JNK regulates MCP-1 expression in adenovirus type 19-infected human corneal fibroblasts.
OBJECTIVE
Previous studies indicate that adenovirus type 19 (Ad19) infection of human corneal fibroblasts (HCFs) induces the expression of several proinflammatory mediators, including IL-8 and monocyte chemoattractant protein-1 (MCP-1), and that the tyrosine kinase c-Src and its downstream target, the mitogen-activated protein kinase ERK1/2, mediate IL-8 expression. In this context, the authors sought to investigate the potential role of another mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), in adenoviral ocular pathogenesis.


METHODS
Ad19- and mock-infected HCFs were solubilized at various time points after infection, and cell lysates were subjected to SDS-PAGE followed by immunoblot analysis with a panel of antibodies against components of the MKK7/JNK/c-Jun pathway or immunoprecipitated for JNK assay. The induction of chemokine mRNA and protein was determined by real-time PCR and ELISA, respectively.


RESULTS
Ad19 induced the phosphorylation of MKK7, JNK, and the downstream transcription factor c-Jun in HCFs at 15 and 30 minutes after infection. JNK activity was demonstrated at 30 minutes after infection using the GST-c-Jun fusion protein as a target substrate. SP600125, a specific pharmacologic inhibitor of JNK, blocked MCP-1 but not IL-8 mRNA and protein expression. Finally, PP2, a specific inhibitor of c-Src previously shown to inhibit the expression of both IL-8 and MCP-1 in Ad19-infected HCFs, also blocked JNK phosphorylation after infection.


CONCLUSIONS
The MKK7/JNK/c-Jun cascade is rapidly activated and mediates MCP-1 expression in Ad19-infected HCFs. Furthermore, the activation of c-Src on Ad19 infection appears to regulate both the ERK and the JNK pathways.
Xiao
Jingnan
J
Department of Ophthalmology, University of Oklahoma Health Sciences Center, 73104, USA.
Chodosh
James
J
D000260:Adenoviruses, Human / Q000502:physiology*; D018929:Cell Culture Techniques; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism* / Q000821:virology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D048688:MAP Kinase Kinase 7 / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D048031:JNK Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; C482569:MAP2K7 protein, human