Korean J Physiol Pharmacol.  2017 Jul;21(4):377-384. 10.4196/kjpp.2017.21.4.377.

Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells

• Joo HK
• Lee YR
• Choi S
• Park MS
• Kang G
• Kim CS
• Jeon BH

• KMID: 2384451
• DOI: http://doi.org/10.4196/kjpp.2017.21.4.377

Abstract

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

Keyword

Endothelial cells; Mitochondria; Protein kinase C; p66shc; Reactive oxygen species

MeSH Terms

Endothelial Cells*
Gene Silencing
Inflammation
Intercellular Adhesion Molecule-1
Mitochondria
Monocytes
Phosphorylation
Protein Kinase C beta*
Protein Kinase C*
Protein Kinases*
Reactive Oxygen Species
Superoxide Dismutase
Intercellular Adhesion Molecule-1
Protein Kinase C
Protein Kinases
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 PKCβII inhibition suppressed PMA-induced mitochondrial ROS generation and endothelial activation in cultured endothelial cells. The effects of PKCβi, a specific PKCβII inhibitor, on PMA-induced (A) ICAM-1 expression, and (B) monocyte adhesion (C), mitochondrial ROS, (D) p66shc phosphorylation are shown. After pretreatment of HUVECs with PKCβi (10 nM), cells were exposed to 250 nM PMA for 6 h (A~C). (A) Following PMA treatment, cell lysates were analyzed by Western blotting for ICAM-1 expression. The β-actin was used as a loading control. (B) Monocyte adhesion was assessed by a monocyte-endothelial cell adhesion assay. Data are expressed as percentage values relative to monocyte adhesion induced by PMA. Each bar represents the mean±S.E.M., n=4. *p<0.05 vs. PMA alone. (C) Relative mitochondrial ROS generation was determined from MitoSOX red fluorescence assays. Fluorescence emission was measured at 590 nm after excitation at 530 nm. Values are expressed as mean fold-change in arbitrary fluorescence values over baseline values. Each bar represents the mean±S.E.M., n=4. #p<0.05 vs. baseline value, *p<0.05 vs. PMA alone. (D) After pretreatment of HUVECs with PKCβi (10 nM), cells were exposed to 250 nM PMA for 5 to 30 min. Phosphorylation of p66shc (S36) and total shc expression were measured by Western blotting.

• 

  Fig. 2 PKCβII inhibition suppressed PMA-induced CREB phosphorylation and MnSOD expression in cultured endothelial cells. (A) Induction of CREB phosphorylation by PMA treatment. HUVECs exposed to 250 nM PMA for 5 to 180 min were analyzed by Western blotting for CREB (S133) phosphorylation and total CREB expression. GAPDH was used as a loading control. (B) Effect of PKCβi on PMA-induced phosphorylation of CREB (S133). After pretreatment with PKCβi (10 nM), cells were exposed to 250 nM PMA for 15 min. Phosphorylation of CREB and total CREB expression were measured by Western blotting. (C) Effect of PKCβi on PMA-induced MnSOD expression. After treatment with PKCβi (10 nM), followed by 250 nM PMA for 6 h, MnSOD expression was measured using Western blotting. β-actin was used as a loading control. (D) Effect of down-regulation of CREB on PMA-induced MnSOD expression. After transfection (48 h) with control siRNA or CREB-targeting siRNA (100 nM), cells were exposed to 250 nM PMA for 6 h. Down-regulated CREB using siCREB and PMA-induced MnSOD expression levels were confirmed by Western blotting.

• 

  Fig. 3 Down-regulation of PKCβII inhibited PMA-induced mitochondrial ROS generation and ICAM-1 expression. (A) At 48 h post-transfection with control siRNA or PKCβII-targeting siRNA (100 nM), HUVECs were exposed to 250 nM PMA for 6 h. Relative mitochondrial ROS generation was determined from MitoSOX red fluorescence assays. Fluorescence emission was measured at 590 nm after excitation at 530 nm. Values are expressed as the mean fold-change in arbitrary fluorescence values over baseline values. Each bar shows the mean±S.E.M., n=3. #p<0.05 vs. baseline value, *p<0.05 vs. PMA alone. (B) After transfection with control siRNA or PKCβII-targeting siRNA (100 nM), HUVECs were exposed to 250 nM PMA for 3 or 6 h. Down-regulated PKCβII using siPKCβII, and PMA-induced MnSOD and ICAM-1 expressions were analyzed by Western blotting. β-actin was used as a loading control.

• 

  Fig. 4 Overexpression of PKCβII increased mitochondrial ROS generation and ICAM-1 expression. (A) At 24 h post-transduction with Adβgal or AdPKCβII, HUVECs were analyzed for mitochondrial ROS generation using MitoSOX red fluorescence assays. Fluorescence emission was measured at 590 nm with excitation at 530 nm. Values are expressed as the mean fold-change in arbitrary fluorescence values over basal value. Each bar shows the mean±S.E. (n=5). #p<0.05 vs. Adβgal. (B) Effect of PKCβII overexpression on p66shc (S36) phosphorylation. At 24 h post-transduction with Adβgal or AdPKCβII, HUVECs were analyzed for p66shc (S36) phosphorylation and total shc expression using Western blot analysis. (C) ICAM-1 expression in PKCβII-overexpressed HUVECs. Following transduction with Adβgal or AdPKCβII, cells were analyzed using Western blotting for ICAM-1 expression. (D) Effect of Mito-TEMPO, mitochondrial ROS inhibitor, on PKCβII-induced ICAM-1 expression. After treatment with Mito-TEMPO (10~500 nM) in AdPKCβII-overexpressed cells, the change in ICAM-1 level was analyzed by Western blotting. Flag-tagged PKCβII overexpression was confirmed by anti-Flag antibody detection. β-actin was used as a loading control. (E) MnSOD expression in PKCβII-overexpressed HUVECs. Following transduction with Adβgal or AdPKCβII, cells were analyzed using Western blotting for MnSOD expression. Quantitative analysis of MnSOD is shown in bottom of Fig. 4E. The total adenoviral MOI of 100 was balanced in the Adβgal control. Flag-tagged PKCβII overexpression was confirmed by anti-Flag antibody detection. β-actin was used as a loading control. #p<0.05 vs. Adβgal.

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