Articles in PresS. Am J Physiol Lung Cell Mol Physiol (February 17, 2006). doi:10.1152/ajplung.00547.2005
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Differentiation of Human Airway Epithelia Is Dependent on ErbB2 Paola D. Vermeer1, Lacey Panko1, Philip Karp1, John H. Lee2 and Joseph Zabner1#
1
Department of Internal Medicine,
University of Iowa Roy J. and Lucille A. Carver College of Medicine, 2
Department of Otolaryngology, Head and Neck Surgery,
University of Iowa Hospitals and Clinics, Iowa City, IA 52242
#Corresponding author: Joseph Zabner, MD University of Iowa Roy J. and Lucille A. Carver College of Medicine 440 EMRB Iowa City, IA 52242 Phone 319 335 7608 FAX
319 335 7623
Email
[email protected]
Running title: ErbB2 Epithelial Differentiation
Copyright © 2006 by the American Physiological Society.
2 ABSTRACT A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, an in vitro model of well-differentiated human airway epithelia was treated with trastuzumab or heregulin-α, an erbB ligand. In addition, co-culturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-α treatment increased epithelial height, decreased the number of metaplastic and non-ciliated columnar cells while it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factorα, heparin binding-epidermal-like growth factor, epiregulin, heregulin-α, and amphiregulin, all of which are erbB ligands. Co-cultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-α treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that might direct various pathways of epithelial differentiation.
Keywords: airway, trastuzumab, fibroblasts
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CASE REPORT A 56-year-old woman underwent a left mastectomy with axillary node dissection. Pathological examination revealed an infiltrating ductal carcinoma grade II-III with involvement of the nipple. Immunophenotyping revealed Her2/Neu positive cells suggesting it may respond to anti-ErbB2 directed antibody therapy. A survey for metastasis was negative. Following chemotherapy and radiation therapy, the patient elected to participate in a clinical research protocol studying the effects of weekly trastuzumab (Herceptin, Genentech, San Francisco, CA) on patients with breast cancer without metastasis at presentation (NSABP clinical trial B31). She was started on trastuzumab (4 mg/kg IV) followed by 2 mg/kg weekly for 51 weeks. Three months after initiating trastuzumab, the patient developed a non-productive cough that persisted despite several courses of antibiotics and got worse through the year of trastuzumab treatment. A flexible fiberoptic bronchoscopy revealed no endobronchial lesions; endobronchial biopsies from the carina and contralateral main bronchus revealed severe metaplasia of the bronchial and inflammation (Figure 1a and b). The patient’s cough improved 3 months after discontinuing trastuzumab and 6 months later her nasal mucosa appeared normal on physical examination. A repeat flexible fiberoptic bronchoscopy with endobronchial biopsies revealed normal airway epithelial morphology (Figure 1c and d).
4 INTRODUCTION This clinical presentation and course suggested a reversible change in airway epithelial differentiation that coincided with initiation and discontinuation of trastuzumab. We demonstrated a novel mechanism of receptor:ligand interaction in airway epithelia that regulates injury and repair (Vermeer et al. 2003). In this model, the epithelium secretes heregulin-α into the apical airway surface liquid (ASL), and restricts erbB receptors to the basolateral membrane. Compromising epithelial integrity results in immediate receptor: ligand interactions that enhance wound repair.
ErbB receptors belong to a family of tyrosine kinase receptors (Casalini et al. 2004), whose prototype is EGFR (erbB1). These receptors are implicated in a variety of cancers and are associated with poor prognoses, which has led to molecular targeting of erbB receptors in anticancer therapy (Fischer et al. 2003; Ranson and Sliwkowski 2002; Rowinsky 2004). Trastuzumab, a humanized anti-erbB2 antibody, accelerates receptor internalization and degradation (Carter et al. 1992; Fendly et al. 1990) and has become the standard of care in patients with increased erbB2 breast cancer (Baselga et al. 1998; Elkin et al. 2004; Gelmon et al. 2004; Ginestier et al. 2004; Pegram et al. 1998). Importantly, 4.7% of patients experience cardiac dysfunction (Suter et al. 2004), however, the benefits from trastuzumab outweigh this potential risk (Tripathy et al. 2004). Though the mechanism of cardiac toxicity remains unclear, it has been hypothesized that erbB2 may be required for cardiac myocyte regeneration following injury.
5 This case presentation suggests that blocking erbB2 alters airway epithelial differentiation. If trastuzumab played a causal role in de-differentiating the patient’s airway epithelium, heregulin-α and/or other erbB ligands may preserve differentiation. Thus, we hypothesized that a basolateral source of erbB ligands sustains a basal state of differentiation; disruption of signaling by trastuzumab de-differentiates the epithelium.
6 METHODS Primary human airway epithelial cell culture model Airway epithelial cells were isolated from trachea and bronchi of donor lungs and cultured at the air-liquid interface as previously described (Karp et al. 2002; Smith et al. 1996; Yamaya et al. 1992; Zabner et al. 1996). Experiments were performed on epithelia from 7 different donors. Co-cultures of primary human airway epithelia and normal human lung fibroblasts were grown on 6.5 mm clear Transwells (Corning Costar, Corning, NY). One day prior to seeding, the epithelial sides of Transwells were coated with human placental collagen type IV (Sigma, St. Louis, MO) as previously described (Choe et al. 2003; Karp et al. 2002; Mio et al. 1998). The next day, the collagen was aspirated and Transwells washed twice with phosphate buffered saline. On the day of seeding, a mixture of 80% Vitrogen (bovine dermal collagen; Cohesion Technologies, Palo Alto, CA), 10% DME/F12 and 10% 0.1N NaOH was titrated to a pH of 7.2. Passage 5 normal human lung fibroblasts (Cambrex Corporation, East Rutherford, NJ) were added to this working Vitrogen solution at a density of 2.0-3.8 X 105 fibroblasts per ml of gel. The underside of the Transwells was fitted with plastic insert ring attachment holder (Jim’s Instruments, Iowa City, IA) onto which aliquots of fibroblasts and Vitrogen mixture were seeded. Inserts were maintained upside-down in a 37°C incubator without CO2 for at least one hour to allow the Vitrogen matrix to gel. Once gelled, Transwells were flipped right-side-up and primary human airway epithelia seeded on top. Co-cultures were initially maintained in 5% fetal calf serum in DME/F12. One day post-seeding, co-culture medium was switched to 2% USG, 2% fetal calf serum (needed for fibroblasts) in DME/F12 and continuously maintained in this medium; 2% serum added to USG had no appreciable effect on epithelial differentiation.
7 Primary cultures were treated with: recombinant human heregulin-α (R&D Systems) or trastuzumab (Genentech) added to the media. Control cultures were not treated. Morphometry Hematoxylin and eosin stained epithelial sections were chosen at random and photographs of different regions randomly selected for analysis. PAS-stained sections for some epithelial cultures were also analyzed and the number of goblet cells counted in PAS vs hematoxylin and eosin stained sections correlated. However not all cultures were PAS stained. The photographs were enlarged uniformly, overlaid with transparent grids, and used for morphometric analysis (Weibel 1979). The volume densities of the filter and tissue were determined using point counting; the length of the filter was measured using an eyepiece measuring grid. Two independent observers blinded to experimental conditions made all morphometric measurements. Two donors were analyzed per condition and six Transwells per donor were treated; 4-6 sections per Transwell were evaluated. The height of the airway epithelia was normalized to the height of the filter to correct for possible variability in the sectioning of the epithelia (the filters are 20 µm thick). Airway epithelial cell number was normalized per length of the filter and area of the epithelia. We trained two independent observers to identify the following cell types: basal, ciliated, goblet, columnar non-ciliated. Dysplastic cells were identified as those cells that did not resemble any of the above mentioned cell types. The correlation coefficients between observers for height, area and cell density were 0.99, 0.99, and 0.98, respectively. The correlation coefficients for basal, ciliated, goblet, and non-ciliated columnar were 0.90, 0.87, 0.97, and 0.99, respectively.
8 Western blot Lysate was made by incubation in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3V04, 10 mM NaPPi, 100 mM NaF, PMSF, leupeptin, pepstatin, aprotonin) with 1% Triton X-100 at 4°C. Lysate was collected, homogenized, membranes pelleted and the soluble fraction incubated with antibody for erbB2 (DAKO corp). Immunoprecipiated protein was complexed with protein G Sepharose (PAS, Pierce), the complex pelleted and washed. Protein was eluted by incubation in 2X sample buffer (4% SDS, 100 mM DTT, 20% glycerol, 0.005% bromphenol blue, 0.065 M Tris, pH 6.8), boiled, separated by SDSPAGE and transferred to PVDF membrane (Immobilon-P, Millipore Corporation). PVDF membrane was blocked in 5% BSA, washed and incubated with anti-phosphotyrosine antibody (Upstate Biotech). Following washes, incubation in HRP-secondary antibody (1:10,000, Amersham Pharmacia) allowed for bound antibody detection with SuperSignal solution (Pierce) and exposure to film (X-OMAT AR, Kodak Scientific Imaging Film).
RT-PCR of erbB ligands Fibroblasts embedded in Vitrogen matrix were pulled away from Transwells and incubated in 1% collagenase (Sigma, St. Louis, MO) for 30 minutes at 37°C to digest the matrix. Fibroblasts were then harvested by centrifugation and washed with phosphate buffered saline. RNA isolation was performed using the RNAqueous-4PCR kit (Ambion, Austin, TX) as per manufacturer’s instructions. RNA was reverse transcribed with Thermoscript RT (Invitrogen, Carlsbad, CA). Controls included the omission of RT. The generated cDNA served as the template in PCR reactions using erbB ligand specific forward and reverse primers for betacellulin, TGF-α, HB-EGF, epiregulin, heregulin-α, and amphiregulin. The forward and
9 reverse primers used were as follows: Betacellulin: GAA TAT GTC CCT GGG TGT GG, CTA CAA GGC AGG ACA CC; TGF-α : TAG GCA TTT CAG GCC AAA TC, GCA TTT CCT CAC ATA AGG AGT TT; HB-EGF: CTC TCC CTG CCA AGT CTC AG, TGA ACC AGG TTT GGA AAT ACA; Epiregulin: CCA AGG ACG GAA AAT GCT TA, AAA GGG CAT AGT GCT TGC AT; Heregulin-α: ATA TCC ACC ACT GGG ACA AGC CAT, TAT CAC GGG TGG AGA CAT TTC CGA; Amphiregulin: TGG ATT GGA CCT CAA TGA CA, CGT TCA CCG AAA TAT TCT TGC. Following heat denaturation, DNA was amplified in an Eppendorf Mastercycler thermocycler (Westbury, NY) as follows: 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 60 seconds for 40 cycles, followed by a 72°C post-dwell for 2 minutes. Amplified DNA was separated by agarose gel electrophoresis and imaged (Polaroid, Cambridge, MA) under UV light.
10 RESULTS Trastuzumab treatment causes de-differentiation of human airway epithelia grown at the air-liquid interface Trastuzumab was added to an in vitro model of well-differentiated human airway epithelia (Karp et al. 2002). Hematoxylin and eosin stained sections (Figure 2a and b) demonstrate that trastuzumab treatment decreased the number of ciliated cells by 49% and increased the number of metaplastic cells by 640% (Figure 2c). These data suggest that erbB2 is important in airway epithelial differentiation and that receptor activation is required.
Heregulin-α stimulates human airway epithelia differentiation Heregulin-α and erbB2 are important in epithelial differentiation and wound repair (Vermeer et al. 2003). The current data further suggests that when erbB2 is blocked by trastuzumab, the epithelium de-differentiates. Whether trastuzumab treatment alters erbB2 receptor mass or leads to loss of receptor signaling without changing receptor mass is not clear from these studies. These data suggested that an erbB ligand, heregulin-α, might have the opposite effect of trastuzumab that is to stimulate epithelial differentiation. To quantitatively examine the effects of heregulin-α on human airway epithelia, we added increasing concentrations of recombinant human heregulin-α to the basolateral medium. Figure 3a shows examples of hematoxylin and eosin stained sections. In contrast to trastuzumab, heregulin-α treatment increased epithelial height, a characteristic of well-differentiated airway epithelia (Shen et al. 1997) and decreased the number of cells per area (Figure 3b and c). Interestingly, heregulin-α treatment appeared to make the cilia longer, however this effect was not quantified.
11 Heregulin-α stimulates differentiation of airway epithelial cell types We asked whether heregulin-α treatment had an effect on the cell types present in the epithelia. We found that the percentage of ciliated and basal cells was not affected (Figure 3d and g). However, there was a significant increase in the number of goblet cells and a decrease in the number of non-ciliated columnar cells (Figure 3e and f). Interestingly, the number of metaplastic cells decreased at the highest dose of heregulin-α tested (Figure 3h).
Lung fibroblasts express erbB ligands and stimulate human airway epithelial differentiation Since the airway epithelium de-differentiates when exposed to trastuzumab, erbB receptor stimulation may be required to maintain a basal level of differentiation in the epithelium. In vitro, erbB ligands may leak from the apical side of the epithelia or some ligands may be provided by the Ultroser G serum substitute in the medium. However, in vivo, subepithelial lung fibroblasts are a likely source of ligand. Their localization basal to the epithelia would be optimal for direct and constitutive epithelial erbB receptor stimulation. Therefore, pulmonary fibroblasts were assayed for expression of erbB ligands by RT-PCR. Human lung fibroblasts express TGF-α, HB-EGF, epiregulin, heregulin-α, and amphiregulin, all erbB ligands (Marmor et al. 2004) (Figure 4a). Betacellulin was the only ligand tested that was not expressed. We then asked if pulmonary fibroblasts could alter the differentiation of human airway epithelia in a manner similar to that of basolateral recombinant heregulin-α treatment. Figures 4b and c show qualitative changes in epithelial morphology in co-cultures (Figure 4c) compared to untreated primary cultures of epithelia grown in isolation (Figure 4b). These changes resemble those of adding recombinant heregulin-α to epithelial cultures in isolation
12 (Figure 3a). Epithelia grown in isolation reach a height of 33.7 with a standard deviation of ±3.7 µm. However, when grown in the presence of pulmonary fibroblasts, epithelial height increased to 43.2 ±8.8 µm, comparable to the epithelial height achieved following basolateral treatment with 3 nM heregulin-α (45.1 ± 4.9 µm). Figure 4d demonstrates Western blot analysis of erbB2 receptor phosphorylation. In primary epithelial cultures erbB2 is not phosphorylated. As expected however, basolateral treatment of primary epithelial cultures with recombinant heregulin-α as well as epithelia grown in co-culture with fibroblasts both result in erbB2 receptor phosphorylation. In addition when epithelial-fibroblast co-cultures were treated with trastuzumab, erbB receptor phosphorylation was inhibited (data not shown).
13 DISCUSSION In the case study, we describe a patient with HER2/Neu3 positive infiltrating ductal carcinoma grade II-III on trastuzumab therapy. Three months after initiating trastuzumab therapy, the patient developed a non-productive cough that persisted despite several courses of antibiotics and worsened throughout the year of trastuzumab treatment. Although the presence of cough may be explained by a number of factors (a combination of chemotherapy, radiation therapy and environmental or other causes), that it was concurrent with trastuzumab therapy warranted further investigation. ErbB receptors belong to a family of four related tyrosine kinase receptors the prototype of which is EGFR also known as erbB1. The extracellular domain of these receptors can bind ligands, resulting in a conformational change allowing for receptor homo- or hetero-dimer formation. Differential ligand affinity for monomers and the resulting receptor dimer combinations allow for a complex and promiscuous signaling capacity. Two of these monomers are uniquely handicapped: erbB2 is an orphan receptor (ligand for erbB2 has not yet been identified) and erbB3 has a dead kinase. However, these two monomers can conveniently come together and complement each other’s limitations. ErbB3 binds heregulin resulting in dimerization with erbB2 which, once engaged, autophosphorylates and transphosphorylates erbB3. ErbB2 can also dimerize with erbB1 and erbB4, resulting in complex, coordinated and specific signaling cascades in response to all known erbB ligands (EGF, amphiregulin, TGF-α, betacellulin, heparin binding EGF-like growth factor, epiregulin). In an in vitro model of human airway epithelia, incubation with trastuzumab led to epithelial de-differentiation. The number of flat, metaplastic cells increased, a finding that closely resembled the patient’s airway biopsies. Though not tested in this study, trastuzumab treatment may also result in cell death, a possibility that cannot be excluded. Incubation with
14 heregulin-α increased epithelial cell height and decreased the number of metaplastic cells. Interestingly, heregulin-α treatment also increased the number of goblet cells while trastuzumab did not alter the goblet cell population. These data suggest that increasing concentrations of heregulin-α induce trans-differentiation of non-ciliated columnar cells into goblet cells. Transdifferentiation is well documented (Bishop 2004). In addition, this paradox suggests that while heregulin-α stimulates epithelial differentiation increasing goblet cell numbers, other ligands acting via erbB2-containing heterodimers must regulate other differentiation lineages. Alternatively, altering erbB2 activation may result in Clara cell and goblet cell differentiation rather than global epithelial de-differentiation; the present study cannot distinguish between the two possibilities. Interestingly, while our data suggest that erbB2 activation stimulates both cellular proliferation and differentiation, others demonstrated that activation of erbB1 leads to goblet cell hyperplasia, without altering the total number of epithelial cells (Takeyama et al. 1999). These data suggest a role for erbB1 in differentiation but not proliferation. Similarly, Shimizu et al (Shimizu et al. 1996) found that inhaling endotoxin led to an increase in the goblet cell population without changing the total cell number. Taken together, these data suggest that multiple ligands activate a variety of erbB receptors and that this complexity likely maintains both epithelial cell numbers and differentiation status, factors critical for normal airway barrier function. This is also consistent with our data showing that pulmonary fibroblasts express all but one of the erbB ligands and that human airway epithelia express all four erbB receptors (Aida et al. 1994; al Moustafa et al. 1999; Polosa et al. 1999; Vermeer et al. 2003). Importantly, Western blot analysis (figure 4d) demonstrates that epithelial-fibroblast co-cultures result in erbB2 receptor activation. Such interactions between epithelia and sub-epithelial cells are not new. In fact, epithelial/mesenchymal communications have been widely studied, and their roles in
15 development and carcinogenesis recognized for decades (Kalluri and Neilson 2003; Kang and Massague 2004; Parmar and Cunha 2004).
Despite being cultured in the absence of fibroblast, the in vitro model of human airway epithelia appears morphologically differentiated. One explanation for this is that apical ligand may constitutively diffuse basolaterally at low levels. Moreover, normal turnover of cells may allow ASL ligands to diffuse into the basolateral compartment and stimulate cellular differentiation in a precise temporal and localized fashion. Finally, an additional artificial source of ligand may be the serum substitute (Ultroser G) present in the medium. Ultroser G is a largely undefined serum substitute that Widdicombe and others have shown to promote airway epithelial differentiation (Sachs et al. 2003; Widdicombe et al. 2003).
The data presented here suggest a central but complex role for erbB receptors and ligands for epithelial differentiation. They further suggest that mesenchymal-epithelial communication modulates this process. Our initial studies focused on only one ligand, heregulin-α, and serve as proof of principle from which to model future studies aimed at unraveling this complexity.
16 ACKNOWLEDGMENTS We thank Jan Launspach, Pary Weber, Tamara Nesselhauf, Daniel Vermeer, Thomas O. Moninger, Theresa Mayhew, and Jamie Kesselring for excellent assistance. We thank Michael Welsh and Jeanne Snyder for insightful discussions. We appreciate the support of the University of Iowa Central Microscopy Research Facility, the In Vitro Cell Models Core (supported by the National Heart, Lung and Blood Institute, the Cystic Fibrosis Foundation, and the National Institutes of Diabetes and Digestive and Kidney Diseases), and the Iowa Statewide Organ Procurement.
This work was supported by the National Institutes of Health, HL61234 and HL075276-01A
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22 FIGURE LEGENDS Figure 1 Hematoxylin and eosin stained sections of bronchial biopsies taken while on trastuzumab (a,b) and 6 months following termination of trastuzumab (c,d). A metaplastic epithelium lacking cilia is evident in biopsies obtained while on trastuzumab. Ciliated cells were restored following termination of trastuzumab. Scale bars are 20 µm.
Figure 2 Primary cultures of human airway epithelia were treated basolaterally with 0 or 1 mg/ml trastuzumab for 2 weeks. Cultures were fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Loss of differentiation is evident in trastuzumab treated cultures (b) as compared to untreated controls (a). When analyzed by morphometry, trastuzumab treatment resulted in a decrease in the percentage of ciliated and an increase in metaplastic cells (c, p
