Protein kinase C-delta regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase

Abstract

The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).

FIG. 1

(A) Thrombin induces phosphorylation of PKC-δ. Confluent HUVEC monolayers were challenged with thrombin (2.5 U/ml) for the indicated time periods. Total cell lysates (10 μg/lane) were separated by SDS-PAGE and immunoblotted with an antibody against the phosphorylated (Thr505) form of PKC-δ. The blots were subsequently stripped and reprobed with an antibody against PKC-δ. (B and C) Thrombin induces PKC-δ activity. Confluent HUVEC monolayers were pretreated without (B) or with (C) rottlerin (5 and 10 μM) and LY379196 (10 nM) for 30 min prior to challenge with thrombin (2.5 U/ml) for 5 min. −, absence of rottlerin or LY379196; +, presence of thrombin. Cell lysates were immunoprecipitated with an antibody against PKC-δ, and in vitro kinase assays were carried out on immunoprecipitates using histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and a phosphorylated form of histone H1 was detected by autoradiography.

FIG. 2

(A and B) Inhibitors of PKC prevent thrombin-induced ICAM-1 mRNA expression. Confluent HUVEC monolayers were pretreated with calphostin C (A) or with staurosporine (B) prior to challenge with thrombin for 3 h. Total RNA was isolated and analyzed by Northern hybridization with a human ICAM-1 cDNA, which hybridizes to a 3.3-kb transcript. Blots were stripped and reprobed to determine GAPDH mRNA expression as a measure of RNA loading. DMSO, dimethyl sulfoxide. (C) Phorbol ester-induced depletion of cPKC and nPKC isoforms prevents thrombin-induced ICAM-1 mRNA expression. Confluent HUVEC monolayers were treated without (−) or with (+) PMA (500 nM in 10% FBS–EBM2) for 24 h followed by stimulation with thrombin (2.5 U/ml), PAR-1-activating peptide (TRAP; 25 μM), or PMA (100 nM) for 3 h. ICAM-1 and GAPDH mRNA expression was determined by Northern blotting as described in Materials and Methods.

FIG. 3

Inhibition of PKC-ζ fails to prevent thrombin-induced ICAM-1 mRNA expression. HUVEC were transfected with sense (S) or antisense (AS) oligonucleotide to PKC-ζ as described in Materials and Methods. After 36 to 48 h, cells were stimulated for 3 h with thrombin (2.5 U/ml). ICAM-1 and GAPDH mRNA expression was determined by Northern blotting as described in Materials and Methods. (A), autoradiogram; (B), bar graph showing the relative intensities of ICAM-1 mRNA signals.

FIG. 4

Inhibition of PKC-δ reduces thrombin-induced ICAM-1 mRNA expression. Confluent HUVEC monolayers were pretreated with rottlerin (A) or with LY379196 (B) prior to challenge with thrombin for 3 h. ICAM-1 and GAPDH mRNA expression was determined by Northern blotting as described in Materials and Methods. (A and B) Autoradiograms. Bottom of panel A contains a bar graph showing the effects of rottlerin on relative intensities of ICAM-1 mRNA signals.

FIG. 5

(A and B) Inhibition of PKC-δ prevents thrombin-induced ICAM-1 protein expression. Confluent HUVEC monolayers were pretreated with rottlerin or LY379196 (A) and peptide antagonist of PKC-θ (B) at the indicated concentrations prior to challenge with thrombin for 8 h. Expression of ICAM-1 protein was determined by Western blotting as described in Materials and Methods. The blots were subsequently stripped and reprobed with an antibody against PKC-δ or IκBβ to indicate equal loading of the gel. (C) Inhibition of PKC-δ prevents thrombin-induced endothelial adhesivity towards PMN. Confluent HUVEC monolayers were pretreated with rottlerin or LY379196 at the indicated concentrations prior to challenge with thrombin. Expression of endothelial adhesivity was determined by PMN adhesion assays as described in Materials and Methods.

FIG. 6

Inhibition of NF-κB activity by expression of a kinase-defective mutant of PKC-δ. HUVEC were cotransfected with plasmid pNF-κB-LUC and the constructs encoding kinase-defective mutants of PKC-δ (PKC-δ^mut), -ɛ (PKC-ɛ^mut), or -α (PKC-α^mut) isoform using the DEAE-dextran method as described previously (47). In some experiments pcDNA3 alone was used as the vector control. Cells were stimulated for 8 h with thrombin (2.5 U/ml) before being harvested. Cytoplasmic extracts were prepared, and luciferase activity was determined. Firefly luciferase activity normalized to Renilla luciferase activity is expressed in RLU per microgram of protein. Data are mean ± standard error (n = 3 for each condition).

FIG. 7

(A) Involvement of IKKβ in thrombin-induced NF-κB activity. HUVEC were cotransfected with pNF-κB-LUC and a construct encoding a kinase-defective mutant of IKKβ (IKKβ^mut) using the DEAE-dextran method as previously described (47). In some experiments pcDNA3 alone was used as the vector control. Cells were stimulated for 8 h with thrombin (2.5 U/ml) before being harvested. Cytoplasmic extracts were prepared, and luciferase activity was determined. Firefly luciferase activity normalized to Renilla luciferase activity was expressed in RLU per microgram of protein. Data are mean ± standard error (n = 3 for each condition). (B) Inhibition of IKKβ prevents PKC-δ-mediated NF-κB activity. HUVEC were cotransfected with pNF-κB-LUC in combination with the constructs encoding a kinase-defective mutant of IKKβ (IKKβ^mut) and a constitutively active PKC-δ mutant (PKCδ^CAT) using Superfect as described previously (45). In some experiments pcDNA3 alone was used as the vector control. Twenty-four hours later, cytoplasmic extracts were prepared and luciferase activity was determined. Firefly luciferase activity normalized to Renilla luciferase activity was expressed in RLU per microgram of protein. Data are mean ± standard error (n = 3 for each condition). +, presence of pcDNA3 or IKKβ^mut; −, absence of either plasmid.

FIG. 8

Inhibition of PKC-δ prevents thrombin-induced IκBα degradation and NF-κB DNA binding activity. Confluent HUVEC monolayers were pretreated with calphostin C and staurosporine (A) or rottlerin (B) at the indicated concentrations prior to challenge with thrombin for 1 h. Cytoplasmic (A) and nuclear (B) extracts were prepared and assayed for IκBα degradation by Western blot analysis (A) and for NF-κB DNA binding activity by EMSA (B) as described in Materials and Methods.

FIG. 9

(A) Thrombin induces phosphorylation of p38 MAP kinase. Confluent HUVEC monolayers were challenged with thrombin (2.5 U/ml) for the indicated periods. Total cell lysates (10 μg/lane) were separated by SDS-PAGE and immunoblotted with an antibody against a phosphorylated (Thr180/Tyr182) form of p38 MAP kinase. The blots were subsequently stripped and reprobed with an antibody against p38 MAP kinase. (B) Thrombin induces MAPKAP kinase 2 activity. Confluent HUVEC monolayers were pretreated for 30 min with SB203580 prior to challenge with thrombin for 5 min. Cell lysates were immunoprecipitated with an antibody against MAPKAP kinase 2, and in vitro kinase assays were carried out on immunoprecipitates using Hsp-25 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of Hsp-25 was detected by autoradiography.

FIG. 10

Inhibition of PKC-δ prevents thrombin-induced phosphorylation of p38 MAP kinase. Confluent HUVEC monolayers were pretreated for 30 min with rottlerin prior to challenge with thrombin for 5 min. Total cell lysates (10 μg/lane) were resolved in SDS-PAGE and were immunoblotted with an antibody against the phosphorylated form of p38 MAP kinase.

FIG. 11

Inhibition of p38 MAP kinase reduces thrombin-induced ICAM-1 mRNA expression. Confluent HUVEC monolayers were pretreated with SB203580 (10 μM) (A) or PD98059 (50 μM) (B) prior to challenge with thrombin for 3 h. ICAM-1 and GAPDH mRNA expression was determined by Northern blotting as described in Materials and Methods.

FIG. 12

Inhibition of NF-κB activity by expression of the dominant negative mutant of p38 MAP kinase. HUVEC were cotransfected with pNF-κB-LUC and a construct encoding the dominant negative mutant of p38 MAP kinase (p38^mut) using the DEAE-dextran method as described previously (47). Cells were stimulated for 6 h with thrombin (2.5 U/ml) before being harvested. Cytoplasmic extracts were prepared, and luciferase activity was determined. Firefly luciferase activity normalized to Renilla luciferase activity was expressed in RLU per microgram of protein. Data are mean ± standard error (n = 3 for each condition).

FIG. 13

Inhibition of p38 MAP kinase fails to prevent thrombin-induced IκBα degradation, nuclear translocation, and NF-κB DNA binding activity. Confluent HUVEC monolayers were pretreated for 30 min with SB203580 prior to challenge with thrombin for 1 h. Cytoplasmic (A) and nuclear (B and C) extracts were prepared and assayed for IκBα degradation (A) and NF-κB nuclear translocation (B) by Western blot analysis and for NF-κB DNA binding activity (C) by EMSA as described in Materials and Methods.

FIG. 14

Signaling events regulating thrombin-induced NF-κB activation and ICAM-1 transcription in endothelial cells. Thrombin challenge of endothelial cells results in PKC-δ activation, which in turn activates IKKβ and p38 MAP kinase. Activation of IKKβ contributes to thrombin-induced ICAM-1 gene transcription by promoting IκBα degradation and subsequently NF-κB binding to the ICAM-1 promoter. Activation of p38 MAP kinase contributes to ICAM-1 transcription, possibly by increasing the transactivation potential of bound NF-κB p65 through its phosphorylation. Alternatively, p38 MAP kinase may contribute to the response by promoting the interaction of NF-κB with the basal transcription machinery through phosphorylation of TBP.