Neurotrophic Tyrosine Kinase Receptor B ... - ATS Journals

Neurotrophic Tyrosine Kinase Receptor B/Neurotrophin 4 Signaling Axis Is Perturbed in Clinical and Experimental Pulmonary Fibrosis ¨ nther1, Werner Seeger1, Sibel Avcuoglu1*, Ma1gorzata Wygrecka2*, Leigh M. Marsh3, Andreas Gu 1 4 1 _ Norbert Weissmann , Ludger Fink , Rory E. Morty , and Grazyna Kwapiszewska1 1

Department of Internal Medicine, and 2Department of Biochemistry, University of Giessen Lung Center, Justus Liebig University, Giessen, Germany; 3Department of Clinical Chemistry and Molecular Diagnostics, University Hospital Giessen and Marburg, Marburg, Germany; and 4¨ Uberregionale Gemeinschaftspraxis Institute for Pathology and Cytology, Wetzlar, Germany

The neurotrophins (NTs) are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in diseased lungs. In this study, the increased expression of NT4/5 and of its cognate receptor, the neurotrophic tyrosine kinase receptor Type 2 (TrkB), was observed in human lungs explanted from patients with idiopathic pulmonary fibrosis (IPF), and in lungs from mice with bleomycin-induced pulmonary fibrosis. The expression of NT4/5 and TrkB localized to hyperplastic alveolar Type II cells (ATII) and fibroblastic foci in affected lungs. Increased concentrations of NT4/5 and TrkB were evident in ATII isolated from the lungs of bleomycin-treated mice. Primary ATII were shown to secrete NT4/ 5 into the cell culture medium. The profibrotic cytokine transforming growth factor–b1, stimulated TrkB, but not NT4/5 gene expression, suggesting that perturbed profibrotic growth factor signaling in affected lungs may drive the expression of TrkB. NT4/5 enhanced the proliferation of ATII through a TrkB/extracellular–regulated kinase/protein kinase B pathway, and could also drive the proliferation of primary human and murine lung fibroblasts, through TrkB-dependent and protein kinase B–dependent pathways. Taken together, these data suggest that a dysregulated TrkB/NT4/5 axis may contribute to several of the pathological lesions associated with pulmonary fibrosis, including ATII hyperplasia and the proliferation of fibroblasts, and we would add IPF to the list of disorders, such as pain and cancer, for which therapeutic targeting of the TrkB/neurotrophin axis has been proposed for further investigation. Keywords: proliferation; neurotrophin; alveolar Type II; fibroblast; idiopathic pulmonary fibrosis

Brain-derived neurotrophic factor (BDNF) and neurotrophins (NTs) 4 and 5 are members of the neurotrophin family of growth factors, initially described to be involved in neuronal cell differentiation, morphology, and function (1, 2). Neurotrophin signaling is effected by the binding of NT ligands to their cognate high-affinity receptors, the so-called neurotrophic tyrosine kinase (Trk) receptors, which constitute a family of single-pass transmembrane receptors, currently numbering three members: TrkA, TrkB, and TrkC (3, 4). Ligand–receptor interactions are (Received in original form May 11, 2010 and in final form February 15, 2011) * These authors contributed equally to this work. This work was supported by research grants from the University Medical Center Giessen and Marburg (G.K. and M.W.), Clinical Research Group 118 ‘‘Pulmonary Fibrosis’’ (A.G. and W.S.), and Excellence Cluster 147 ‘‘Cardio-Pulmonary System’’ (G.K., M.W., A.G., W.S., N.W., and R.E.M.). Correspondence and requests for reprints should be addressed to Graz_ yna Kwapiszewska, Ph.D., Department of Internal Medicine, University of Giessen Lung Center, Justus Liebig University, Klinikstrasse 36, D-35392 Giessen, Germany. E-mail: [email protected]

specific, with TrkA mediating nerve growth factor (NGF) signaling, TrkB mediating BDNF and NT4 signaling, and TrkC mediating NT3 signaling. A low-affinity receptor, p75NTR, also exists, which binds all neurotrophins with similar affinity (3, 4). Neurotrophins and their cognate receptors are expressed and are functionally active in several non-neuronal tissues, including the non-neuronal tissues of the lung. All NT ligands were detected in the peripheral lung tissues of mice (5, 6). Similarly, both NT ligands and their cognate receptors were detected in adult human lung tissues. High concentrations of TrkB and BDNF and low concentrations of p75NTR were detected at the mRNA and protein levels in human whole-lung extracts (7). Histological studies indicate that NT ligands are produced primarily by lung epithelial cells and alveolar macrophages (and other inflammatory leukocytes), whereas the NT receptors are expressed to varying degrees in all lung cell types (5–9). This pattern of expression suggests that autocrine and paracrine signaling pathways are operational in the lung for the three NT/NT receptor axes. A growing body of evidence implicates NT signaling pathways in lung disease, where the expression of NTs and of their cognate receptors is altered. Concentrations of neurotrophin are elevated in the plasma and bronchoalveolar lavage fluid of patients with rhinitis and asthma (10), and NTs were directly implicated in eosinophilic recruitment in allergic disorders (11). Concentrations of NT3 are suppressed in chronic obstructive pulmonary disease (12), and the expression of NTs and NT receptors is dysregulated in tumors of the lower respiratory tract (9). Evidence also exists that NTs play a role in the pathogenesis of pulmonary arterial hypertension (13). A preliminary study in patients with idiopathic pulmonary fibrosis (IPF) indicated that TrkB and BDNF staining was evident in fibroblast foci in the lungs of affected patients (14). IPF is a disease characterized by the proliferation, activation, and transdifferentiation of fibroblast, as well as by alveolar Type II cell hyperplasia. Because the TrkB/NT axis is a potent regulator of cell proliferation and fate (1, 2), dysregulation of the TrkB/NT axis may underlie, at least in part, some of the pathogenic processes at play in IPF, including profibrotic signaling and cell hyperplasia. To explore this idea, we investigated the expression and activity of the TrkB/NT axis in human IPF and in the bleomycin murine model of pulmonary fibrosis, paying particular attention to TrkB/NT signaling in the epithelial cell, which is emerging as an important contributor to profibrotic processes in the lung.

MATERIALS AND METHODS

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Reagents

Am J Respir Cell Mol Biol Vol 45. pp 768–780, 2011 Originally Published in Press as DOI: 10.1165/rcmb.2010-0195OC on February 17, 2011 Internet address: www.atsjournals.org

Dulbecco’s modified Eagle’s medium (DMEM) and FCS were from Invitrogen (Carlsbad, CA). Murine and human recombinant neurotrophin 4/5 (NT4/5; 10 ng/ml), transforming growth factor (TGF)–b1

Avcuoglu, Wygrecka, Marsh, et al.: TrkB/NT4 Axis Is Dysregulated in Lung Fibrosis

(5 ng/ml), insulin-like growth factor (IGF)–I (50 ng/ml), and or plateletderived growth factor (PDGF)–BB (10 ng/ml) were obtained from R&D Systems (Wiesbaden, Germany). K252a (100 nM), an inhibitor of TrkA/ TrkB phosphorylation by NGF and NTs, was obtained from Alamone Labs (Jerusalem, Israel). U0126 (20 mM), an inhibitor of Mitogenactivated protein kinase kinase 1 (MEK1) phosphorylation, was acquired from Calbiochem (San Diego, CA). Wortmannin (10 mM), an inhibitor of PI3K/Akt phosphorylation, was obtained from Sigma-Aldrich (Taufkirchen, Germany). The antibodies used in this study included TrkB (794) (catalogue number sc-12), BDNF (N-20) (catalogue number sc-546), NT-4 (N-20) (catalogue number sc-545; all from Santa Cruz Biotechnology, Santa Cruz, CA), b-actin (Sigma-Aldrich, St. Louis, MO), proliferating cell nuclear antigen (PCNA; Abcam, Cambridge, UK), phospho-p38 Mitogen-activated protein kinase (MAPK) (Thr180/ Tyr182), total-p38 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), total-p44/42 MAPK, phospho-Akt (Tyr326), total-Akt (all from Cell Signaling Technology, Beverly, MA), prosurfactant protein C (proSP-C) (Chemicon, Hampshire, UK), S100A4 (Abcam), vimentin (Santa Cruz), von Willebrand factor (vWF), widespread cytokeratin (WSCK) (both from Dako, Hamburg, Germany), E-cadherin (BD Transduction, Heidelberg, Germany), rabbit IgG (Upstate, Chemicon), and goat IgG (R&D Systems).

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three independent experiments. The DDCt values for each target gene were calculated as a difference between DCttreatment and DCtcontrol, where DCt 5 Ctreference 2 Cttarget. For reaction coditions and primer sequences, please refer to the online supplement.

NT4/5 Enzyme-Linked Immunosorbent Assay A DuoSet ELISA kit was used to measure mouse NT4 in cell culture supernatants, according to the manufacturer’s protocol (R&D Systems).

Proliferation Assay Cell proliferation was assessed by the incorporation of [3H]thymidine. For more details, please refere to the online supplement.

Statistical Analysis Values are presented as mean 6 SEM. Groups were compared with a two-tailed t test. Paired samples were analyzed using a paired Student t test. All experiments were designed with matched control conditions, to enable statistical comparisons. For multiple analyses, ANOVA with Dunnett post hoc tests was applied to compare each time-point to the control. P < 0.05 was considered significant.

Human Material Lung tissue from nine patients with IPF (mean age 6 SD, 53.6 6 4.1 years; seven were male, and two were female) who underwent lung transplantation was kindly provided by Professor Walter Klepetko (Department of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria). The diagnosis of IPF was based on both clinical criteria and proof of an Usual Interstitial Pneumonia pattern in histopathological specimens from the explanted lungs. Tissue isolated from six donor lungs (mean age, 43.8 6 6.1 years; four were male, and two were female) served as control samples (n 5 6). The processing of tissue from lung donors and explanted lungs from patients with IPF was described previously (15). The study protocol was approved by the Ethics Committee of the University of Giessen School of Medicine (AZ 31/93), and informed consent was obtained from each subject for the study protocol.

Animal Studies Bleomycin (Almirall Prodesfarma, Barcelona, Spain) was administered as an aerosol through a microsprayer device, as recently described (16, 17). Age-matched and sex-matched control mice received saline.

Cell Isolation and Cell Culture Primary murine alveolar epithelial type II cells (ATII) were isolated from bleomycin-treated or saline-treated C57BL/6 mice as originally described by Corti and colleagues (18) and Marsh and colleagues (19), with modifications. Cells were then cultured without any additional matrix for up to 3 days. Murine and human fibroblasts were isolated as described previously (20). Briefly, fibroblasts were grown out and cultured in DMEM supplemented with 10% FCS and used during passage 3. For more details, please refer to the online supplement.

Immunocytochemistry and Immunohistochemistry Immunocytochemistry and immunohistochemistry were performed as described in the online supplement.

Immunoblotting Immunoblotting was performed as outlined in the online supplement.

RNA Isolation and cDNA Synthesis Cell and tissue RNA (whole murine lungs, or samples from donors and IPF lungs) was isolated using an RNeasy Miniprep Kit (QIAgen, Hilden, Germany), according to the manufacturer’s protocol. For cDNA synthesis, please refer to the online supplement and (21).

Relative mRNA Quantification by RT-PCR Real-time PCR was performed in an ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), using SYBR–Green I as fluorescent probe. Each gene was measured in duplicate in at least

RESULTS TrkB and NT4/5 Localize to Pathogenic Lesions in Fibrotic Human Lungs

The localization of the tyrosine kinase receptor TrkB and its ligands NT4/5 and BDNF was examined in lung sections from donors and patients with IPF. In donor lungs, the expression of TrkB was generally restricted to the vascular compartment, with weak immunostaining visible in the alveolar septa (Figure 1A). In fibrotic lungs, the expression of TrkB was also detected in fibrotic foci (Figure 1B). The expression of NT4/5 in healthy lungs was restricted to the bronchial epithelium (Figure 1A), whereas in fibrotic lungs, the expression of NT4/5 was also detected in hyperplasic alveolar epithelial cells (Figure 1B). Staining for BDNF was localized to the vasculature in healthy lungs (Figure 1A), and was weakly visible in fibrotic foci and epithelial cells in fibrotic lungs, as well as in mononuclear cells (Figure 1B). Because these proteins localized to areas of the lung that are remodeled during the development of IPF, changes in the levels of mRNA expression were assessed in healthy and fibrotic lungs. Using real-time RT-PCR, a significantly elevated expression of TrkB and NT4/5 mRNA was evident in samples from fibrotic lungs compared with donor lungs (Figure 1C). In contrast, levels of BDNF mRNA were unchanged (Figure 1C). Together, these data demonstrate the increased expression and altered localization of TrkB and NT4/5 in lungs from patients affected by IPF. Expression of NT4/5 and TrkB Is Elevated in Experimental Pulmonary Fibrosis

The altered expression of TrkB and NT4/5 in pathological lesions of human fibrotic lungs suggested that the TrkB/NT4/5 signaling axis may contribute to processes associated with the development of IPF. Therefore, the localization and expression of TrkB, NT4/5, and BDNF were also assessed in a murine model of bleomycin-induced pulmonary fibrosis. In control salinetreated mice, immunoreactivity for TrkB, NT4/5, and BDNF was observed in the conducting airway epithelium (Figure 2A). After the challenge with bleomycin, strong TrkB, NT4/5, and BDNF immunoreactivity was visible in the remodeled areas of affected lungs (Figure 2B). Double immunofluorescence staining of lung sections from bleomycin-treated mice demonstrated the localization of TrkB and NT4/5 in ATII (Figure 2C). An analysis of expression levels after different time-points of

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011

Figure 1. Expression and localization of neurotrophic tyrosine kinase B (TrkB) and its ligands neurotrophins (NTs) 4 and 5 and brain-derived neurotrophic factor (BDNF) in lung tissues from donors and patients with idiopathic pulmonary fibrosis (IPF). (A and B) Immunohistochemical staining (alkaline phosphatase, red ) for TrkB, NT4/5, and BDNF was performed on lung-tissue sections from (A) donors or (B) patients with IPF. Inset shows negative control staining with isotype-matched IgG. Arrows indicate alveolar Type II cells. (C ) Levels of mRNA expression in donor or IPF lung tissue were assessed by real-time RT-PCR for TrkB, NT4/5, and BDNF. Values represent the mean DDCt 6 SEM (n 5 6 for control tissue, n 5 9 for IPF). **P , 0.01. ***P , 0.001. ns, not significant.

bleomycin application revealed an increase in concntrations of TrkB (Figure 2D) and NT4/5 mRNA (Figure 2E), but no change in levels of BDNF mRNA (Figure 2F). Supporting these observations, protein expression levels for TrkB and NT4/5 were also elevated after treatment with bleomycin (Figures 2G and 2H), whereas BDNF protein expression levels were unchanged (Figures 2G and 2H). Expression of NT4/5 and TrkB Is Upregulated in ATII from Bleomycin-Treated Mice

Because of the strong increase in expression levels of TrkB and NT4/5, and the localization of TrkB and NT4/5 to the fibrotic regions of affected murine and human lungs, ATII were isolated from both saline-treated control mice and bleomycin-treated mice. The purity of ATII was assessed by staining for the epithelial markers WSCK and E-cadherin and the fibroblast marker vimentin (Figure 3A). The ATII were then assessed for the expression of TrkB, NT4/5, and BDNF by immunocytochemistry, real-time RT-PCR, and immunoblot analysis. The expression of TrkB and NT4/5 appeared to be more pronounced in ATII isolated from bleomycin-treated mice (Figure 3B, right), compared with ATII isolated from saline-treated

control mice (Figure 3B, left), as assessed by immunocytochemical staining, where the levels of BDNF expression appeared unchanged. Supporting these data, concentrations of mRNA for TrkB and NT4/5 (Figure 3C) were elevated in ATII from bleomycin-treated mice, compared with ATII isolated from saline-treated control mice, whereas concentrations of BDNF mRNA were unchanged between the two groups (Figure 3C). These data were confirmed by immunoblotting, where protein extracts from ATII isolated from bleomycin-treated mice exhibited higher levels of TrkB and NT4/5, compared with protein extracts from ATII isolated from saline-treated control mice (Figures 3D and 3E). Consistent with the mRNA data, levels of BDNF protein were not elevated in ATII isolated from bleomycin-treated mice compared with saline-treated control mice (Figures 3D and 3E). Interestingly, NT4/5 was not only expressed by ATII, but was also secreted into the culture medium (Figure 3F). Supernatants collected from control ATII after 24 hours of culture revealed significantly higher concentrations of NT4/5 compared with naive medium (Figure 3F). These data indicate that the expression of the TrkB and NT4/5 signaling machinery is elevated in ATII from bleomycin-treated mice, and that NT4/5 is secreted by ATII, and thus may


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Figure 2. Expression and localization of TrkB and its ligands NT4/5 and BDNF in lungs from saline-treated control mice or mice with bleomycin-induced pulmonary fibrosis. (A and B) Immunohistochemical staining (alkaline phosphatase, red) for TrkB, NT4/5, and BDNF was performed on lung tissue sections from (A) saline-treated control mice or (B) mice 14 days after treatment with bleomycin. Inset shows negative control staining with isotype-matched IgG. (C ) Colocalization of TrkB or NT4/5 with proSPC, as assessed by double immunofluorecence staining of lung sections from bleomycin-treated mice. Arrows indicate colocalization of both antigens. Insets show negative control staining with isotypematched IgG. (D–F) Levels of mRNA expression in lung homogenates from saline-treated mice (n 5 8) or bleomycin-treated mice for 7 (n 5 8), 14 (n 5 7), 21 (n 5 12), and 28 (n 5 3) days were assessed by real-time RT-PCR for (D) TrkB, (E) NT4/5, and (F) BDNF. Values represent the mean DDCt 6 SEM. (G) Representative immunoblots of protein expression levels of TrkB, NT4/5, and BDNF from lung homogenates of saline-treated or bleomycintreated mice. b-actin served as a loading control. (H) Densitometric analyses of G. Values represent mean relative intensity 6 SEM (n 5 6, per group). *P , 0.05. **P , 0.01. ***P , 0.001. ns, not significant.

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Figure 3. Expression and localization of TrkB and its ligands NT4/5 and BDNF in alveolar Type II cells (ATII) isolated from lungs of saline-treated control mice or mice with bleomycin-induced pulmonary fibrosis. (A) Assessment of cell purity, using the ATII markers widespread cytokeratin (WSCK) and E-cadherin, and the fibroblast marker vimentin. Upper right inset gives an example of a positive-stained cell (fibroblast). (B) Immunocytochemical staining for TrkB, NT4/ 5, and BDNF was performed on ATII isolated from saline-treated control mice or from mice 14 days after treatment with bleomycin. Isotype-matched nonimmune IgG provided negative controls (lower left insets). (C ) Levels of mRNA expression in ATII isolated from saline or bleomycin-treated mice were assessed by real-time RT-PCR for TrkB, NT4/5, and BDNF (n 5 4). (D ) Protein expression levels of TrkB, NT4/5, and BDNF were assessed in ATII from salinetreated or bleomycin-treated mice by immunoblotting. b-actin is shown as a loading control. (E ) Densitometric analyses of the data presented in D (n 5 3 per group). (F ) Concentrations of NT4/5 were assessed in serum-free cell-culture supernatants from ATII isolated from control (healthy) mice (n 5 5). Serum-free medium (naive) served as a control (medium). *P , 0.05. **P , 0.01. ns, not significant.

participate in autocrine and paracrine control of TrkB-mediated processes in the lung. Regulation of TrkB, NT4/5, and BDNF Expression in ATII by Growth Factors

To elucidate how the expression of TrkB, NT4/5, and BDNF may be regulated in pulmonary fibrosis, murine primary ATII

were stimulated with TGF-b1, PDGF-BB, and IGF-I, the key profibrotic growth factors, and the expression of TrkB, NT4/5, and BDNF mRNA was assessed by real-time RT-PCR. TGF-b1 and PDGF-BB were able to upregulate the expression of TrkB on an mRNA level, whereas TGF-b1 exhibited effects only 5 and 10 hours after stimulation, and the effect of PDGF-BB was evident only 5 hours after stimulation (Figure 4A). In contrast, the


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Figure 4. Growth-factor regulated expression of components of the TrkB/NT4/BDNF signaling system in murine alveolar Type II cells. (A–C) Murine alveolar Type II cells (ATII) were serum-starved (0.1% FCS) for 6 hours and then stimulated with transforming growth factor (TGF)–b1 (5 ng/ml), insulin-like growth factor (IGF)–I (50 ng/ml), or platelet-derived growth factor (PDGF)-BB (10 ng/ml) for 5, 10, and 24 hours. RNA was then isolated and analyzed for the expression of (A) TrkB, (B ) NT4/5, and (C ) BDNF by real-time RT-PCR (n 5 3). (D and E) Evaluation of TrkB, NT4/5, and BDNF expression after 10 hours of stimulation with TGF-b1, IGF-I, and PDGF-BB, with respective densitometric analyses from multiple immunoblots (n 5 3). *P , 0.05. **P , 0.01. ns, not significant.

expression of NT4/5 and BDNF was unaffected by any of these three profibrotic growth factors (Figures 4B and 4C). Changes in expression were next assessed at the protein level by immunoblotting (Figures 4D, and 4E). On the protein level, we observed an increase in the expression of TrkB after 10 hours of TGF-b1 stimulation (Figures 4D and 4E). These data suggest that the increase in pulmonary TrkB expression observed in clinical and experimental pulmonary fibrosis may be driven, at least in part, by TGF-b1, which has well-characterized profibrotic activity. NT4/5 Drives the Proliferation of ATII

Because the expression of TrkB and NT4/5 (but not of BDNF) was upregulated both in whole-lung homogenates and in extracts from ATII, and TrkB and NT4/5 staining was evident in hyperplastic ATII in lung sections from humans, the effects of NT4/5 treatment on the proliferation of ATII were studied via PCNA staining, and by the incorporation of [3H]thymidine. Increased expression of PCNA (Figure 5A), and elevated uptake of [3H]thymidine (Figure 5B) by ATII were evident after stimulation with NT4/5. These data indicate that NT4/5 can drive the proliferation of ATII, which makes for an interesting correlate with the elevated expression of TrkB and NT4/5 in

clinical and experimental lung fibrosis, which is characterized by hyperplastic (and thus proliferating) ATII. NT4/5 Drives the Proliferation of ATII through TrkB and p44/42 Signaling

To identify the signaling pathways underlying the pro-proliferative effects of NT4/5 on ATII, cells were stimulated with NT4/5, and extracts were examined for changes in the phosphorylation status of candidate signaling molecules. NT4/5 induced a rapid and short-lived phosphorylation of TrkB within 5 minutes of ligand application (Figures 5C and 5D). The phosphorylation of TrkB was accompanied by a transient activation of p44/42 (Figures 5C and 5D). However, no activation of AKT and p38 kinases was evident, as assessed by densitometric analysis (data not shown). Pretreatment of ATII with K252a, an inhibitor of TrkA/ TrkB activation, blocked the phosphorylation of TrkB by NT4/5 (Figures 5E and 5F). Both K252a and U0126 blocked the phosphorylation of p44/42, indicating that p44/42 is downstream of TrkB in the NT4/5 signaling cascade in ATII (Figures 5G and 5H). Wortmannin did not affect the phosphorylation of p44/42 kinase, according to densitometric analysis (Figure 5H). The stimulation of ATII with NT4/5 did not appear to induce the phosphorylation of AKT, although phosphorylation may not have

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Figure 5. Effects of NT4/5 on proliferation and signaling pathways in murine alveolar Type II cells. (A and B) The effect of NT4/5 (10 ng/ml; 16 hours) on the proliferation of quiescent alveolar Type II cells (ATII) was assessed by (A) proliferating cell nuclear antigen (PCNA) staining and (B) the incorporation of [3H]thymidine (n 5 6, in triplicate). Inset represents control staining with isotype-matched IgG. Medium containing 1% (vol/vol) FCS served as a positive control. (C ) ATII stimulated with NT4/5 for the indicated time-points were analyzed for the activation of downstream signaling molecules by immunoblotting. p, phospho. (D) Densitometric analysis of selected signaling molecules (n 5 3). (E–H) ATII were stimulated with NT4/5 for 10 minutes in the absence or presence of the kinase inhibitors K252a (TrkB; 100 nM), U0126 (p44/42; 20 mM), and wortmannin (PI3K/AKT; 10 mM). Cells were harvested and analyzed for the activation of downstream signaling molecules (E and F) TrkB, (G and H) p44/42, and (I and J) AKT. Representative blots and densitometric analysis (n 5 3) are shown. (K) The effect of NT4/5 on the proliferation of ATII in the presence of kinase inhibitors was assessed by the incorporation of [3H]thymidine (n 5 5, in triplicate). Medium containing 1% (vol/vol) FCS served as a positive control. (L) Localization of phospho-Trk (alkaline phosphatase, red) in lung sections from donors and patients with IPF. Inset represents control staining with isotypematched IgG. (M and N) Determination of phospho-TrkB concentrations in lung homogenates from human donors and from patients with IPF by immunoblotting and respective densitometric analyses (n 5 4 for donors, n 5 7 for IPF). *P , 0.05. **P , 0.01. ***P , 0.001. ns, not significant.

been evident because ofthe high basal level of AKT activation (Figures 5C and 5I). In support of this idea, the TrkB inhibitor K252a did reduce the phosphorylation of AKT after stimulation with NT4/5 (Figures 5I and 5J). Furthermore, the phosphorylation of AKT was completely blocked by wortmannin (Figures 5I and 5J). Both K252a and U0126 inhibited the pro-proliferative effects of NT4/5 on ATII (Figure 5K), demonstrating the importance of the TrkB/p44/42 cascade in the mitogenic response to NT4/5. Interestingly, wortmannin also antagonized the pro-proliferative effects of NT4/5 on ATII (Figure 5K), implicating an involvement of the PI3K/AKT pathway in the mitogenic effects mediated by NT4/5. However, we cannot rule out that the impact of wortmannin on cell proliferation could be NT4/5-independent, bexcause of the presence of other factors in FCS. To examine whether this signaling pathway was activated in patients with IPF, lung sections from patients with IPF were stained for phospho-TrkB, and lung homogenates were assessed for the phosphorylation of TrkB by immunoblotting. Indeed, more pronounced phospho-TrkB staining was evident in hyperplastic ATII and interstitial fibroblasts in the lungs of patients with IPF, compared with donor lungs (Figure 5L). In support of this observation, elevated concentrations of phospho-TrkB were evident in the lung homogenates of patients with IPF (Figures 5M and 5N). NT4/5 Stimulates the Proliferation of Primary Murine and Human Lung Fibroblasts

Because the expression of both TrkB and NT4/5, but not BDNF, was increased in fibrotic regions of the lung in clinical

and experimental pulmonary fibrosis, we examined the impact of NT4/5 on the proliferation of lung fibroblasts. Isolated murine (Figure 6A, left) and human (Figure 6A, right) lung fibroblasts were first assessed for purity by staining with fibronectin and S100A4 (fibroblast markers), pro-SPC (an ATII marker), and vWF (an endothelial cell marker). Isolated murine (Figure 6B, left) and human (Figure 6B, right) lung fibroblasts were then assessed for the expression of TrkB, NT4/5, and BDNF by immunocytochemistry. All three proteins were detected in murine and human lung fibroblasts, suggesting intact TrkB/NT4/5 signaling in lung fibroblasts. Therefore, the effects of treatment with NT4/5 on the proliferation of lung fibroblasts was studied in human and murine lung fibroblasts by the incorporation of [3H]thymidine (Figures 6C and 6D). The increased uptake of [3H]thymidine by murine (Figure 6C) and human (Figure 6D) lung fibroblasts was evident after stimulation with NT4/5. These data indicate that NT4/5 can also drive the cell proliferation of lung fibroblasts, which makes for an additional interesting correlate with the elevated expression of TrkB and NT4/5 in clinical and experimental fibrosis, which is characterized by the activation, proliferation, and transdifferentiation of fibroblasts. NT4/5 Drives the Proliferation of Lung Fibroblasts through TrkB, p44/42, and AKT Signaling

To identify the signaling pathways underlying the pro-proliferative effects of NT4/5 on lung fibroblasts, primary human lung fibroblasts were stimulated with NT4/5, with or without preincubation with the inhibitors K252a, U0126, or wortmannin. NT4/5 induced the phosphorylation of p44/42 in human lung fibro-


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Figure 5. (Continued).

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Figure 5. (Continued).

blasts (Figures 7A and 7B). However, in contrast to what we observed in ATII (Figure 5), the pre-exposure of human lung fibroblasts to K252a did not affect the phosphorylation of p44/42 by NT4/5 (Figures 7A and 7B). As expected, U0126 prevented the phosphorylation of p44/42 (Figures 7A and 7B). These data suggest that another NT4/5 receptor, such as p75 NTR , could be responsible for the activation of p44/42 in human lung fibroblasts. In contrast to ATII (Figure 5), the stimulation of human lung fibroblasts with NT4/5 resulted in the activation of AKT. This effect was abrogated by the preincubation of fibroblasts with K252a. Pretreatment of human lung fibroblasts with wortmannin diminished the phosphorylation of AKT after treatment with NT4/5 (Figures 7C and 7D). This outcome suggests that wortmannin-sensitive kinases were induced by NT4/5 to phosphorylate AKT. Together, these data suggest that the NT4/5 signaling pathways in human fibroblasts and ATII are not identical. All three inhibitors (K252a, U0126, and wortmannin) inhibited the pro-proliferative effects of NT4/5 on human lung fibroblasts (Figure 7E), implicating the TrkB/p44/42/AKT cascade in the pro-proliferative signaling pathway of NT4/5 in fibroblasts. The possible contribution of elevated NT signaling by lung fibroblasts in pulmonary fibrosis is underscored by the increased expression of components of the NT signaling axis (Figure 7F) in lung fibroblasts from bleomycin-treated mice, compared with control mice. Because no increase in the expression of a–smooth muscle actin protein was observed after the stimulation of murine (Figures 7G and 7H) or human (Figures 7I and 7J) lung fibroblasts with NT4/5, no indication of fibroblast-to-myofibroblast differentation was apparent.

DISCUSSION The neurotrophins are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in lung disease. The importance of the neurotrophin system in the lung is evident early in ontogeny, as indicated by the dynamic regulation of the low-affinity p75NTR receptor during fetal growth (22) and by evidence of an essential role for TrkB in airway branching and alveolarization, as revealed by studies in TrkB2/2 mice (6). Similarly, neurotrophin ligands are also precisely regulated during embryonic lung development in mice (23). In early postnatal life, NTs and NT receptors are also implicated in several adult lung diseases, including chronic obstructive pulmonary disease (12), lung cancer (9), and pulmonary arterial hypertension (13). Together, these reports highlight neuronal as well as non-neuronal roles for the NT/NT receptor system in lung pathophysiology. A recent study also suggested increased expression of the NT ligand BDNF and its cognate receptor, TrkB, in fibroblast foci of patients with IPF (14). In the present study, a pronounced increase in the expression of NT4/5 and its cognate receptor TrkB was evident in lungs of patients with IPF. The increased expression of NT4/5 and TrkB was evident in hyperplastic ATII, as well as of TrkB in fibrotic areas of affected lungs. A similar pattern of NT4/5 and TrkB expression was observed in the lungs of mice in which pulmonary fibrosis was induced with bleomycin, whereas in murine lungs, we cannot rule out that inflammatory cells may also express NTs and NT receptors. These findings indicate a potential role for the NT4/5/TrkB axis in pathological processes associated with pulmonary fibrosis. Idiopathic pulmonary fibrosis is a devastating disease with a poor prognosis, characterized by excessive deposition of


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Figure 6. NT4/5 effects a proliferation response by murine and human lung fibroblasts. (A) Assessment of fibroblast purity, using fibronectin and S100A4 (fibroblast markers) as well as pro-SPC (alveolar Type II cell marker) and von Willebrand factor (vWF; endothelial cell marker). (B) Murine and human lung fibroblasts were examined for the expression of TrkB, NT4/5, and BDNF proteins. Inset shows respective isotype control stainings. (C and D) The effect of NT4/5 stimulation on the proliferation of (C ) murine and (D) donor lung fibroblasts, as assessed by the incorporation of [3H]thymidine (n 5 3, in triplicate) disintegrations per minute (dpm). *P , 0.05. **P , 0.01.

extracellular matrix, originally explained as a result of fibroblast proliferation and the accumulation of myofibroblasts, resulting in extensive remodeling of lung parenchyma (24). More recently, several concepts have emerged that highlight roles for ATII dysfunction, encompassing ATII hyperplasia, apoptosis, and an epithelial-to-mesenchymal transition in disease pathogenesis (15, 25). The documented roles of NT and NT receptor signaling in promoting the survival, resistance to apoptosis, and prolifera-

tion of epithelial cells suggest that the elevated expression of NTs and NT receptors in lungs affected by pulmonary fibrosis may underlie, at least in part, the hyperplasia of ATII that characterizes IPF. In the context of lung epithelial cell biology, the TrkB pathway is widely implicated in cancer growth (26), including the malignant transformation of lung epithelial cells, where TrkB is overexpressed (27). The stimulation of lung adenocarcinoma cells with neurotrophins activates an AKTdependent cell-survival program, highlighting a prosurvival role

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Figure 7. NT4/5 signaling in human lung fibroblasts. (A–D) Human lung fibroblasts were stimulated with NT4/5 in the absence or presence of the kinase inhibitors K252a (TrkB; 100 nM), U0126 (p44/42; 20 mM), and wortmannin (PI3K/AKT; 10 mM). Cells were harvested after 10 minutes and analyzed for the activation of downstream signaling molecules (A and B) p44/42 and (C and D) AKT. Densitometric analysis was performed after three independent experiments. (E ) The effect of NT4/5 on cell proliferation of quiescent donor lung fibroblasts in the presence of kinase inhibitors was assessed by the incorporation of [3H]thymidine. Values represent the mean 6 SEM (n 5 5, in triplicate). (F ) Levels of mRNA expression of TrkB, NT4/5, and BDNF were assessed in fibroblasts from control and bleomycintreated mice by real-time RT-PCR (n 5 3, per group). (G–J ) Assessment of a–smooth muscle actin (a-SMA) expression after NT4/5 stimulation by immunoblotting and corresponding densitometric analyses (n 5 3) for (G and H ) murine and (I and J ) human lung fibroblasts. *P , 0.05. **P , 0.01. ***P , 0.001. ns, not significant.


for neurotrophins in cancerous lung epithelial cells. Intervention with neurotrophin receptor signaling, using K252a in lung adenocarcinoma cells, prevented the activation of AKT in response to NGF or BDNF, induced apoptotic cell death, and diminished the growth properties of the cancer cells (27). These observations led to the proposal that TrkB/neurotrophin signaling represents an attractive new signaling pathway that may be targeted therapeutically in the lung cancer (28). Indeed, the high level of expression of Trk receptors was proposed as a proliferation marker for cancer cells (29). The data reported in the present study reveal that TrkB, NT4/5, and BDNF are expressed in ATII, and that the expression of TrkB and NT4/5 is dysregulated in ATII from bleomycin-treated mice, compared with ATII isolated from saline-treated control mice. The overexpression of TrkB and NT4/5 by ATII in bleomycin-treated mice is reminiscent of the elevated expression of the TrkB/neurotrophin system in lung epithelial cell cancers, and we speculate that overactive TrkB/ NT4/5 signaling may underlie, at least in part, the hyperproliferation of ATII in pulmonary fibrosis. In support of this idea, the stimulation of ATII with NT4/5 enhanced the proliferation of ATII, an effect that was blocked by the pretreatment of ATII with the TrkB kinase inhibitor K252a. The observations in the present study documenting the upregulation of TrkB expression in ATII by the key profibrotic growth factor TGF-b1 (30) hint at a mechanism that may drive the expression of TrkB in clinical and experimental pulmonary fibrosis. Thus, the dysregulated activity of TGF-b1 in lungs affected by pulmonary fibrosis may promote the aberrant expression (and signaling) of TrkB in ATII, contributing to the proliferation of this cell population. The secretion of NT4/5 by ATII reported in the present study suggests that NT4/5 may act in an autocrine and paracrine fashion in the lung. Indeed, the juxtaposition of cell types expressing NTs and cell types expressing Trk receptors in the developing murine lung makes a strong case for paracrine and autocrine signaling by NTs. In this study, strong TrkB staining was evident in fibrotic foci in lungs affected by pulmonary fibrosis, suggesting that the response by fibroblasts to NTs may also be relevant in clinical and experimental fibrosis. This idea is supported by observations of a TrkB/NT proliferation pathway in murine embryonic fibroblasts (31), a potentially important new pathogenic concept in IPF (32). In the present study, we documented that NT4/5 drove the proliferation of murine and human lung fibroblasts through both TrkB-dependent and TrkB-independent pathways, in contrast with the NT4/5 signaling pathways operative in ATII. Together, these observations suggest that the dysregulated expression and signaling of TrkB/ NT4/5 in the fibrotic lung may affect not only ATII hyperplasia but also the proliferation of fibroblasts. Altogether, the data reported in the present study suggest that a dysregulated TrkB/NT4/5 axis may contribute to several of the pathological lesions associated with pulmonary fibrosis, and we would add IPF to the list of disorders (such as pain and cancer) for which the therapeutic targeting of the TrkB/neurotrophin axis was proposed for further investigation (28). These studies should be facilitated by the intensive efforts currently focused on the development of potent and specific TrkB kinase inhibitors, such as CEP-751 (33) and the more recently described, orally bioavailable AZ-23 compound (34). Author Disclosure: W.S. received consulantacy fees from Bayer Schering Pharma AG and Lung Rx, Inc., and lecture fees from Encysive/Pfizer Pharma GmbH and Bayer Schering Pharma AG. W.S. received an industry-sponsored grant from United Therapeutics. A.G. received consultancy fees from Nycomed and Activaero, and serves on the advisory boards of GlaxoSmithKline, Actelion, Intermune, Pfizer, and Novartis. None of the other authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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Acknowledgments: The authors thank Miriam Schmidt, Alice Vohmann, and Gisella Mu¨ller for their expert technical assistance. The authors also thank Dr. Melanie Ko¨nigshoff and Andreas Jahn for instructing them in the isolation of murine alveolar Type II cells.

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