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

Bernard M. Fischer^*, Jacob G. Cuellar^*, Angela S. Byrd^*, Annette B. Rice, James C. Bonner^,1, Linda D. Martin and Judith A. Voynow^*,2

^* Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA;
Laboratory of Respiratory Biology, National Institute for Environmental Health Sciences, Research Triangle Park, North Carolina, USA; and
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA

²Correspondence: Division of Pediatric Pulmonology, Box 2994, Duke University Medical Center, Durham, NC 27710, USA. E-mail: voyno001{at}mc.duke.edu

SPECIFIC AIMS

The aim of this study was to determine the epithelial response to neutrophil elastase (NE), a major airway inflammatory mediator in cystic fibrosis and chronic bronchitis. We observed that immediately after NE exposure, normal human bronchial epithelial (NHBE) cells significantly decrease DNA synthesis and proliferation. We investigated the effect of NE on the receptor tyrosine kinase ErbB2, a potential regulator of epithelial DNA synthesis.

PRINCIPAL FINDINGS

1. NE significantly reduces NHBE DNA synthesis and proliferation
NHBE cells were treated with NE (50 nM, 24 h) or control vehicle, then followed for 72 h (chase period) to determine the effect of NE on immunostaining for Ki67, a nuclear proliferation antigen, and on ³H-thymidine uptake, a measure of DNA synthesis. NE profoundly reduced Ki67 immunostaining immediately after NE exposure and during the 72 h after NE treatment. Thymidine uptake was also significantly reduced after NE exposure, but increased during the 72 h chase period, suggesting that DNA synthesis is an early reparative process.

2. NE activates the digestion of ErbB2 but not epidermal growth factor receptor (EGFR)
ErbB2 has been reported to regulate epithelial repair after injury and mediate cancer cell proliferation. Therefore, our initial hypothesis was that expression and phosphorylation of ErbB2 would be required for epithelial repair in response to NE. Cell lysates from NHBE cells, treated with NE (50 nM, 24 h, treatment period) or control vehicle, then followed for 48 h (chase period), were immunoprecipitated with anti-ErbB2 antibody and evaluated by Western blot analysis for total phosphotyrosine and total ErbB2. Surprisingly, NE exposure significantly reduced full-length (185 kDa) ErbB2 content at the end of the treatment period (long arrow, Fig. 1 B, IB: ErbB2 lane 2, and Fig. 1C ). Smaller bands (bracket, molecular masses 23–40 kDa) were detected by the anti-ErbB2 antibody, suggesting that the loss of full-length ErbB2 was due to a proteolytic event. In contrast, control cells maintained the same level of full-length ErbB2 (Fig. 1A , IB: ErbB2 lane 2 and Fig. 1C ). These results suggest that reduction of total ErbB2 protein levels by NE is a mechanism to down-regulate ErbB2 activity and suppress DNA synthesis. As a control, we also evaluated EGFR protein levels in these cell lysates by Western blot analysis, which revealed that EGFR protein content was similar under control or NE treatment conditions. Thus, the effect of NE on reducing full-length ErbB2 levels appears to be specific.

View larger version (45K): [in this window] [in a new window]   Figure 1. NE activates the digestion of full-length ErbB2 in NHBE cells. Confluent NHBE cells were washed and the medium replaced with serum-free defined medium without EGF and BPE for 16–24 h before the addition of neutrophil elastase. Cells were treated with NE, 50 nM for 24 h, then followed over time (chase, 24–48 h; B). As a control for decreased cell density after NE treatment, subconfluent control cells (control) were handled in a manner similar to NE-treated cells, treated with control vehicle for 24 h, and followed over time (chase, 24–48 h; A). Immunoprecipitation of cell lysate (100 µg) was performed with an anti-ErbB2 antibody. Immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody (IB: P-Tyr; upper panel, arrowhead) or an anti-ErbB2 antibody (IB: ErbB2, lower panel, long arrow, full-length ErbB2; bracket, cleaved ErbB2 bands). Representative blots from 4 separate experiments are shown. Lane 1: pretreatment; lane 2: end of treatment period; lane 3: 24 h chase; lane 4: 48 h chase. Molecular mass markers in kilodaltons are on the right of each lower blot. The graph (C) summarizes densitometry results for total full-length Erb2 (long arrow) from control (white bars) or NE-treated cultures (black bars) of 4 separate experiments; mean±SE). Densitometry results are expressed as a percentage of pretreatment ErbB2 expression. *Significantly different from total ErbB2 expression at the end of the treatment period (chase time zero, P<0.05). Significantly different from pretreatment total ErbB2 expression (P<0.05).

3. Following NE treatment of NHBE cells, recovery of full-length ErbB2 expression correlates with increased DNA synthesis
To determine the course of ErbB2 protein expression during epithelial recovery after NE treatment, ErbB2 was evaluated by immunoprecipitation and Western blot analysis during the 48 h chase period in NE-treated and control-treated NHBE cells. Levels of full-length 185 kDa ErbB2 increased over the 48 h after NE treatment reaching pretreatment levels (long arrow, Fig. 1B , IB: ErbB2 lanes 3 and 4, and Fig.1C ). Levels of ErbB2-phosphotyrosine correlated directly with the total levels of ErbB2 under all culture conditions (Fig. 1A, B , IB: P-Tyr; upper panel, arrowhead), suggesting that regulation of ErbB2 activity under these culture conditions was primarily related to expression of full-length protein.

The increase in ErbB2 protein levels also correlated directly with the increase in thymidine uptake during the chase period supporting the hypothesis that after NE exposure, ErbB2 expression was required for DNA synthesis.

4. Following NE treatment of NHBE cells, the ErbB2-specific inhibitor, AG825, or the anti-ErbB2 neutralizing antibody, Herceptin, block the increase in DNA synthesis
To determine whether ErbB2 function is critical for epithelial repair after NE exposure, we treated cells with the ErbB2 inhibitor AG825 (5 µM) before, during, and after NE treatment. Although AG825 had no effect on thymidine uptake in control cells, it significantly reduced thymidine incorporation in NE-treated cells during the chase period (Fig. 2 A). To confirm these observations, NHBE were also treated with Herceptin (3.1 µM), a neutralizing monoclonal anti-ErbB2 antibody, before, during, and after NE exposure. Herceptin also inhibited thymidine uptake by NE-treated cells, but had no effect on control cells (Fig. 2B ). Together, these results suggest that after NE exposure, the recovery of ErbB2 expression and activity are critical for epithelial repair since inhibition of receptor activity at this vulnerable stage inhibits DNA synthesis (Fig. 3 ).

View larger version (14K): [in this window] [in a new window]   Figure 2. AG825, an ErbB2 receptor tyrosine kinase inhibitor, or Herceptin, an anti-ErbB2 monoclonal antibody, reduce DNA synthesis in NHBE cells after NE exposure. Confluent NHBE cells were washed and the medium replaced with serum-free defined medium without EGF and BPE for 16–24 h before the addition of neutrophil elastase. Cells were pre- and coincubated with the ErbB2 receptor tyrosine kinase inhibitor, AG825 (5 µM; A), or the anti-ErbB2 monoclonal antibody Herceptin (3.1 µM; B), treated with NE (50 nM, 22 h), rinsed, then followed over time (chase; 24–48 h) in the presence or absence of AG825 or Herceptin, for 3H-thymidine incorporation, a marker of DNA synthesis and proliferation. As a control for decreased cell density after NE treatment, subconfluent cells were handled in a manner similar to NE-treated cells, pre- and coincubated with the ErbB2 inhibitors, and followed over time for 3H-thymidine incorporation in the presence or absence of AG825 or Herceptin. Each graph summarizes n = 9–12 (mean±SE). Results are expressed as a percent of the corresponding first chase point. *Significantly different from the corresponding NE chase point (P<0.01). AG825 and Herceptin had no significant effect on control cells.

View larger version (20K): [in this window] [in a new window]   Figure 3. Postulated mechanism of NE regulation of epithelial DNA synthesis mediated by ErbB2. NE causes epithelial injury in part by activating the digestion of ErbB2 (1) resulting in decreased glycoprotein expression of ErbB2 and reduced DNA synthesis (Injury). 48 h after NE treatment, ErbB2 full-length protein levels recover to pretreatment levels and DNA synthesis significantly increases (Repair). However, after NE treatment during the early recovery of ErbB2 full-length protein levels, inhibition of ErbB2 activity with AG825 or Herceptin (2) results in reduced DNA synthesis (Injury). Thus, both ErbB2 expression and activity are required for DNA synthesis after NE exposure to airway epithelial cells.

CONCLUSIONS AND SIGNIFICANCE

NE is an abundant unopposed protease found in the airways of patients with cystic fibrosis and chronic bronchitis. Although this observation was made several decades ago, our understanding of the impact of proteolytically active NE on airway epithelial cells is limited. In this report, we demonstrate that NE has a profound, previously unrecognized influence on epithelial cell homeostasis by significantly reducing DNA synthesis and epithelial proliferation. We have made two major observations concerning the mechanism of NE-reduced epithelial proliferation: 1) NE directly or indirectly decreases full-length ErbB2 content in human airway epithelial cells; and 2) ErbB2 expression and activity are required for DNA synthesis after NE exposure. Our results highlight the importance of measuring total receptor protein expression, not just receptor phosphorylation, in considering mechanisms of receptor regulation. These findings shift the current paradigm of epithelial repair processes to recognize that inflammatory proteases affect growth factor receptor expression, not just growth factor ligand release. Finally, these results confirm the importance of ErbB2 expression and activation in epithelial repair processes.

Several key questions are raised by this report. 1) What is the mechanism by which NE reduces expression of full-length ErbB2? It is possible that NE activates a similar post-translational regulation of ErbB2 as has been reported after exposure to geldanamycin in tumor cells, mediated by separation from its chaperone, and caspase-dependent degradation. 2) How do changes in ErbB2 expression signal downstream to reduce DNA synthesis? ErbB2 activates several downstream signaling pathways depending on its receptor tyrosine kinase heterodimerization partner and on the activating ligands. Reduced ErbB2 expression has been associated with increased apoptosis in tumor cells, another potential outcome that would correlate with decreased DNA synthesis and proliferation. 3) How does recovery of ErbB2 influence epithelial repair mechanisms in the long term? Are these cells destined for proliferation and transdifferentiation to secretory cells as seen in chronic inflammatory airway diseases? Experiments are ongoing to investigate the regulatory mechanisms and long-term consequences of ErbB2 regulation and activation in airway epithelial cells both in vitro and in vivo after NE exposure.

FOOTNOTES

¹ Current address: CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, NC 27709-2137, USA.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2675fje;

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This Article Full Text (PDF) All Versions of this Article: 19/10/1374 04-2675fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fischer, B. M. Articles by Voynow, J. A. Search for Related Content PubMed PubMed Citation Articles by Fischer, B. M. Articles by Voynow, J. A.