ErbB2 activity is required for airway epithelial repair following neutrophil elastase exposure

Abstract

In cystic fibrosis and chronic bronchitis, airways are chronically injured by exposure to neutrophil elastase (NE). We sought to identify factors required for epithelial repair following NE exposure. Normal human bronchial epithelial cells were treated with NE (50 nM, 22 h) or control vehicle. Following NE treatment, we found a marked and sustained decrease in epithelial proliferation as detected by Ki67 immunostaining. 3H-thymidine incorporation was also initially depressed but increased over 72 h in NE-treated cells, which suggests that DNA synthesis constitutes an early repair process following NE exposure. We hypothesized that ErbB2 receptor tyrosine kinase, a regulator of cancer cell proliferation, was required for epithelial DNA synthesis following NE exposure. Immediately following NE treatment, by flow cytometry analysis, we found a decrease in ErbB2 surface expression. Protein levels of the full-length 185 kD ErbB2 receptor significantly decreased following NE treatment and smaller ErbB2-positive bands, ranging in size from 23 to 40 kD, appeared, which suggests that NE caused ErbB2 degradation. By real-time RT-PCR analysis, we found no change in ErbB2 mRNA expression following NE treatment, which suggests that changes in ErbB2 protein levels were regulated at the post-translational level. Following NE treatment, full-length 185 kD ErbB2 levels increased to pretreatment levels, correlating with the increase in thymidine incorporation during the same time period. Importantly, inhibition of ErbB2 activity with AG825 (5 microM) or Herceptin (3.1 microM), an ErbB2-neutralizing antibody, blocked thymidine incorporation only in NE-treated cells. These results suggest ErbB2 is a critical factor for epithelial recovery following NE exposure.

Neutrophil elastase inhibition of cell cycle progression in airway epithelial cells in vitro is mediated by p27kip1

“…p27 has also been reported to have an anti-apoptotic role; p27 is required for inhibition of proliferation and apoptosis following inflammatory injury of renal epithelial cells (31). We have shown that NE treatment of airway epithelial cells reduces proliferation (12), and now in this report, we present evidence that p27 is a key molecule regulating NE-induced cell cycle arrest. Collectively, the evidence presented here and in the literature suggests that p27 serves an important function in mediating cell cycle arrest and regulation of apoptosis in response to inflammatory injury.…”

“…Results were expressed as a ratio of p27 protein expression to ␤-actin protein expression (mean Ϯ SE) and then expressed as a percentage of the corresponding control. *NE significantly different from control, P Ͻ 0.05. proliferation (12). In this report, we present evidence that p27 is upregulated by NE and is critical for NE-induced inhibition of cell cycle progression.…”

“…Flow cytometry was used to assess DNA content as a surrogate measure for cell cycle phase. NHBE cells were synchronized in G 0/G1 by incubation for 48 h in epidermal growth factor (EGF)-free and bovine pituitary extract (BPE)-free media (12). Growth factors (25 ng/ml EGF and 0.13 mg/ml BPE) were added to initiate cell cycling, and at the same time cells were treated with NE (50 nM, 22-24 h) or control vehicle (50 M sodium acetate, pH 5, 100 M sodium chloride).…”

“…Growth factors (25 ng/ml EGF and 0.13 mg/ml BPE) were added to initiate cell cycling, and at the same time cells were treated with NE (50 nM, 22-24 h) or control vehicle (50 M sodium acetate, pH 5, 100 M sodium chloride). We have previously reported that this noncytotoxic concentration of NE decreases airway epithelial DNA synthesis and proliferation (12,13). At the end of the treatment period, cells were collected by trypsinization and resuspended in Hanks' balanced salt solution (Invitrogen).…”

“…CKIs bind to the cyclin complex, inhibit its activity, and arrest the cell cycle. We have previously reported that NE treatment results in a decrease in DNA synthesis (12). Because cyclin E-cdk2 complex activity is necessary for entry into S phase (54), and the Cip/Kip CKI family inhibits cyclin E-cdk2 complex activity, we have investigated the effect of NE on cell cycle arrest and have hypothesized that CKIs, specifically p27, mediate this arrest.…”