Figure 1.
The JNK inhibitor SP600125 dose-dependently increases Rgs4 expression in rabbit colonic smooth muscle cells.
Cultured and serum-starved muscle cells were treated with indicated concentration of SP600125 1 h before treatment with IL-1β (10 ng/ml) for 3 h, followed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) (A, B) and Western blot analysis (C, D). The relative level of Rgs4 mRNA expression (fold induction) was presented as compared with the control without SP600125 pretreatment after GAPDH normalization (A, B). Levels of β-actin and GAPDH were used as a loading control (C). The relative optical density (fold change) was presented as compared with the vehicle control (DMSO) after β-actin or GAPDH normalization (D). Values are means ± SE of 3 experiments. * (p<0.05) indicates significant increase after IL-1β treatment as compared with the control. + (p<0.05) indicate significant increase by ANOVA and Newman-Keuls comparison of SP600125 treatment with the vehicle control.
Figure 2.
Knockdown of JNK protein expression by shRNA (A) increased Rgs4 protein expression (B) in rabbit colonic smooth muscle cells.
Cultured cells were transfected with pLL3.7 empty vector or indicated JNK shRNA expression vectors. After 48 h, cells were starved for 24 h and treated with vehicle control or IL-1β (10 ng/ml) for 3 h, followed by Western blotting with anti-JNK (A) or anti-RGS4 (B) antibodies. The β-actin was used for loading control. Short and long exposures of the blot are shown. Similar results were observed in 3 experiments.
Figure 3.
Inhibition of JNK pathway potentiates Rgs4 transcription in rabbit colonic smooth muscle cells.
A. Transcriptional inhibition prevents Rgs4 mRNA upregulation by IL-1β and SP600125. Cultured muscle cells were starved for 24 h and pretreated with actinomycin D (10 µM) for 1 h and SP600125 (10 µM) for 30 min before exposure to IL-1β (10 ng/ml) for 3 h. Expression level (fold change) of Rgs4 mRNA was determined by RT-qPCR using GAPDH for normalization. B. SP600125 potentiates constitutive and IL-1β-induced promoter activity of rabbit Rgs4. Cultured muscle cells were cotransfected with promoter-less pMlu3 empty vector or Rgs4 promoter vector carrying secreted renilla luciferase and pGL4-CMV vector carrying firefly luciferase (for normalization). After 24 h, cells were serum-starved for 24 h and treated with IL-1β (10 ng/ml) and SP600125 (10 µM) for 24 h. The renilla and firefly luciferases were measured separately. The relative fold changes in renilla luciferase activity after normalization by firefly luciferase were expressed as compared with the empty vector and vehicle DMSO treatment. Data represents the mean ± SEM of 4 experiments, each with quadruplicate. ** P<0.01 and * P<0.05 indicate statistically significant increase by student's t test compared with corresponding DMSO treatment. ++ (p<0.01) indicates significant decrease after actinomycin D treatment compared with corresponding vehicle control.
Figure 4.
IL-1β promoted AP1-binding activity within proximal Rgs4 promoter. A. Inhibitory function of AP1 binding site within Rgs4 promoter for activation of reporter gene.
Site-directed mutant of AP1 site from Rgs4 promoter was co-transfected with normalization vector into cultured smooth muscle cells. After 24 h, cells were serum-starved for 24 h and treated with IL-1β (10 ng/ml) for 24 h before relative luciferase activity was determined. * P<0.05 indicates a statistically significant increase by student's t test compared with control treatment. ++ P<0.01 indicates a significant increase by AP1 site mutation in promoter activity compared with the wild-type Rgs4 promoter. Values represent the mean ± SEM of 4 individual experiments. B. Induction of AP1-DNA binding activity by IL-1β that is blocked by JNK or NFκB inhibition. Serum-starved muscle cells were treated with or without IL-1β (10 ng/ml) for 3 h in the absence or presence of JNK inhibitor SP600125 (10 µM) or NFκB inhibitor IKK2-IV (10 µM), and nuclear extracts were prepared for electrophoretic mobility shift assay using AP1-binding motif of rabbit Rgs4 promoter. The number under each panel indicates the relative fold of optical density compared with the corresponding control. n.s. for non-specific band. C. Induction of endogenous c-Fos- and c-Jun-DNA binding activity but inhibition of ATF-2-DNA binding within Rgs4 promoter by IL-1β. Serum-starved muscle cells were treated with IL-1β for 3 h before chromatin immunoprecipitation assay with indicated antibodies. Input indicates the DNA from supernatant after precipitation without IgG. Data are representative of 3 experiments.
Figure 5.
IL-1β induces a rapid and transient phosphorylation of JNK and sustained phosphorylation of ATF-2 and c-Jun in rabbit colonic smooth muscle cells.
Cultured and serum-starved muscle cells were treated with IL-1β (10 ng/ml) for the indicated time period, followed by Western blot analysis with indicated anti-phospho antibodies. The β-actin was used for the loading control.
Figure 6.
MEKK1-MKK4-JNK pathway inhibits Rgs4 expression in rabbit colonic smooth muscle cells.
A. Overexpression of MEKK1 inhibited constitutive and IL-1β-induced Rgs4 expression. Cultured muscle cells were transfected with pCMV empty vector or MEKK1 vector for 24 h. After serum starvation for 24 h, cells were treated with or without IL-1β (10 ng/ml) for 3 h before Western blot analysis with anti-Rgs4 antibody. The β-actin was used for the loading control. The number between each blot indicates the relative fold of optical density compared to the corresponding control. B. Inhibitory effect of MEKK1 on RGS4 expression was blocked by the dominant mutants of MKK4, JNK1 and JNK2. Cells were cotransfected with indicated vectors for 24 h. After serum starvation for 24 h, Western blot analysis with anti-Rgs4 and anti-β-actin antibodies was performed. The number between each blot indicates the relative fold of optical density compared with the pCMV empty control. The number below the lower blot indicates the fold change related to MEKK1 group. C. Inhibitory effect of MEKK1 on RGS4 expression was partially reversed by the shRNA of JNK1B and JNK2A. Cells were cotransfected with indicated vectors for 48 h, followed by 24 h serum-starvation and Western blot analysis with anti-Rgs4 and anti-β-actin antibodies. Short (5 second) and longer (5 minute) exposures are presented. The number between each blot indicates the relative fold of optical density compared with the pCMV empty control.
Figure 7.
JNK pathway interacts with NFκB and p38 MAPK pathways.
A. SP600125 enhances IL-1β-induced activation of canonical IKK2/IκBα/NFκB signaling. Cultured muscle cells after serum starvation for 24 h were pretreated with vehicle DMSO or JNK inhibitor SP600125 (10 µM) for 1 h before treatment with or without IL-1β (10 ng/ml) for 15 min. Activation of NFκB signaling was determined by Western blot analysis using indicated specific antibodies. B. IL-1β-induced phosphorylation of JNK (Thr183/Tyr185) is blocked by SP600125, enhanced by p38 MAPK inhibitor but not affected by MEK1 inhibitor. Cultured and serum-starved muscle cells were pretreated with p38 MAPK inhibitor SB203580 (1 µM) or MEK1 inhibitor PD98059 (20 µM) or JNK inhibitor SP600125 for 1 h before exposure to IL-1β (10 ng/ml) for 15 min. Activation of JNK pathway was determined by Western blot analysis using anti-phospho JNK antibody. The antibodies against GAPDH and β-actin were used for the loading control.
Figure 8.
Schematic model for IL-1β-induced upregulation of Rgs4 expression in colonic smooth muscle cells via canonical IKK2/IκBα/NFκB signaling differentially modulated by MAPK pathways.
IL-1β induces NFκB activation involving phosphorylation of IKK2, degradation of IκBα and nuclear translocation of p65/p50 leading to upregulation of Rgs4 mRNA expression. IL-1β also activates three MAPKs. ERK1/2 and p38 MAPK enhance while JNK inhibits IL-1β-induced Rgs4 upregulation. The effect of ERK1/2 is exerted on the canonical IKK2/IκBα/p65 pathway of NFκB activation and p38 MAPK may target at the chromatin level. The p38 may also inhibit JNK activity. Activation of the MEKK1-MKK4-JNK pathway down-regulates Rgs4 expression through transcriptional repression at the chromatin level (via AP1 binding) and also signal inhibition of NFκB activation at the level of IKK2. The intricate interactions across various transcription factors and chromatin remodeling need further investigation. The solid arrows indicate the activation while the spotted arrows represent the inhibition.