Early immune response and regulation of IL-2 receptor subunits.
Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.
Northern California Institute for Research and Education, San Francisco, CA, United States. milliehf@aol.com
Millie
M
Hughes-Fulford
Eiko
E
Sugano
Thomas
T
Schopper
Chai-Fei
CF
Li
J B
JB
Boonyaratanakornkit
Augusto
A
Cogoli
D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D053631:Interleukin Receptor Common gamma Subunit; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D053645:Interleukin-2 Receptor alpha Subunit; D053646:Interleukin-2 Receptor beta Subunit; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000235:genetics; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
C508017:IL2RA protein, human; C508020:IL2RB protein, human; C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D053646:Interleukin-2 Receptor beta Subunit; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D015375:Receptors, Interleukin-2; D018808:Transcription Factor AP-1; C015482:protein kinase modulator; D007371:Interferon-gamma; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases