Dual regulation of tumor necrosis factor-α on myosin light chain phosphorylation in vascular smooth muscle

Abstract

We previously demonstrated that inhibitor κB kinase 2 (IKK2) is a myosin light chain kinase (MLCK). In the present study, we assess whether the prototypical activator of IKK2 tumor necrosis factor-α (TNF-α) regulates the MLCK activity of IKK2 and thus MLC phosphorylation in vascular smooth muscle cells (VSMCs). Kinase activity assay revealed that TNF-α downregulated the MLCK activity of IKK2 in human VSMCs (HVSMCs). However, Western blot analysis did not demonstrate a significant effect of TNF-α on MLC phosphorylation in HVSMCs, and myograph analysis did not reveal a significant effect of TNF-α on the contraction of the aorta from Sprague-Dawley rats and C57Bl/6j mice, suggesting a dual regulation of MLC phosphorylation by TNF-α. Confirming this notion, TNF-α significantly increased MLC phosphorylation in IKK2(-/-) but not wild-type cells. Furthermore, our results show that TNF-α increased GTP-bound RhoA and MLC phosphatase subunit MYPT1 phosphorylation and markedly reduced MLC phosphorylation in the presence of Rho-kinase inhibitor Y-27632, suggesting that downregulation of MLCK activity of IKK2 by TNF-α is antagonized by simultaneous RhoA/Rho-kinase activation. These results indicate that TNF-α dually regulates MLC phosphorylation through both IKK2 and RhoA/Rho-kinase pathways.

Keywords: IKK2; MLC phosphorylation; blood pressure; inflammation; vasoconstriction.

Fig. 1.

Tumor necrosis factor-α (TNF-α) downregulates the myosin light chain kinase (MLCK) activity of inhibitor κB kinase 2 (IKK2). A and B: human vascular smooth muscle cells (HVSMCs) were treated with TNF-α (10 ng/ml) for the indicated time. IKK2 was precipitated from the cell lysates with anti-IKK2 antibody and used to phosphorylate MLC and IκBα in vitro. Phospho-MLC, phospho-IκBα, total IκBα, and IKK2 in products were analyzed by Western blot. Total MLC was stained with Ponseau S solution. The representative images (A) and the summary (B) of 6 independent experiments are presented. *P < 0.05 vs. 0, one-way ANOVA. C: HVSMCs were treated with TNF-α (10 ng/ml) for the indicated time, and the MLC phosphorylation levels were determined by Western blot. One representative result and the summary of 8 independent experiments are presented. D: HVSMCs were treated with TNF-α (10 ng/ml) for the indicated time, and immunoprecipitation with anti-IKK2 was performed on cell lysates. Both the pellets and supernatants were subjected to Western blot with the indicated antibody.

Fig. 2.

Dual regulation of TNF-α on MLC phosphorylation. A and B: wild-type (WT) and IKK2^−/− mouse aortic smooth muscle cells were treated with TNF-α (10 ng/ml) for 5 min, and MLC phosphorylation and IκBα expression were analyzed by Western blot; n = 3. *P < 0.05 vs. WT control; #P < 0.05 vs. IKK2^−/− control, two-way ANOVA. C and D: aortic rings were prepared from C57Bl/6j mice and mounted onto myograph. After precontraction by phenylephrine (PE; 1 μM), TNF-α (10 ng/ml) was added. One representative recording (C) and the summary (D) of 4 independent experiments are presented. E and F: endothelium-denuded rat aortic rings were contracted by PE (1 μM), and then the indicated concentration of TNF-α was added in a cumulative manner. A representative recording (E) and the summary (F) from 3 independent experiments is present. G and H: aortic rings were prepared from smooth muscle specific IKK2 knockout (SM22-CreIKK2^flox/flox) mice and mounted onto myograph. After precontraction by PE (1 μM), TNF-α (10 ng/ml) was added. One representative recording (C) and the summary (D) of 4 independent experiments are presented.

Fig. 3.

TNF-α activates RhoA/Rho-kinase pathway. A and B: HVSMCs were pretreated with AM-BAPTA (10 mM) or calyculin A (0.3 μM) for 15 min, followed by treatment with TNF-α (10 ng/ml) for the indicated time and then subjected to MLC phosphorylation analysis by Western blot. The representative images (A) and summary (B) of 4 independent experiments are presented. *P < 0.05 vs. vehicle of the same time point; #P < 0.05 vs. calyculin A of 0 min, two-way ANOVA. C–E: HVSMCs were treated with TNF-α (10 ng/ml) for the indicated time. GTP-bound RhoA (C; n = 4) was assessed by the RhoA G-LISA activation assay (cytoskeleton); MLC and MYPT1 phosphorylation (D and E; n = 3) was analyzed by Western blot. *P < 0.05 vs. 0, one-way ANOVA. F and G: HVSMCs were pretreated with vehicle or Y-27632 (10 μM) for 30 min and followed by treatment with TNF-α (10 ng/ml) for the indicated time. MLC phosphorylation was then analyzed by Western blot; n = 3. *P < 0.05 vs. 0; #P < 0.05 vs. vehicle, two-way ANOVA. H and I: HVSMCs were transfected with vector or plasmid that expresses HA-tagged dominant negative RhoA (DN-RhoA) and treated with TNF-α (10 ng/ml) for 5 min. MLC phosphorylation was then analyzed by Western blot; n = 3. *P < 0.05 vs. vehicle, two-way ANOVA. J and K: vascular smooth muscle cells were isolated from SM22-CreIKK2^flox/flox mice and treated with TNF-α (10 ng/ml) for 5 min in the presence of calyculin A (0.3 μM) or Y-27632 (10 μM); n = 3. *P < 0.05 vs. vehicle, two-way ANOVA.

Fig. 4.

The MLCK activity of IKK2 is coprecipitated with NF-κB essential modulator (NEMO). A and B: HVSMCs were treated with TNF-α (10 ng//ml) for 5 min. IKK complex was immunoprecipitated with anti-NEMO and subjected to in vitro phosphorylation reaction. The products were then analyzed by Western blot. The representative result (A) and summary (B) of 3 independent experiments are presented. *P < 0.05 vs. control, one-way ANOVA. C and D: cell lysates were prepared from wild-type and IKK^−/− mouse aortic smooth muscle cells. IKK complex was immunoprecipitated with anti-NEMO and subjected to in vitro phosphorylation reaction. The products were then analyzed by Western blot. The representative result (C) and summary (D) of 3 independent experiments are presented. *P < 0.05 vs. control, one-way ANOVA.

Fig. 5.

NEMO mediates TNF-α-induced downregulation of the MLCK activity of IKK2. A–C: HVSMCs were pretreated with NBD (a cell-permeable cytoplasmic transduction peptide YGRRARRRARR fused to the NEMO binding domain peptide TALDWSWLQTE; Calbiochem) or control peptide [a cell-permeable Antennapedia-NBD mutated (Trp739 and Trp741 replaced with Ala) fusion peptide analog of NEMO-binding domain binding peptide] for 15 min and then stimulated with TNF-α (10 ng/ml) for 5 min. IKK complex was immunoprecipitated from cell lysates with anti-IKK2 and used to perform in vitro phosphorylation reaction. The products were then analyzed by Western blot. The representative result (A) and summary (B) of 5 independent experiments are presented. *P < 0.05 vs. control, one-way ANOVA. The cellular MLC phopshorylation levels (C) were also analyzed by Western blot, and the representative image and summary of 3 independent experiments are presented. *P < 0.05 vs. control peptide, one-way ANOVA. D and E: endothelium-denuded aortic rings from C57Bl/6J mice were mounted to myograph and pretreated with NBD or control peptide for 15 min and then stimulated with TNF-α (10 ng/ml). The maximal contraction was expressed as percentage of contraction induced by KCl (120 mM); n = 3. *P < 0.05 vs. control peptide; Student's t-test.