 48 h.^**P < 0.01 vs. 0 μM,^***P < 0.001 vs. 0 μM, (n = 3). d CCK-8 kit detects the cell viability of bromocriptine on MMQ and GH3 cells. e, f The expression of PRL protein in the supernatant of GH3 and MMQ cells treated with bromocriptine for 48 h was detected by ELISA.^*p < 0.05 vs. 0 μM,^***p < 0.001 vs. 0 μM,^****p < 0.0001 vs. 0 μM, (n = 6)

Fig. 3

Comparison of the effects of bromocriptine on MAPK11/12/13/14 and PRL in GH3 cells and MMQ cells. a, b Western blot analysis of the protein expression of MAPK11, MAPK12, MAPK13, MAPK14 and PRL after treating GH3 cells and MMQ cells with different concentrations of bromocriptine(12.5 μM, 25 μM, 50 μM, 100 μM) for 48 h.^*p < 0.05 vs. 0 μM,^**p < 0.01 vs. 0 μM,^***p < 0.001 vs. 0 μM,^****p < 0.0001 vs. 0 μM, (n = 3). c RT-qPCR analysis of MAPK11, MAPK14 and PRL mRNA expression after bromocriptine treatment of GH3 cells and MMQ cells for 48 h.^**p < 0.01 vs. 0 μM,^***p < 0.001 vs. 0 μM,^****p < 0.0001 vs. 0 μM, (n = 3)

Fig. 4

Comparison of the effects of bromocriptine on NF-κB p65, Bcl2 and Bax in GH3 cells and MMQ cells. a, b The apoptosis rate of GH3 and MMQ cells treated with bromocriptine (12.5 μM, 25 μM, 50 μM) for 48 hours was detected by Annexin V-FITC and PI double staining.^**p < 0.01 vs. Control,^***p < 0.001 vs. Control, ^****p < 0.0001 vs. control, (n = 3). c, d Western blot analysis of NF-κB p65, Bcl2 and Bax protein expression after bromocriptine treatment of GH3 cells and MMQ cells for 48 h.^**p < 0.01 vs. 0 μM,^***p < 0.001 vs. 0 μM,^****p < 0.0001 vs. 0 μM, (n = 3)

Fig. 5

MAPK14 siRNA was transfected into GH3 cells to detect the effect of MAPK14 protein on the expression of PRL, NF-κB p65, Bcl2 and Bax in GH3 cells regulated by bromocriptine. a Western blotting was used to analyse the knockdown efficiency of three MAPK14 siRNA (100 nM) transfected GH3 cells for 72 h. b The level of PRL in the supernatant of GH3 cells was detected by ELISA.^**p < 0.01, ^****p < 0.0001 (n = 6). c, d The protein expression levels of PRL, NF-κB p65, Bcl2 and Bax in GH3 cells were analysed by western blotting.^*p < 0.05, ^**p < 0.01, ^***p < 0.001,^****p < 0.0001 (n = 3)

Fig. 6

MAPK11 siRNA was transfected into GH3 cells to detect the effect of MAPK11 protein on the expression of PRL, NF-KB p65, Bcl2 and Bax in GH3 cells regulated by bromocriptine. a Western blotting was used to analyse the knockdown efficiency of three MAPK11 siRNA (50 nM, 100 nM) transfected into GH3 cells for 72 h. b The level of PRL in the supernatant of GH3 cells was detected by ELISA.^*p < 0.05, ^****p < 0.0001 (n = 6). c, d The protein expression levels of PRL, NF-κB p65, Bcl2 and Bax in GH3 cells were analysed by western blotting.^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 (n = 3)

Fig. 7

Haloperidol blocks the effect of bromocriptine on MMQ cells. a, b Western blot analysis of DRD2, MAPK14, MAPK11 and PRL protein expression in MMQ cells pretreated with haloperidol for 4 h with or without bromocriptine for 48 h.^**p < 0.01 (n = 3). c The expression of PRL in the supernatant of MMQ cells was detected by ELISA.***p < 0.001 (n = 6). d The expression of PRL mRNA in MMQ cells was detected by RT-qPCR assays.***p < 0.001 (n = 3)

Fig. 8

Molecular mechanism of bromocriptine resistance in prolactinomas. Bromocriptine binds to DRD2 on the surface of prolactin cells, upregulates the expression of DRD2, downregulates the expression of MAPK11 and MAPK14, promotes cell apoptosis, and inhibits PRL secretion, thereby reversing the bromocriptine resistance of prolactinomas and restoring the sensitivity of prolactinoma to bromocriptine. The lack or low expression of DRD2, bromocriptine had no significant effect on MAPK11 and MAPK14 proteins, leading to bromocriptine resistance in prolactinomas