Figure 1.
Effect of MAP kinase and MSK1 inhibitors on IL-1β-induced p65, MSK1 and CBP binding to the κB site of the SCF intronic enhancer.
Human lung fibroblasts in culture were pre-incubated for 1 h with a combination of the p38 inhibitor SB202190 (SB; 3.5 µM) and the MEK inhibitor PD98059 (PD; 20 µM) or with the MSK1-PKA inhibitor H89 (10 µM) and treated with IL-1β (20 U/ml) for 30 min. The ChIP experiment was performed with anti-p65, MSK1, CBP, phospho-Ser10 histone H3 and control Ig antibodies. Co-immunoprecipitated genomic DNA fragments were amplified by PCR with SCF intronic enhancer-specific primers. Input reflects the relative amounts of sonicated DNA fragments before immunoprecipitation. Results are representative of 3 independent experiments performed in fibroblasts from 3 different donors.
Figure 2.
Effect of MAP kinase and MSK1 inhibition on IL-1β-induced NF-κB activation.
A. Human lung fibroblasts in culture were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) and treated with IL-1β for the indicated time. Western blot analysis used anti-phospho-Ser276-p65, anti-phospho-Ser536-p65, anti-IκB and anti-p65 antibodies. B. Cells were transfected with 1 µg of pHis-p65 plasmid (WT p65 subunit with a N-terminal His-Tag) and pre-incubated with a combination of SB and PD, or H89 (10 µM) for 1 h before IL-1ß treatment. C. Cells were co-transfected with His-p65 plasmid and WT or “kinase-dead” (KD) MSK1 plasmid (1 µM), and treated with IL-1β (20 U/ml). Results are representative of three independent experiments performed in fibroblasts from three different donors.
Figure 3.
Effect of MSK1-mediated p65 Ser276 phosphorylation on IL-1β-induced p65-CBP interaction.
A. Human lung fibroblasts in culture were transfected with 1 µg of pHis-p65 plasmid (WT p65 subunit with a N-terminal His-Tag), and pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM), or with H89 (10 µM) and treated with IL-1β (20 U/ml). B. Human lung fibroblasts in culture were transfected with pHis-p65 or pHis-p65 S276C plasmids (1 µM), and treated with IL-1β (20 U/ml). His-p65 and co-precipitating CBP were revealed by immunoblotting using anti-p65 and anti-CBP antibodies. IP: immunoprecipitation; IB: immunoblot. Results are representative of three independent experiments performed in fibroblasts from three different donors.
Figure 4.
Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression.
A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C. Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
Figure 5.
Pathway leading to SCF expression in primary human lung fibroblasts upon IL-1β treatment.
After treatment with IL-1β, both p38 and ERK MAPK activate MSK1 which phosphorylates nuclear p65 at Ser276. This leads to binding of NF-κB p65 to the κB site of the SCF intronic enhancer, and to interaction with the coactivator CBP. Phosphorylation of histone H3 at Ser10 upon IL-1β-treatment is mediated through a MSK1-independent pathway. IL-1β: interleukin-1β; IL-1R: interleukin-1 receptor; ERK: extracellular signal regulated kinase; MAPK: mitogen-activated protein kinase; NF-κB: nuclear factor-κB; IκB: inhibitor of NF-κB; MSK1: mitogen- and stress-activated protein kinase 1; CBP: CREB binding protein; H3: histone 3.