Elsevier

Cellular Signalling

Volume 18, Issue 5, May 2006, Pages 621-627
Cellular Signalling

TrkA signalling pathways in human airway smooth muscle cell proliferation

https://doi.org/10.1016/j.cellsig.2005.06.007Get rights and content

Abstract

NGF may play a role in airway inflammation and hyperresponsiveness. We studied its possible involvement in airway remodelling and report here its proliferative effect and its receptor and signalling pathways in human airway smooth muscle cells in culture (HASMC). Proliferation of HASMC induced by NGF (0.1–10 pM) was assessed by the XTT and BrdU techniques with and without kinase inhibitors. Immunoprecipitation and Western blotting were used to study phosphorylation of TrkA and MAPK. NGF caused dose-dependent proliferation of HASMC and induced TrkA phosphorylation, both abolished by the tyrosine-kinase inhibitor K252a. PI3K and JNK inhibitors had no effect. PKC inhibitors partially inhibited NGF-induced proliferation and totally abolished p38 phosphorylation but did not affect ERK1/2 phosphorylation. The rafK inhibitor decreased NGF-induced proliferation, and totally abolished ERK1/2 phosphorylation, but did not affect p38 phosphorylation. This finding was confirmed by the decrease of NGF-induced proliferation after treatment with inhibitors of the p38 or of ERK1/2 pathways. In conclusion, NGF activation of the TrkA receptor involves two distinct signalling pathways: PKC selectively activates p38, and the ras/raf pathway selectively activates ERK1/2. Both are necessary to induce HASMC proliferation.

Introduction

The nerve growth factor NGF has been identified in the airways, located in and released from both inflammatory [1], [2], [3] and structural cells: fibroblasts and epithelial and smooth muscle cells in culture express NGF mRNA and protein [4], [5], [6], [7]. NGF synthesized in the airways is thought to play a role in inflammation and hyperresponsiveness. Animal studies show that it is involved in the development and persistence of inflammation and can induce bronchial hyperresponsiveness by itself [for reviews [8], [9], [10]]. A recent study reported that it may also do so in isolated human bronchus [11].

The biological action of NGF is mediated through activation of the high-affinity tropomyosin-receptor kinase A (TrkA) at picomolar concentrations [12], [13]. TrkA signalling pathways have been described in neural cells: transphosphorylation on tyrosine of the intracellular domain activates the small G protein ras, phospholipase Cγ (PLCγ) and protein kinase C (PKC), which in turn activate the mitogen-activated protein kinase (MAPK) cascade [[14], [15] for review [16]]. TrkA activation also initiates the phosphoinositol signalling pathway through PI3-kinase (PI3K) [17]. These transduction mechanisms are involved in the proliferation of airway smooth muscle cells induced by growth factors such as EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) for instance [for review 18]. Moreover, airway smooth muscle expresses an immunoreactive TrkA receptor in human bronchial biopsy sections [19]. Because these findings suggest that NGF plays a role in remodelling these muscles, we studied its effects on proliferation of human airway smooth muscle cells (HASMC). We found that NGF at picomolar concentrations activates the TrkA receptor and induces HASMC proliferation through two simultaneous signalling pathways: PKC selectively activates the p38 MAPK pathway, and ras/raf selectively activates the ERK1/2 pathway.

Section snippets

Isolation of HASMC

Primary cultures were created as previously described [7], [20] with human airway smooth muscle obtained from healthy lung transplant donors after sudden death (Center for Biological Resources (CRB), N. Martinet, Nancy, France). Cells at passage 7 were seeded in 96-well culture plates for proliferation studies and in 25-cm2 flasks for mRNA, protein and phosphorylation studies.

Cell treatment

Cells were treated with human β-NGF (0.001–0.1 ng/ml, i.e., 0.1–10 pM, R&D Systems, Lille, France) or its solvent for 4

NGF induces proliferation of HASMC

NGF (0.1–10 pM) significantly and dose-dependently increased proliferation of HASMC (Fig. 1), as both the XTT (Fig. 1A) and the BrdU (Fig. 1B) techniques showed. Proliferation was greatest at 3 pM (12.1 ± 0.7% increase over baseline proliferation, P < 0.01) and then decreased as the NGF concentration increased.

Involvement of the TrkA receptor in NGF-induced proliferation of HASMC

Compound K252a, a tyrosine-kinase inhibitor, totally abolished the proliferation induced by NGF at 3 pM (P < 0.001) (Fig. 1A and B), whereas a p75NTR blocking antibody did not modify it (Fig. 1

Discussion

We report here that NGF at picomolar concentrations induces proliferation of cultured HASMC by activating a functional TrkA receptor expressed on these cells. Our study also shows that the signalling pathways of the TrkA receptor inducing this proliferation involve phosphorylation of both p38 MAPK, activated by the PKC pathway, and ERK1/2 MAPK, activated by the ras/raf pathway.

First, our study reveals interesting new aspects of NGF induction of airway smooth muscle proliferation. This

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