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. 2008 Apr;294(4):L778-86.
doi: 10.1152/ajplung.00410.2007. Epub 2008 Feb 1.

Hepatocyte growth factor regulates cyclooxygenase-2 expression via beta-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells

Young H Lee  1 , Yuichiro J Suzuki, Autumn J Griffin, Regina M Day
Affiliations

Hepatocyte growth factor regulates cyclooxygenase-2 expression via beta-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells

Young H Lee et al. Am J Physiol Lung Cell Mol Physiol. 2008 Apr.

Abstract

Hepatocyte growth factor (HGF) is upregulated in response to lung injury and has been implicated in tissue repair through its antiapoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play a role in cell growth. Here, we report that HGF induces gene transcription of COX-2 in human bronchial epithelial cells (HBEpC). Treatment of HBEpC with HGF resulted in phosphorylation of the HGF receptor (c-Met), activation of Akt, and upregulation of COX-2 mRNA. Adenovirus-mediated gene transfer of a dominant negative (DN) Akt mutant revealed that HGF increased COX-2 mRNA in an Akt-dependent manner. COX-2 promoter analysis in luciferase reporter constructs showed that HGF regulation required the beta-catenin-responsive T cell factor-4 binding element (TBE). The HGF activation of the COX-2 gene transcription was blocked by DN mutant of beta-catenin or by inhibitors that blocked activation of Akt. Inhibition of p42/p44 MAPK pathway blocked HGF-mediated activation of beta-catenin gene transcription but not Akt activation, suggesting that p42/p44 MAPK acts in a parallel mechanism for beta-catenin activation. We also found that inhibition of COX-2 with NS-398 blocked HGF-induced growth in HBEpC. Together, the results show that the HGF increases COX-2 gene expression via an Akt-, MAPK-, and beta-catenin-dependent pathway in HBEpC.

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Figures

Fig. 1
Fig. 1
Role of Akt in hepatocyte growth factor (HGF)-induced gene expression in human bronchial epithelial cells (HBEpC). HBEpC were grown to 80% confluence and placed in serum-free medium for 16 h. Cells were treated with 25 ng/ml HGF for the indicated times for each experiment. A: cell lysates were prepared, and equal amounts of protein were used for Western blots for phosphorylated c-Met protein; blots were stripped and probed for total c-Met protein. Experiments were repeated at least 3 times; representative results are shown. Band densitometry at left shows the means ± SD, *P ≤ 0.05. B: equal amounts of mRNA from each time point were used for semiquantitative RT-PCR of COX-2 (bottom band), with a competing internal standard (top band). Densitometry shows average of several experiments of COX-2 mRNA induced by HGF treatment, normalized to the internal standard (IS), *P ≤ 0.05. Experiments were repeated at least 3 times; representative results are shown. C: cell lysates were prepared, and equal amounts of protein from each time point were used in Western blot analysis for phosphorylated Akt; the blot was stripped and probed for total Akt protein as a control. Experiments were repeated at least 3 times; representative results are shown. Densitometry at left shows means ± SD, *P ≤ 0.05. D and E: HBEpC were grown to 80% confluence and infected with either control adenovirus (AdCont) or adenovirus expressing DN Akt (AdDnAkt). Following infection, cells were placed in serum-free medium for 16 h before treatment with 25 ng/ml HGF for the indicated times. D: cell lysates were made, and Western blots were performed on equal amounts of protein to detect Akt levels. E: equal amounts of mRNA from each time point were used for semiquantitative RT-PCR of COX-2, with a competing IS. Densitometry was performed as for B. Experiments were repeated at least 3 times; representative results are shown.
Fig. 2
Fig. 2
HGF regulation of the COX-2 promoter and the role of the TBE element. A: schematics of the COX-2 promoter: top, with predicted regulatory elements indicated (1, 2, 22); bottom, with the segments of the promoter used for luciferase reporter constructs indicated. B: HBEpC were grown to 80% confluence and transiently transfected with a COX-2-luciferase reporter construct: WT promoter −1432 bp to +59 bp, truncated WT promoter −327 bp to +59 bp, −1432 bp to +59 bp with either a deletion mutation in the TBE element, or −1432 bp to +59 bp with a point mutation in the TBE element. Six hours following transfection, cells were placed in serum-free medium for 1 h before treatment with 25 ng/ml HGF for 17 h. Luciferase assays were performed on equal amounts of protein. Firefly luciferase activity was normalized to Renilla activity to correct for transfection differences between samples. Results show means of 3 replicates ± SD; *P < 0.05. Experiments were performed at least 3 times, and representative results are shown.
Fig. 3
Fig. 3
Role of β-catenin for COX-2 promoter activation by HGF. A and B: HBEpC were grown to 80% confluence and placed in serum-free medium overnight. Cells were treated with 25 ng/ml HGF for the indicated time points. A: cell lysates were prepared, and equal amounts of protein were subjected to SDS-PAGE. Western blots were performed for phosphorylated GSK-3β protein; the blot was stripped and probed for total GSK-3β protein. Densitometry, below, shows means ± SD, *P ≤ 0.05. B: nuclear extracts were prepared, and equal amounts of protein were subjected to SDS-PAGE followed by Western blotting for β-catenin; the blot was stripped and probed for SP3 and stripped again and probed for β-actin. Densitometry, below, shows means ± SD, *P ≤ 0.05. C: HBEpC were grown to 80% confluence and transfected with the COX-2-luciferase reporter construct from −1432 bp to +59 bp cotransfected with either a plasmid expressing the mutant Y654/670 β-catenin (DN β-cat) or the p3XFlagCMV control (empty vector). Lysates were made 17 h following treatment with HGF (25 ng/ml). Luciferase assays were performed on equal amounts of protein, and firefly luciferase activity was normalized to Renilla activity to correct for transfection variations between samples. Results show means of 3 replicates ± SD; *P < 0.05.
Fig. 4
Fig. 4
Role of phosphatidylinositol 3-kinase (PI3K) for HGF-induced β-catenin-responsive promoter activity. HBEpC were grown to 80% confluence and transfected with the either the COX-2-luciferase reporter construct from −1432 bp to +59 bp (A) or with the TOPFLASH β-catenin-responsive reporter (B). Left: 6 h following transfection, cells were placed in serum-free medium for 1 h before treatment with 25 ng/ml HGF for the indicated times ± 20 min pretreatment with 10 μM LY-294002 (LY). Right: 6 h after transfections, cells were placed in serum-free medium for 1 h before treatment with 10 μM LY-294002 alone. Luciferase assays were performed on equal amounts of protein. Firefly luciferase activity was normalized to Renilla activity to correct for transfection differences between samples. Results show means of 3 replicates ± SD; *P < 0.05.
Fig. 5
Fig. 5
Role of MAPK for HGF-induced β-catenin-responsive promoter activity. A and B: HBEpC were grown to 80% confluence and placed in serum-free medium overnight. Cells were treated with 25 ng/ml HGF for the indicated time points, and cell lysates were prepared; where indicated, cells were treated with 20 μM U0126 or 20 μM LY-294002 for 20 min before addition of HGF. Equal amounts of protein were subjected to SDS-PAGE, and Western blots were performed for phosphorylated p42/p44 MAPK (B) or phosphorylated Akt (C); the blots were stripped and reprobed for the respective total protein. Densitometry for MAPK shows means ± SD, *P ≤ 0.05. C: HBEpC were grown to 80% confluence and transfected with the TOPFLASH β-catenin-responsive reporter. Six hours following transfection, cells were placed in serum-free medium for 1 h before treatment with 25 ng/ml HGF for 6 h ± pretreatment with U0126 (20 μM, left); control experiments used transfected cells treated with U0126 alone (right). Luciferase assays were performed on equal amounts of protein, and firefly luciferase activity was normalized to Renilla activity to correct for transfection differences between samples. Results show means of 3 replicates ± SD; *P < 0.05.
Fig. 6
Fig. 6
COX-2 activity is required for HGF-induced proliferation in HBEpC. HBEpC were split on day 1 to 5 × 104 cells/35-mm dish in defined medium. HGF was added (25 ng/ml) to the medium on days 1, 2, and 4. The NS-398 COX-2 inhibitor (10 μM) was added to cells before the HGF. Cells were counted on days 3 or 6. Experiments were done in triplicate; data show means ± SD; *P < 0.05. Representative data are shown.
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