TrkA signalling pathways in human airway smooth muscle cell 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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