Transcriptional regulation of lysophosphatidic acid-induced interleukin-8 expression and secretion by p38 MAPK and JNK in human bronchial epithelial cells

Abstract

HBEpCs (human bronchial epithelial cells) contribute to airway inflammation by secreting a variety of cytokines and chemokines in response to allergens, pathogens, viruses and environmental toxins and pollutants. The potent neutrophil chemoattractant, IL-8 (interleukin-8), is a major cytokine secreted by HBEpCs. We have recently demonstrated that LPA (lysophosphatidic acid) stimulated IL-8 production in HBEpCs via protein kinase C delta dependent signal transduction. However, mechanisms of IL-8 expression and secretion are complex and involve multiple protein kinases and transcriptional factors. The present study was undertaken to investigate MAPK (mitogen-activated protein kinase) signalling in the transcriptional regulation of IL-8 expression and secretion in HBEpCs. Exposure of HBEpCs to LPA (1 microM) enhanced expression and secretion of IL-8 by 5-8-fold and stimulated threonine/tyrosine phosphorylation of ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase). The LPA-induced secretion of IL-8 was blocked by the p38 MAPK inhibitor SB203580, by p38 MAPK siRNA (small interfering RNA), and by the JNK inhibitor JNK(i) II, but not by the MEK (MAPK/ERK kinase) inhibitor, PD98059. LPA enhanced the transcriptional activity of the IL-8 gene; that effect relied on activation of the transcriptional factors NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein-1). Furthermore, SB203580 attenuated LPA-dependent phosphorylation of IkappaB (inhibitory kappaB), NF-kappaB and phospho-p38 translocation to the nucleus, NF-kappaB transcription and IL-8 promoter-mediated luciferase reporter activity, without affecting the JNK pathway and AP-1 transcription. Similarly, JNK(i) II only blocked LPA-mediated phosphorylation of JNK and c-Jun, AP-1 transcription and IL-8 promoter-mediated luciferase reporter activity, without blocking p38 MAPK-dependent NF-kappaB transcription. Additionally, siRNA for LPA(1-3) receptors partially blocked LPA-induced IL-8 production and activation of MAPKs. The LPA1 and LPA3 receptors, as compared with LPA2, were most efficient in transducing LPA-mediated IL-8 production. These results show an independent role for p38 MAPK and JNK in LPA-induced IL-8 expression and secretion via NF-kappaB and AP-1 transcription respectively in HBEpCs.

Figure 1. LPA stimulates IL-8 gene expression and secretion in HBEpCs

(A) HBEpCs (approx. 80% confluence in 35 mm dishes) were treated with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 30–180 min. IL-8 gene expression was evaluated by real-time RT-PCR as described in the Experimental procedures section. Results are representative of at least three independent experiments. (B) HBEpCs (approx. 80% confluence in 35 mm dishes) were treated with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 1–24 h. IL-8 in the medium was quantified by ELISA as described in the Experimental procedures section. The results are expressed as the means±S.D. (n=3) performed in triplicate. *, statistically different from control (P<0.05).

Figure 2. LPA stimulates phosphorylation of MAPKs in HBEpCs

HBEpCs (approx. 80% confluence in 35 mm dishes) were treated with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 0–15 min. Cell lysates (10 μg) were subjected to SDS/PAGE and Western blotted for phospho- (p) and total-(T) ERK (A) or phospho- and the total p38 MAPK (B) or phospho- and total JNK (C). Shown are representative immunoblots of three independent experiments performed in duplicate. Fold changes in phospho-ERK/ERK or phospho-p38 MAPK/p38 MAPK or phospho-JNK/JNK were calculated from the Western blots by image analysis and results were normalized to total ERK or p38 MAPK or JNK. *, significantly different from 0 min (P<0.05).

Figure 3. Effect of MAPK inhibitors on LPA-induced IL-8 secretion

HBEpCs (approx. 80% confluence in 35 mm dishes) were pretreated with BEGM (vehicle) or PD89059 (25 μM) or SB 203580 (25 μM) or JNK_i II (25 μM) for 1 h. The cells were then challenged with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 2 h. IL-8 secreted into medium was quantified by ELISA as described in the Experimental procedures section. The results are expressed as the means±S.D. (n=3) performed in triplicate. *, significantly different from control (P<0.05), **, significantly different from cells treated with LPA alone (P<0.05).

Figure 4. p38 MAPK siRNA and SB-203580 attenuate LPA-induced IL-8 secretion in HBEpCs

HBEpCs (approx. 80% confluence in 35 mm dishes) were pretreated with various concentrations of SB203580 for 1 h prior to LPA (1 μM) challenge for either 15 min or 2 h. (A) Cell lysates (10 μg) from 15 min treatment were subjected to SDS/PAGE and Western blotted with phospho- and pan p38 MAPK and MAPKAPK-2 antibodies. (B) The medium was collected after 2 h LPA exposure and analysed for IL-8 by ELISA as described in the Experimental procedures section. Values are means of three independent experiments in triplicate. (C) HBEpCs (approx. 50% confluence in 35 mm dishes) were transfected with control siRNA or p38 MAPK siRNA (100 nM) for 72 h as described in the Experimental procedures section. Cell lysates (10 μg) were subjected to SDS/PAGE and Western blotted with p38 MAPK or β-actin or ERK or JNK1 antibodies. Shown is a representative blot of three independent experiments. (D) HBEpCs (approx. 50% confluence in 35 mm dishes) were transfected with control siRNA or p38 MAPK siRNA (100 nM) for 72 hours prior to LPA (1 μM) challenge for 2 h. The results are expressed as the means±S.D. (n=3) performed in triplicate. *, significantly different from control (P<0.05), **, significantly different from LPA treatment (P<0.05).

Figure 5. Effect of JNK_i II on LPA-induced phosphorylation of JNK and c-Jun and IL-8 secretion in HBEpCs

HBEpCs (approx. 80% confluence in 35 mm dishes) were pretreated with various concentration of JNK_i II for 1 h prior to LPA (1 μM) challenge for either 15 min or 2 h. (A) Cell lysates (10 μg) from 15 min treatment were subjected to SDS/PAGE and Western blotted with phosph-JNK1/2 or total JNK1 antibodies. (B) and (C) HBEpCs (approx. 80% confluence in 35 mm dishes) were pretreated with either JNK_i II (25 μM) (B) or SB203580 (25 μM) (C) for 1 h prior to challenge with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% BSA plus LPA (1 μM) for 15 min. Cell lysates (10 μg) were subjected to SDS/PAGE and Western blotted with phospho-c-Jun or total c-Jun antibodies. (D) Cells were exposed to LPA for 2 h and medium was collected and analysed for IL-8 by ELISA as described in the Experimental procedures section. Values are means of two independent experiments performed in triplicate.

Figure 6. p38 MAPK activation and NFκB transcription in HBEpCs

HBEpCs (approx. 80% confluence in 35 mm dishes) were pretreated with SB 203580 (25 μM) for 1 h prior to exposure to BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 15 min. (A) Cell lysates (10 μg) were subjected to SDS/PAGE and Western blotted with phospho-IκB or phospho-p38 MAPK or phospho-MAPKAPK-2 or pan p38 MAPK or MAPKAPK-2 or β-actin antibodies. (B) as in A, cells were pretreated with JNK_i II (25 μM) for 1 h prior to challenge with LPA and cell lysates (10 μg protein) were subjected to SDS/PAGE and Western blotted with phospho-IκB and β-actin antibodies. (C) and (D) HBEpCs were grown on 8-well chamber slides to approx. 70% confluence and pretreated with SB203580 (25 μM; SB) or JNK_i II (25 μM) for 1 h prior to challenge with LPA (1 μM) for 15 min. Cells were then subjected to immuno-staining for NF-κB (p65) (C) or p-p38 MAPK (D) and examined by fluorescence microscopy as described in the Experimental procedures section. The immunofluorescent image is representative of two independent experiments.

Figure 7. Effect of MAPK inhibitors on LPA-induced NF-κB activation

A, HBEpCs (approx. 60% confluence in 24-well plates) were transiently transfected with 100 ng of NF-κB reporter construct along with 1 ng of pRL-TK reference plasmid. After overnight incubation, cells were pretreated with SB203580 (25 μM) or JNK_i II (25 μM) for 1 h prior to LPA challenge (1 μM) for 3 h. Luciferase activity was measured using a commercially available kit. The results are expressed as the means±S.D. (n=3). * significantly different from vehicle controls (p < 0.05), **, significantly different from LPA treatment alone (p < 0.05). B, Nuclear extracts isolated from control (lanes1, 5) and LPA-stimulated HBEpCs from different times (lanes 2–4) were incubated with ³²P end-labelled NF-κB. Lanes 6–7 contain extracts from cells pre-incubated with vehicle or SB203580 prior to LPA challenge for 60 min. Shown is a representative gel shift assay of three independent experiments.

Figure 8. JNK_i II and c-Jun siRNA block p-JNK nuclear translocation, and LPA-induced AP-1 activation

A, HBEpCs were grown on 8-well chamber slides to approx. 70% confluence and pretreated JNK_i II (25 μM) for 1 h prior to challenge with vehicle or LPA (1 μM) for 15 min. Cells were then subjected to immuno-staining with anti-phospho-JNK antibody and examined by fluorescence microscopy as described in the Experimental procedures section. The immunofluorescent image is representative of three independent experiments. (B) Nuclear extracts (N. Ext) and post-nuclear supernatant (PNS) from control (vehicle) and LPA stimulated cells (15 min) were Western blotted with anti-phospho-specific-JNK- and anti-total-JNK1- antibodies. (C) HBEpCs (approx. 60% confluence in 24-well plates) were transiently transfected with 100 ng of AP-1 reporter construct along with 1 ng of pRL-TK reference plasmid. After overnight incubation, cells pretreated with JNK_i II or SB203580 for 1 h prior to LPA challenge (1 μM) for 3 h. Luciferase activity was measured using a commercially available kit. The results are expressed as the means±S.D. (n=3). * significantly different from controls (P<0.05), **, significantly different from LPA treatment (P<0.05). (D) Nuclear extracts isolated from control (lanes 1, 6, 9) and LPA-stimulated for different times (lanes 2–4) were incubated with the ³²P end-labelled AP-1. Lanes 8 and 11 contain extracts from cells pretreated with JNK_i II and c-Jun siRNA respectively prior to LPA challenge. Nuclear extract in lane 5 was incubated with 100-fold molar excess of unlabelled double-stranded AP-1. Shown is a representative of three independent experiments.

Figure 9. Effect of c-Jun siRNA on LPA-induced IL-8 secretion

HBEpCs (approx. 50% confluence in 35 mm dishes) were transfected with control siRNA or c-Jun siRNA (100 nM) for 72 h and then challenged with BEGM containing 0.1% (w/v) BSA (vehicle) or BEGM containing 0.1% (w/v) BSA plus LPA (1 μM) for 2 h. (A) Cell lysates (10 μg protein) were subjected to SDS/PAGE and Western blotted for c-Jun or ERK or β-actin. (B) Cells were treated with vehicle or LPA for 2 h and IL-8 secreted into medium was quantified by ELISA as described in the Experimental procedures section. The results are expressed as the means±S.D. (n=3) performed in triplicate. *, significantly different from controls (P<0.05), **, significantly different from LPA treatment (P<0.05).

Figure 10. Effect of LPA receptor siRNA on LPA-induced phosphorylation of MAPKs

HBEpCs (approx. 50% confluence in 35 mm dishes) were transfected with control siRNA or LPA_1–3 receptor siRNA (200 nM) for 72 h and then harvested (A, B, C) or challenged with BEGM with 0.1% (w/v) BSA (vehicle) or BEGM with 0.1% (w/v) BSA plus LPA (1 μM) for 15 min (D, E, F). Cell lysates were subjected to SDS/PAGE and Western blotted with LPA₁, LPA₂ or LPA₃ or β-actin antibodies (A, B, C) or anti-phospho-specific- and pan-antibodies for ERK, p38 MAPK, JNK (D, E, F).

Figure 11. Proposed MAPK signalling involved in LPA-induced IL-8 secretion

Ligation of LPA to its G-protein-coupled receptors LPA_1–3 results in activation of ERK, p38 MAPK and JNK pathways. LPA-induced activation of p38 MAPK and JNK (heavy arrows), but not ERK (dashed arrow) regulates NF-κB and AP-1 transactivation and IL-8 secretion.