Biochemical and Biophysical Research Communications
Wound-induced TGF-β1 and TGF-β2 enhance airway epithelial repair via HB-EGF and TGF-α
Highlights
► Production of TGF-β1 and TGF-β2 is induced by wound in airway epithelial cells. ► The TGF-β1 and TGF-β2 enhance EGFR phosphorylation and airway epithelial repair. ► The TGF-β1 and TGF-β2 effects on wound repair required a release of HB-EGF and TGF-α. ► Collaboration between TGF-β and EGFR contribute to maintaining airway homeostasis.
Introduction
The airway epithelium is continuously exposed to inhaled gaseous and particulate components and therefore is subject to injury. A rapid repair after injury is crucial for restoring epithelial barrier function. In response to environmental challenges, bronchial epithelial cells produce a number of growth factors [1], [2], [3], including transforming growth factor-beta (TGF-β), which is a multifunctional regulator of cell growth, differentiation, migration, extracellular matrix formation, and immune responses [4], [5]. Among the three isoforms of TGF-β family members secreted by mammalian cells, both TGF-β1 and TGF-β2 have been implicated in the early stage of skin wound repair [6]. In reference to the airway, some studies demonstrated that TGF-β2, but not TGF-β1, was produced by damaged airway epithelial cells (AECs) [3], [7], [8], but others reported that TGF-β1, but not TGF-β2, was expressed in AECs of fibrotic lungs, which promoted the migration of AECs in damaged monolayers [9], [10]. Thus, the role of TGF-β1 and TGF-β2 in wound repair of airway epithelium is still controversial.
Epidermal growth factor receptor (EGFR) signaling has been implicated in some effects of TGF-β1, such as accumulation of fibronectin in mesangial cells, regulation of the cell cycle in fetal rat hepatocytes, and migration of smooth muscle cells [11], [12], [13]. The main members of EGFR ligands potentially involved in wound repair include epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGF-α), and amphiregulin (AR) [14], [15], [16]. All these ligands are synthesized as transmembrane precursors and proteolytically processed to release the biologically active mature protein. An autocrine activation of the EGFR signaling plays a key role in epithelial wound repair by increasing epithelial cell proliferation, migration, differentiation, and survival [15], [17]. It have been suggested that the EGFR transactivation in response to TGF-β1 may be mediated by the release of a membrane-anchored EGFR ligand [13].
In the present study, we examined the involvement of TGF-β1 and TGF-β2 in airway epithelial repair and the contribution of EGFR transactivation. Our data, for the first time, show that mechanical injury induces secretion of both TGF-β1 and TGF-β2, which enhance EGFR activation and wound repair through production of HB-EGF and TGF-α in an AEC in vitro culture model. These findings suggest that both TGF-β1 and TGF-β2 play an important role in normal airway epithelial repair, which activates EGFR pathway by autocrine HB-EGF and TGF-α.
Section snippets
Reagents
Recombinant soluble human TGF-β1 and TGF-β2 were from Peprotech (Rocky Hill, NJ, USA). Anti-TGF-β1, anti-phosphorylated EGFR (pY845) antibodies, and neutralizing antibodies of EGF, HB-EGF, TGF-α, and AR were obtained from R&D Systems (Minneapolis, MN, USA). ELISA kits for TGF-β1, TGF-β2, EGF, HB-EGF, TGF-α, and AR were also from R&D Systems. Anti-TGF-β2 and anti-α-tublin antibodies, AG1478, SB431542, and Eagle’s minimum essential medium (MEM) were from Sigma Chemicals (St. Louis, MO, USA).
TGF-β1 and TGF-β2 enhance airway epithelial repair
We first investigated whether exogenous TGF-β1 and TGF-β2 could enhance airway epithelial repair. Injured monolayers of NHBE (Fig. 1A) and 1HAEo− (Fig. 1B) cells were treated with different concentrations of TGF-β1 or TGF-β2 (0.1–100 ng/ml). Addition of TGF-β1 significantly enhanced the epithelial repair in a dose-dependent manner. This result is consistent with a previous report that TGF-β1 accelerated bronchial epithelial wound repair [9]. Interestingly, TGF-β2 at 1, 10 and 100 ng/ml also
Discussion
Autocrine activation of EGFR, at least in part, plays a key role in epithelial wound repair [16], [17]. Mechanical damage rapidly induces EGFR phosphorylation in AECs through endogenous mediators. Bronchial epithelial cells produce EGF family, such as HB-EGF and TGF-α [14], [19]. Our previous study has revealed that wound-induced HB-EGF enhances airway epithelial repair [14], and also TGF-α has been shown to induce proliferation of AECs [20]. We now showed that mechanical injury induced
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