Nathan M. Krah. Preliminary Exam. Department of Human Genetics. July Defining the functions of alveolar c-met in late-stage emphysema

Transcription

1 Krah 1 Nathan M. Krah Preliminary Exam Department of Human Genetics July 2014 Defining the functions of alveolar c-met in late-stage emphysema Committee: Gabrielle Kardon Mark Metzstein Carl Thummel Matthew Topham

2 Abstract Krah 2 The alveolus is the functional unit of the lung consisting of two discrete cell populations, type 1 alveolar cells (AEC1s) and type 2 alveolar cells (AEC2s). AEC1s are the site of gas exchange, while AEC2s act as progenitors by differentiating into AEC1s during normal homeostasis and, more prominently, following injury [1,2]. Cigarette smoke (CS) induces apoptosis of alveoli, leading to emphysema, which kills ~100,000 patients in the USA per year. While no cure exists for emphysema, restoring functional alveoli holds promise as a potential therapeutic strategy. However, little is known about the process of alveolar re-growth and any information regarding this process will be essential to the understanding and management of emphysema. Canonical Hgf/c-Met signaling promotes epithelial cell survival and proliferation by activating downstream pro-growth pathways, such as PI3K/AKT and MEK/MAPK [3]. A recent study demonstrated that tissue-specific deletion of c-met from AEC2s causes emphysema, confirming that Hgf/c-Met signaling is necessary to maintain alveolar homeostasis [4]. Furthermore, supplementation of Hgf is sufficient to inhibit emphysema [4,5]. Additionally, other non-canonical c-met functions may influence alveolar survival; for example, c-met inhibits apoptosis by binding to the death receptor, Cd95 [6]. Given the central role of c-met signaling in maintaining lung homeostasis, I hypothesize that c-met is a master regulator of epithelial survival and regeneration following CS-induced emphysema. In this proposal I will address two outstanding questions in the emphysema field: First, which type of lung progenitor cells contribute to alveolar restoration following chronic CS exposure? Second, how does Hgf/c-Met signaling contribute to the maintenance/repair of alveoli following CS-induced injury? I hypothesize that c-met expressing AEC2s repopulate the lung following chronic CS exposure and that canonical activation of c-met is necessary for this expansion. I will also investigate whether c-met sequestration of Cd95 inhibits apoptosis following CS-induced emphysema. Data generated from this proposal will enhance our understanding of lung regeneration and provide avenues for novel treatments of emphysema.

3 Introduction Krah 3 The Lung, COPD, and HGF/c-MET signaling 1 : The lung is a highly branched organ with a large and intricate epithelial surface area, which is organized into terminal units called alveoli. Efficient gas exchange relies on the functions of two prominent cell types that comprise the respiratory epithelium. Very thin type one alveolar epithelial cells (AEC1) cover ~90% of the alveolar surface; these cells reside adjacent to capillaries and are the site of gas exchange. Each alveolus also contains cuboidal type two epithelial cells (AEC2), which produce Surfactant Protein (Sftp-C) to decrease surface tension and prevent fluid accumulation. AEC2s are some of the most important cells in normal pulmonary physiology and disease, as their dysfunction has been implicated in neonatal respiratory distress syndrome, pulmonary fibrosis, and lung cancer [1,2,7]. Other cell types also perform crucial functions in the terminal bronchioles, which are adjacent to alveoli. Clara cells express Secreteoglobin1a1 (Scgb1a1) and produce components of Surfactant. Pulmonary fibroblasts generate extracellular matrix constituents and secrete growth factors, such as Hepatocyte growth factor (Hgf), which aid in maintaining alveolar homeostasis [4,8,9]. The self-renewal and re-differentiation of AEC2s sustains lung epithelium under normal physiological conditions [1,2,10]. Intriguingly, AEC2s rapidly self renew and differentiate into AEC1s following acute injury [1,2] (Fig. 1). AEC2s, however, are not the only progenitor cell population that has been identified in the lung. Additional cells, known as BASCs (bronchioalveolar stem cells), co-express the AEC2 marker Sftp-C and the Clara cell marker Scgb1a1 and have been implicated as alveolar stem cells in vitro. However, it remains unclear what contributions these cells make to alveolar homeostasis and re-growth following injury in vivo [11,12]. In all, recent studies suggest that several discrete cell populations can function as alveolar progenitors based on their capacity to clonally proliferate and differentiate into diverse lineages in culture [1,2,9,12-14]. While Abbreviations: Hgf Hepatocyte Growth Factor, CS Cigarette Smoke, COPD Chronic Obstructive Pulmonary Disease, Sftp-C Surfactant Protein C, AEC1 - Type One Alveolar Epithelial Cell, AEC2 - Type Two Alveolar Epithelial Cell, BASC bronchioalveolar stem cell, Scgb1a1- Secreteoglobin1a1

4 Introduction Krah 4 many of these progenitor populations have been studied widely in vitro, it would be most beneficial if these cells could be manipulated to repopulate the lung epithelium in models of common diseases where alveoli are lost, such as Chronic Obstructive Pulmonary Disease (COPD). The loss of lung epithelium in COPD is commonly due to environmental insults such as cigarette smoke (CS), which creates oxidant stress that drives inflammation and epithelial cell apoptosis [15,16]. Despite the known risks and public campaigns about the dangers of cigarette smoking, it continues to be a global health problem. The World Health Organization estimates that there are currently over 1 billion people who smoke and nearly 30% of smokers will develop a form of COPD [17,18]. According to the U.S. Center for Disease Control, COPD is the third leading cause of death in America, highlighting the need for a deeper understanding of its pathophysiology. It is noteworthy that non-smokers are affected by COPD; about 20% of cases are caused by genetic predisposition or exposure to pollution [17]. Emphysema, the primary subtype of COPD, is characterized by destruction of alveoli via apoptosis [16,19,20]; other features include enlargement of air space and reduced elastic pressure that generates expiratory airflow. As lung epithelium is lost, it becomes increasingly difficult to obtain adequate oxygen. The quest to preserve existing alveoli and regenerate functional lung epithelium once emphysema has progressed is a necessary, albeit elusive medical goal. Over the past two years, however, there has been progress in preventing alveolar apoptosis and restoring alveoli in mice with progressed emphysema [4,21,22]. These studies provide important clues as to the central molecular pathways that promote alveolar survival and restoration, and what cell types are capable of proliferating and differentiating into functional AEC1s. Recent studies demonstrated that Hgf, a molecule crucial for embryonic lung development, is sufficient to inhibit emphysema progression in different experimental models [4,5,23]. Downstream of the Hgf receptor, c-met, canonical signaling activates cascades that are crucial for cell proliferation, survival, and migration, such as PI3K/AKT,

5 Introduction Krah 5 RAC/PAK, and MEK/MAPK (See Fig 2) [3]. Recent studies also suggest that AEC2s act as progenitor cells in the lung, replacing AEC1s following injury [1,2]. However, whether HGF acts specifically on AEC2s or other progenitor cell populations has not been investigated. Because of these observations I hypothesize that HGF/c-MET signaling plays a central role in 1) inducing AEC2s to repair damaged alveoli following CS-induced injury and 2) inhibiting lung epithelial apoptosis following chronic CS epxosure. Alternative HGF isoforms: Full length Hgf is synthesized and secreted from cells of mesenchymal origin, such as pulmonary fibroblasts [3,9,24]. This form of Hgf acts on the epithelial and endothelial cell c-met receptor to promote alveolar homeostasis [4]. The full Hgf transcript contains all 18 of the gene s exons; however, alternatively spliced isoforms of Hgf have also been identified and these variants have opposing biological functions. Nk2 is a truncated Hgf transcript, which lacks exons 8-18 and possesses a different 3 untranslated region (UTR). This isoform, contrary to canonical Hgf, antagonizes the mitogenic activity of c-met signaling by acting as a competitive antagonist on the c-met receptor [25,26]. Thus, the Hgf gene encodes both a potent regenerative growth factor and a competitive c-met inhibitor. One crucial signaling pathway that regulates Hgf splice variants is the Tgf-β signaling cascade. Using MRC-5 fetal human lung fibroblasts, Harrison and colleagues demonstrated that addition of Tgf-β1 promotes the degradation of full length Hgf transcripts, but does not affect the posttranscriptional stability of the truncated Hgf transcript encoding Nk2 [27]. Mungunsukh and Day corroborated these data 13 years later; they demonstrated that Tgf-β1 increases mir-199a, which selectively targets the 3 end of full length Hgf transcripts, but not the Nk2 splice variant in adult pulmonary fibroblasts [28]. Taken together, these data indicate that increased Tgf-β could suppress Hgf/c-Met signaling by increasing the relative abundance of mir199a and Nk2 in lung

6 Introduction Krah 6 fibroblasts. The effects of Nk2 and mir-199a, however, have not been elucidated in the lung in vivo, nor have they been evaluated in emphysema. Independent studies, which do not focus on Hgf/c-Met signaling, demonstrated that Tgf-β signaling is greatly increased in mice subjected to chronic CS [22]. Podowski and colleagues demonstrated that Tgf-β1 and phospho-smad2, a readout of downstream Tgf-β signaling, is abundant in the lungs of CS-exposed mice and of human COPD patients, but not corollary controls. Importantly, inhibition of Tgf-β using either losartan or a pan-selective Tgf-β antibody, was sufficient to attenuate airspace enlargement [22]. Losartan is an angiotension II-type 1 receptor blocker that is approved for the treatment of hypertension and diabetic nephropathy, but is also widely used as a Tgf-β inhibitor [22,29,30]. Because increased Tgf-β signaling favors Nk2 at the expense of Hgf [27,28], I hypothesize that the increased Tgf-β signaling during chronic CS exposure allows Nk2 perdurance in alveoli by increasing mir-199a. I propose that suppression of canonical c-met signaling by Nk2 is a mechanism that prevents recovery from CS-induced injury. Hgf activates lung progenitors: Several reports demonstrate that AEC2s act as progenitors and repopulate the lung during normal homeostasis and, to a much greater extent, following injury [1,2,31]. Desai and colleagues used lineage tracing to show that a single Sftp-C + AEC2 can selfrenew, redifferentiate and give rise to AEC1s that repopulate an alveolus following hyperoxic treatment [1]. Given that Hgf signaling has necessary roles in cell proliferation and migration during lung development and on lung progenitor cells in vitro [9,32], it represents a strong candidate that could propagate AEC2 self-renewal and proliferation in normal physiology and following lung injury. Importantly, Hgf supplementation inhibits genetically induced emphysema in the tight-skinmouse (Fibrillin1 mutant) and the Hgf receptor, c-met, is expressed specifically on AEC2s [4,23]. Lung and systemic Hgf are reduced in models of emphysema as the disease progresses and this observation could explain why AEC2s fail to repopulate the lung once emphysema is progressed to

7 Introduction Krah 7 late stages [4,5]. I, therefore, hypothesize that AEC2s repopulate alveoli during recovery from chronic CS exposure in an Hgf dependent manner. As described above, several categories of lung progenitor cells have been described in the literature [11-14,31]. These cell types represent important candidates that could be explored as alternatives if AEC2s do not aid in epithelial regeneration following CS-induced injury. For example, a subset of cells that express both Clara cell Scgb1a1 and the AEC2 marker Sftp-C proliferate following injury; based on the differentiation potential of these cells in vitro, they have been termed BASCs [12]. However, when their lineage fate was followed in vivo, Rawlins and colleagues found that BASCs did not contribute to the alveolar epithelium during normal tissue maintenance or following hyperoxic injury [11]. However, others still contest that BASCs can give rise to alveolar epithelium following viral infection and other acute injuries [31]. Since the role of BASCs in vivo remains a debated topic within the pulmonary scientific community, I will investigate the importance of BASCs in alveolar restoration following CS-induced injury. c-met mediates apoptosis: Despite acting as an established survival and proliferation pathway, Hgf/c-Met signaling can mediate apoptosis in several cell types [3,6]. This is consistent with this signaling cascade s potential role in emphysema, where the balance of proliferation and apoptosis is dysregulated. Apoptosis is mediated by one of three pathways: the extrinsic, intrinsic, or Perforin/GranzymeB pathway [33]. Data suggests that c-met can mediate extrinsic and intrinsic apoptosis. The extrinsic pathway is mediated by the Cd95 receptor and Fas ligand (FasL) [6,34]. FasL binding to Cd95 causes aggregation of Cd95 receptors, which subsequently recruits intracellular Fadd to directly interact with the intracellular death domain of Cd95 [35]. This interaction recruits Procaspase-8 to form DISC (death-inducing signaling complex). Once DISC is formed, Caspase-8 is cleaved into its active form and it activates executioner caspases-3 and 7 [36]. These effector Caspase proteins activate a number of other enzymes, such as nucleases, which

8 Introduction Krah 8 degrade chromosomal DNA and propagate cell death. Intrinsic apoptosis, however, is mediated by mitochondrial permeability; Bcl-2 proteins are the principal regulator of cytochrome c release from the mitochondria. If Bcl-2 proteins are sequestered away from the mitochondrial membrane, cytochrome C is released and this molecule activates caspase-9, which propagates downstream Caspase signaling [33]. The mechanism by which c-met mediates apoptosis has only been partially elucidated. Studies suggest that the extracellular domain of c-met physically interacts with Cd95; this sequestration of Cd95 restricts FasL from interacting with Cd95, which prevents activation of the extrinsic pathway [3,6]. However, high concentrations of FasL are sufficient to dissociate this complex and sensitize cells to apoptosis [6]. Additionally, TNF-α supplementation is sufficient to cause cleavage of the intracellular domain of c-met, which generates a 40kD fragment that induces apoptosis [37,38]. This fragment localizes to the mitochondria, where it increases mitochondrial permeability and activates the intrinsic pathway [34]. While local Hgf is decreased in emphysema [4,5], local factors that mediate apoptosis, such as TNF-α, are increased in the lung [39]. I hypothesize that chronic CS exposure leads to c-met mediated apoptosis, which propagates epithelial cell loss during late-stage emphysema, even after cessation of CS. Concluding Remarks: c-met is a well-characterized receptor that mediates proliferation, regeneration, and apoptosis. Nk2 inhibits c-met in vitro, however, whether Nk2 suppresses c-met signaling in the lung in vivo, has not been investigated. If Nk2 does act in the lung, it likely suppresses c-met signaling in AEC2s, which are lung progenitors in vivo. It remains unknown, however, if AEC2s repopulate the lung following chronic CS-induced lung injury and whether this process is dependent on Hgf/c-Met. The role of c-met mediated apoptosis is also poorly characterized in the lung, but has been well defined in vitro. I propose that c-met is a safeguard, protecting the lung epithelium from further apoptotic damage following CS-induced injury.

9 Introduction Krah 9 BASC Sftp-C Scgb1a1 Self Renewal Proliferation Self Renewal AEC2 Sftp-C Differentiation Post-Injury AEC1 Cell Death/Emphysema Figure 1: Alveolar cell lineages. AEC2s serve as alveolar progenitors by self-renewing, clonally expanding, and differentiating into AEC1s in injury models and, to a lesser extent, during normal alveolar maintenance [1,2]. BASCs are self-renewing cells that give rise to many lung cell lineages in vitro. However, it remains debated if these cells give rise to AEC1s and/or AEC2s in vivo [11,12]. Pulmonary Fibroblast Hgf c-met Nk2 GAB1 PI3K RAS AKT GAB1 Apoptosis MEK mtor Survival ERK Proliferation Epithelial Cell Figure 2: Major downstream pathways regulated by c-met. Hgf, which is secreted by pulmonary fibroblasts, activates canonical c-met signaling, while Nk2 acts as a competitive antagonist and inhibits downstream signaling. (Blue) Gab1 coordinates cellular responses to c-met and binds to the intracellular region of the receptor. When Gab1 interacts with c-met it becomes phosphorylated, which recruits effector proteins, such as Phophoinositide 3-kinase (PI3K). (Orange) PI3K activates Akt, which phosphorylates several substrates involved in cell proliferation, survival, and growth, such as mtor and GSK3β (not shown). Active Akt also protects cells from apoptosis. (Green) Canonical c-met signaling activates extracellular signal-regulated kinases (ERKs) via the Ras pathway and promotes cell proliferation.

10 Specific Aims Krah 10 Emphysema is a significant cause of morbidity and mortality worldwide and is characterized by apoptosis of respiratory epithelium [19]. Recent studies demonstrate that canonical Hgf/c-Met signaling, which promotes proliferation and regeneration, inhibits emphysema [4,5,23]. Here, I propose that c-met is a master regulator of lung epithelial restoration and survival after chronic CSinduced damage. As such, I hypothesize that the well-characterized endogenous inhibitor of c-met, Nk2, is increased after CS exposure and delays alveolar re-growth [25,26]. Recent studies suggest that the lung has robust regenerative capacity following injury, which is mediated by AEC2s [1,2,40]. Given that AEC2s express c-met and that Hgf supplementation is sufficient for lung progenitor colony formation in vitro, I hypothesize that c-met signaling mediates AEC2 proliferation and differentiation after chronic CS-induced injury [4,9]. While other cell types may function as progenitors in the lung, such as Scgb1a1 + /Sftp-C + BASCs, I predict that these cells make little contribution to c-met mediated alveolar restoration following CS injury [11,12]. Interestingly, c-met can regulate apoptosis by binding directly to the death receptor, Cd95 [6]. This sequestration of Cd95 restricts FasL from interacting with Cd95, which prevents activation of the extrinsic pathway. While signals from the surrounding milieu can disrupt the binding of c- Met to Cd95, the effects of CS on this interaction have not been examined. I will test the hypothesis that CS dissociates c-met and Cd95, thus rendering lung epithelial cells susceptible to apoptosis. I predict that stabilization of c-met/cd95 in vivo will be sufficient to inhibit lung epithelial apoptosis during and after chronic CS exposure. Hypothesis: Following chronic CS exposure, Nk2 antagonism of c-met prevents alveolar restoration, while canonical c-met signaling inhibits emphysema progression by promoting AEC2-mediated alveolar re-growth and inhibiting apoptosis. AIM 1: Test whether AEC2s repopulate the alveolar epithelium in a Hgf/c-Met dependent manner following CS-induced emphysema.

11 Specific Aims Krah 11 Several recent studies suggest that AEC2s repopulate alveoli following injury. I hypothesize that canonical Hgf/c-Met signaling is necessary and sufficient to activate the expansion and redifferentiation of AEC2s, but not BASCs, following chronic CS-induced injury. 1.1 Test whether c-met is necessary for AEC2 proliferation, formation of clonal foci, and differentiation into AEC1s following smoke-induced injury. 1.2 Test whether Hgf supplementation is sufficient to induce clonal proliferation and redifferentiation of AEC2 cells following smoke induced injury. 1.3 Scgb1a1 + BASCs do not contribute to Hgf/c-Met mediated alveolar repair following CS exposure and I will test this hypothesis using lineage tracing. AIM 2: Test whether TGF-β mediated Nk2 persistence induces alveolar loss through a mir- 199a dependent mechanism. Nk2 is a splice variant of Hgf that acts as a competitive inhibitor of canonical c-met signaling. Nk2 is stabilized in response to Tgf-β signaling, which is increased during CS exposure [22,28]. Mechanistically, Tgf-β signaling upregulates mir-199a, which selectively degrades Hgf, but not Nk2. However, it remains unknown if Nk2 plays an important role in the lung in vivo. In Aim 2, I hypothesize that Nk2 is preferentially stabilized in emphysema in a Tgf-β and mir-199a dependent manner. I will also test whether alteration of the mir-199a target site on full-length Hgf will inhibit CS-induced emphysema. 2.1 Test whether Nk2 is increased relative to full-length Hgf after CS-induced emphysema in a Tgfβ dependent manner 2.2 Sustained NK2 expression will prevent recovery from emphysema and I will test this model by exposing MT-1 Nk2 mice to chronic CS-induced injury. 2.3 mir-199a will be upregulated following chronic exposure to CS and I will test this hypothesis using in situ hybridization. 2.4 Mutation of the mir-199a target site on Hgf will preserve lung epithelium during CS-induced exposure and I will test this hypothesis using CRISPR/CAS. AIM 3: Test whether c-met inhibits alveolar cell apoptosis following CS-induced injury by binding to Cd95. c-met directly binds to the Cd95 death receptor to prevent extrinsic apoptosis. I will test the hypothesis that CS disrupts this interaction. c-met can also modulate apoptosis through the proapoptotic fragment, p40-met. I will additionally test is this process contributes to apoptosis in the lung epithelium.

12 Specific Aims Krah Test whether CS extract disrupts the interaction between c-met and Cd95 in lung epithelial cells in vitro. 3.2 Chronic exposure to CS will increase the amount of unbound Cd95 in lung epithelial cells in vivo and I will test this hypothesis by assaying for c-met/cd95 interactions at different CS exposure lengths. 3.3 c-met sequestration of Cd95 inhibits lung epithelial apoptosis following chronic CS exposure and I will test this hypothesis by generating a nanobody that stabilizes this interaction. Conclusions: The experiments planned within this proposal will thoroughly test the various functions Hgf/c-Met signaling in lung epithelium. Testing the proposed hypotheses will lead to a greater understanding of c-met function in promoting lung progenitor cell activity and inhibiting apoptosis in the context of emphysema. These data may provide insights into novel treatment opportunities for this, currently, incurable disease. Model Diagram of Major Hypothesis: Hypothesis Following chronic CS exposure, Nk2 antagonism of c-met prevents alveolar restoration, while canonical c-met signaling inhibits emphysema by promoting AEC2-mediated alveolar renewal and inhibiting apoptosis. Pulmonary Fibroblast AIM 1 AEC2 Hgf HGF c-met Proliferation/Differentiation Hgf Nk2 NK2 AIM 2 Apoptosis

13 Research Strategy Krah 13 AIM1: Test whether AEC2s repopulate the alveolar epithelium in a Hgf/c-Met dependent manner following CS-induced emphysema. While the lung is relatively quiescent, the mouse and human lung have robust regenerative capacity, especially following injury [1,14,40]. Lineage tracing, a powerful tool allowing for observation of all progeny from a single cell, has been crucial in identifying cell types that can regenerate functional alveoli [1,41]. Two studies recently utilized a Confetti reporter to lineage-label AEC2s and demonstrate that these cells form self-renewal foci, where a single AEC2 repopulates an alveolus following injury [1,2]. However, other lung cell types have also been proposed as stem cells based on their regenerative capacity in vitro [9,11-13,31]. It remains unclear what cell type can restore lung epithelium following CS-induced injury and what signals are necessary to modulate expansion of these progenitors. Given that AEC2s act as progenitors and express high levels of c- Met protein [4,5], I hypothesize that damaged alveoli are restored by AEC2s in a Hgf/c-Met dependent manner once emphysema has progressed. 1.1 Test whether c-met is necessary for AEC2 proliferation, formation of clonal foci, and differentiation into AEC1s following CS-induced emphysema. c-met protein is expressed robustly in AEC2s and activation of this receptor is a known inducer of cell proliferation, migration, and regeneration in the lung [4,5,42]. Furthermore, the c-met specific ligand, Hgf, maintains the self-renewal capacity of lung stem cells in vitro and inhibits emphysema in vivo [4,5,9]. Given these data and the defined role of AEC2s in repopulating alveoli following injury [1,2], I will test the hypothesis that Hgf/c-Met signaling is necessary for AEC2-mediated repair of alveoli following CS-induced injury. Procedures: The recent utilization of tissue specific, inducible Cre-recombinase and multicolor reporter constructs makes it possible to examine how a specific cell population contributes to the repair of tissue [41]. c-met will be deleted specifically in the AEC2s, which will also be genetically labeled, by generating SftpC-Cre ERT ;c-met Δ/fl ;R26-Confetti mice and corollary controls. To test whether AEC2s form clonal foci and redifferentiate into AEC1s following CS-induced injury in a c-

14 Research Strategy Krah 14 Met dependent manner, mice will be exposed to CS for 2 months, after which, tamoxifen will be administered to induce c-met deletion. Lungs will be harvested 1 month after cessation of CSexposure (See figure below) and stained for lineage markers. In both experimental groups, the number of reporter-positive clonal foci and the number of lineage labeled AEC1s will be quantified. Additionally, I will quantify the percentage of total alveoli with the AEC2 lineage label, and the number of cells in the cell cycle by staining for Ki67. Experimental Design for Aim1.1 Expected Results: I hypothesize that there will be increased clonal expansion and proliferation of lineage labeled AEC2s following CS exposure in control animals compared to c-met Δ/f mice. If c- Met deficient AEC2s fail to produce lineage marked AEC1s and/or form clonal foci following exposure to CS-induced injury, I will conclude that expression of c-met is necessary for the repopulation of lung epithelium in emphysema. Pitfalls and Alternatives: Persistent injury and limited alveolar re-growth could confound results. Decreasing the length/amount of CS exposure, or increasing the recovery period could resolve these issues. Additionally, other factors could be necessary to induce AEC2 proliferation and differentiation in CS-induced emphysema. Studies suggest that Egf and Vegf play important roles in lung development and in maintaining alveolar homeostasis; these factors may also be important in regeneration [1,43]. Egfr and Vegf have effective pharmacological inhibitors, such as erlotinib, and the monoclonal Vegf antibody, bevacizumab. If the described in vivo studies fail, I propose to isolate reporter + AEC2s and culture them in the presence of Tgf-α (an Egfr ligand) or Vegf with and without their corollary inhibitor in order to determine if these factors are necessary for proliferation, re-differentiation, and/or alveolar sphere formation [2,13,14].

15 Research Strategy Krah Test whether HGF supplementation is sufficient to induce clonal proliferation and redifferentiation of AEC2 cells after CS-induced injury. Hgf levels are significantly reduced in lung tissue and plasma as emphysema progresses [4,5]. While correlative, decreasing Hgf and concurrent increasing emphysema severity is intriguing. Furthermore, Hgf supplementation is sufficient to inhibit genetic emphysema [4,5,23]. While it is clear that Hgf has a role in emphysema, the mechanism of how it restores or preserves alveoli remains unexplained. I hypothesize that Hgf supplementation is sufficient to inhibit emphysema by inducing AEC2s to form clonal foci, undergo proliferation, and differentiate into AEC1s. Procedures: Mice harboring an AEC2 specific Cre and Rosa26R-Confetti reporter (SftpC-Cre ERT ; R26R-Confetti), but no other genetic alterations, will be subjected to the same protocol described in Aim 1.1. Over the final month of CS exposure, mice will be delivered a weekly dose of recombinant Hgf or a vehicle control (See figure, below). Lungs will be harvested and visualized using immunofluorescence. To test whether Hgf activates c-met and AEC2 proliferation, lungs will be co-stained for p-met, the confetti lineage marker, and the Ki67. To determine whether Hgf increases Experimental Design for Aim 1.2 activity of AEC2s following CS exposure, the number of clonal AEC2 foci and the number of lineage derived AEC1s will be quantified in all mice. Expected Results: If Hgf induces clonal proliferation and re-differentiation of AEC2s, I would expect to observe more proliferating AEC2s and an increased number of lineage-labeled AEC1s after Hgf treatment compared with vehicle treatmet. If Hgf does induce lung restoration, I will conclude that Hgf is sufficient to inhibit CS-induced emphysema by stimulating AEC2 mediated proliferation and differentiation. However, if Hgf does not induce AEC2 mediated restoration, I will stain for AEC2 markers, such as Lamp-1 or LysozymeM, in addition to the confetti reporter to determine whether these cells persist after CS-

16 Research Strategy Krah 16 induced injury. If AEC2s are preferentially killed by CS, I would propose to use an alternative model to test this hypothesis, such as genetic emphysema or hyperoxic injury. Pitfalls and Alternatives: While several studies suggest that AEC2s repopulate the lung during normal homeostasis and after injury, others suggest that α6β4 + progenitors repair alveoli following injury [9,14]. One alternative hypothesis is that Hgf acts on α6β4 + cells to induce alveolar regeneration after CS exposure. Importantly, α6β4 + acts as a c-met co-receptor, making these cells plausible candidates to respond to Hgf signaling [3,44]. If I fail to observe AEC2 clonal foci and lineage labeled AEC1s, but do observe restorative growth of the alveoli, these progenitors would be a candidate cell-type to characterize. This could be accomplished by co-staining for β4 integrin, p- Met, and Ki67, and determining whether these cells are 1) increased following injury, 2) have activated (phosphorylated) c-met, and 3) are localized near areas of injury. 1.3 Scgb1a1 + BASCs do not contribute to HGF/c-Met mediated alveolar repair following CS exposure and I will test this hypothesis using lineage tracing. Studies suggest that BASCs have the ability to self-renew and redifferentiate into multiple lineages in vitro [12]. These cells are characterized by the co-expression of the Clara cell marker, Scg1a1 and AEC2 marker, Sftp-C. Many studies predict that these cells are crucial during repair after injury [12,31]. Rawlins et. al. developed an inducible Scg1a1-Cre, which when utilized with a reporter construct, allows for visualization and lineage tracing of Clara cells and BASCs in vivo [11]. I propose to use this tool to test whether BASCs contribute to alveolar repair following chronic CS exposure. Based previous studies, I hypothesize that BASCs contribute little to alveolar restoration after CS-induced injury [1,2,11]. Procedures: In order to lineage label BASCs, CreER TM is expressed under the control of the endogenous Scgb1a1 locus and, in the presence of tamoxifen, can recombine a fluorescent label, such as the Rosa26R-eYFP reporter [11,41]. These mice, Scgb1a1-CreER TM ; Rosa26R-eYFP, will be exposed to CS or ambient air for two months and subsequently administered recombinant Hgf, as

17 Research Strategy Krah 17 described in Aim 1.2. Lungs will be harvested one month following termination of CS exposure and prepared for immunofluorescence (See Figure 1.3). In order to determine whether BASCs contribute to alveolar restoration I will perform immunofluorescence for eyfp, Sftp-C, Ki67, and DAPI. Expected Results: If BASCs do not contribute to alveolar Experimental Design Aim 1.3 regeneration following CS-induced injury I would expected to see similar number of eyfp/sftp-c + cells between treated and untreated groups, little Ki67 staining co-localized with eyfp, and few (if any) lineage labeled AEC1s. However, if abundant Ki67 staining co-localizes with eyfp/sftp-c + cells and/or there are abundant lineage-labeled AEC1s after Hgf treatment, I would conclude that BASCs contribute to alveolar restoration following CS induced emphysema in an Hgf dependent manner. Pitfalls and Alternatives: BASCs do not represent the only alternative progenitor cell population that could repopulate alveoli after CS-induced injury. As discussed in Aim 1.2, α6β4 + cells could respond to Hgf and clonally repopulate in vivo [9,14]. However, if neither of these populations contributed to re-growth, I propose to examine p63 +, Krt5 + distal airway stem cells (DASCs), which have been shown to form alveolar structures in vitro, and repopulate terminal bronchioles in vivo following infection with the H1N1 influenza virus [13]. If I observed negative results throughout this Aim, I would investigate the possibility that AEC1s undergo proliferation in order to maintain lung epithelium following CS-induced injury. Conclusions and Future Directions: Data collected in this Aim will provide insight into which progenitor cells Hgf/c-Met signaling acts on to contribute to the regeneration of lung epithelium following CS-induced injury. If c-met is necessary for AEC2 clonal expansion and redifferentiation to other alveolar cell subtypes during repair, it would be useful to know how these

18 Research Strategy Krah 18 cells are genetically reprogramed in response to c-met activation following injury. Future studies could use whole genome approaches to test what pathways are activated and suppressed by c-met signaling, specifically in AEC2s during and following CS-induced lung injury. These data would provide genetic insight into what regenerative pathways are suppressed in emphysema. AIM 2: Test whether TGF-β mediated Nk2 persistence promotes alveolar loss through a mir- 199a dependent mechanism. While canonical Hgf/c-Met signaling promotes proliferation and survival, the Nk2 splice variant of Hgf acts as a competitive antagonist of c-met signaling [26]. Studies demonstrate that Tgf-β signaling favors stability of the Nk2 transcript and the degradation of full-length Hgf in human lung fibroblasts [27,28]. Mechanistically, Tgf-β1 upregulates mir-199a, which targets full-length Hgf transcripts, while Nk2 transcripts are impervious to mir-199a mediated degradation [28]. Independent studies demonstrated that Tgf-β is greatly increased in mice exposed to CS and in COPD patient samples [22,28]. While recombinant Hgf inhibits emphysema [4], it is not clear what the role of Nk2 is in the pathophysiology of this disease. I propose that increased Tgf-β signaling during emphysema stabilizes Nk2 by upregulating mir-199a; Nk2 inhibits canonical c-met signaling, which could exacerbate the late stages of emphysema. I further hypothesize that emphysema can be attenuated by inhibiting mir-199a mediated Hgf degradation [28]. 2.1 Test whether Nk2 is increased relative to full-length Hgf after CS-induced emphysema in a TGF-β dependent manner Tgf-β is increased following CS-induced emphysema and Tgf-β has been shown to preferentially stabilize Nk2 in lung fibroblasts [22,27,28]. Nk2 acts as a competitive antagonist on c-met, decreasing its downstream mitogenic signaling [25]. Because c-met signaling is necessary to maintain alveoli and inhibit emphysema [4], I hypothesize that Nk2 is increased relative to Hgf following chronic CS-induced injury, and that Tgf-β is necessary for Nk2 perdurance. Procedures: To test whether Nk2 is preferentially stabilized in emphysema in a Tgf-β dependent manner, six week old AKR/J mice [45] will be divided into four cohorts. Mice will be exposed to

19 Research Strategy Krah 19 CS (2 cohorts) or left in ambient air (2 cohorts), and administered either losartan (to inhibit Tgf-β) or vehicle in their drinking water [1,22]. Mice in the CS cohort will be exposed to CS for 2 Experimental Design for Aim 2.1 months; lungs will be harvested and isolated for histology, mrna, and protein (See Figure 2.1). To determine the extent of Tgf-β signaling, I will measure levels of Tgf-β1 expression by ELISA and determine activation of the Tgf-β pathway by phospho-smad levels, using immunohistochemistry. To test whether Nk2 persists after emphysema I will perform qpcr on both Nk2 and full length Hgf; additionally, Nk2 and Hgf protein levels will be assessed via western blot, as previously described [28]. Expected Results: If Nk2 persists after chronic CS injury in a Tgf-β dependent manner, I would predict to observe the following: measurements from room air mice treated with vehicle and losartan will serve as baseline expression levels for Nk2, Hgf, and Tgf-β1 to which comparisons can be made. Mice exposed to CS and treated with vehicle should have elevated Tgf-β1 levels [22], decreased Hgf, and Nk2 levels comparable to (or above) control. Mice exposed to CS and treated with losartan, should express Tgf-β1, Hgf and Nk2 at levels comparable to control. Pitfalls and Alternatives: While losartan inhibits Tgf-β signaling [22,46], it is widely known as an angiotensin II type 1 receptor antagonist used to treat hypertension and diabetic nephropathy [29]. Off target effects could confound these data, but could be resolved by using a pan-selective Tgf-β antibody [22]. Additionally, increased Tgf-β signaling could decrease c-met levels in lung epithelium to drive emphysema independently of Hgf splice variants. If no changes are observed in Nk2 levels, I would test whether Tgf-β signaling directly represses Hgf expression as is seen in skin [48], or targets transcriptional repressors of c-met, such as Daxx [50].

20 Research Strategy Krah Sustained NK2 expression will prevent recovery from emphysema and I will test this model by exposing inducible Nk2 over-expressing mice to chronic CS-induced injury. Deletion of c-met from the lung epithelium is sufficient to cause emphysema [4]. Nk2 is an endogenous inhibitor of c-met that is derived from alternative splicing of the Hgf gene [25,26]. I therefore hypothesize that sustained of Nk2 following chronic CS exposure will decrease alveolar repair by inhibiting canonical Hgf/c-Met signaling. Procedures: Previous studies have utilized a mouse model of Experimental Design for Aim 2.2 sustained Nk2 expression where an Nk2 transgene is driven under an inducible, ubiquitous MT-1 promoter [51-53]. In order to test whether persistent Nk2 expression inhibits alveolar restoration following CS-induced injury, 6-week old mice will be subjected to CS for 2 months. Upon return to ambient air mice will be administered ZnSO 4 (25mM) in drinking water to drive Nk2 expression (See Figure, above). Lungs will be harvested one month after ZnSO 4 treatment is initiated. In order to test whether Nk2 perdurance inhibits alveolar restoration, the amount of alveolar epithelium, relative to control, will be quantified. In order to test whether Nk2 inhibits c- Met signaling in the lung I will perform immunohistochemistry for downstream targets of canonical c-met signaling, such as phosphorylated c-met, p-akt, and p-erk. Expected Results: I hypothesize that persistent Nk2 expression will inhibit alveolar re-growth, as shown by histological quantification of alveoli relative to control animals (those with no persistent Nk2 expression). I predict that Nk2 will inhibit c-met activation, as measured by decreased p-met, p-erk, and p-akt relative to animals without induced Nk2. Pitfalls and Alternatives: While no developmental phenotypes have been reported in these mice Nk2 expression has not been examined in the lung. If Nk2 expression is lower than expected, I propose to perform tracheal injections of purified Nk2 protein. If persistence of Nk2 is not sufficient to inhibit alveolar regeneration in emphysema, I propose to test whether sustained Nk2 exacerbate

21 Research Strategy Krah 21 other lung injury models, such as bleomycin induced pulmonary fibrosis. Such results would not be unprecedented, as Nk2 perdurance exacerbates chemically-induced liver injury [52]. 2.3 mir-199a will be upregulated following chronic exposure to CS and I will test this hypothesis using in situ hybridization. mir-199a degrades full-length Hgf transcripts, but it is unable to target Nk2 in pulmonary fibroblasts in vitro [28]. Furthermore, mir-199a is increased in response to Tgf-β signaling, which is increased in chronic CS exposure [22,54]. These data suggest that mir-199a inhibits canonical c- Met signaling by degrading transcripts of ligands that stimulate canonical c-met signaling, but not inhibitors. Since canonical c-met signaling is decreased in emphysema, I will test whether mir199a is upregulated following chronic CS exposure. Procedures: To test whether mir199a expression is upregulated, mice will be exposed to CS or ambient air for 2 months and lungs will be harvested and fixed for in situ staining (See Figure, below). In situ hybridization will be performed on formaldehyde-fixed, paraffin-embedded sections from both groups using mircury LNA microrna in situ optimization kits and the mir199a-3p biotin-conjugated detection probe [ Probes for U6 and scrambled sequence will be used as positive and negative controls, as previously described [55]. Experimental Design Aim 2.3 Expected Results: If mir-199a is upregulated in response to CS-induced injury, I would expect to see an increase in biotin labeling in alveoli (pulmonary fibroblasts, specifically) from mice exposed to CS, relative to those in ambient air. Pitfalls and Alternatives: Since mirnas are only nucleotides in length, the antisense probes are also small, which could lead to high background or non-specific staining. Programs, such as ImageJ or Photoshop will be utilized to detect signal vs. noise. The possibility also exists that mir- 199a will not be highly regulated in lung following CS exposure. In this case I would propose that

22 Research Strategy Krah 22 Hgf is regulated transcriptionally by other mechanisms, such as Smad-mediated suppression, or low PPARγ activation (see alternatives to Aim 2.4) [21,48]. 2.4 Mutation of the mir-199a target site on Hgf will preserve lung epithelium during CSinduced exposure and I will test this hypothesis using CRISPR/CAS. mir-199a targets Hgf for degradation, but Nk2 is impervious to mir-199a mediated degradation. I hypothesize that an imbalance in these transcripts contributes to decreased c-met signaling. If mir- 199a is indeed a crucial regulator of Hgf, then introducing mutations that inhibit mir-199a from targeting full-length Hgf transcripts should expedite recovery from CS-induced injury. Procedures: I propose to utilize CRISPR-CAS in order to mutate the 3 UTR of the Hgf gene at the proposed mir199a target cite [28]. Briefly, mouse zygotes will be microinjected with Cas9 and single guide RNA (sgrna) against the ACTACTG mir-199a target site and transferred to a mature female. Previous studies suggest that this method generates mice with biallelic mutations in target genes with an efficiency of ~80% [56,57]. Progeny and controls with WT Hgf will be subjected to CS for 2 months as described in Aim 1 and emphysema will be assessed/quantified via histology and by measuring lung volume [21]. Expected Results: If mir199a has a significant effect on the degradation of Hgf in lung fibroblasts in vivo, then I would expect to observe a more rapid recovery of mice with mutated Hgf, compared to animals with wild-type Hgf following chronic CS exposure. Pitfalls and Alternatives: Since the level of Hgf decreases as emphysema progresses, it might be necessary to decrease the length of CS exposure in these cohorts in order to observe an effect [4,5]. Aside from mir-199a, other pathways or transcription factors are likely to mediate Hgf expression levels in lung fibroblasts, such as PPARγ [21,24]. If Nk2 and Hgf are not regulated by mir199a or TGF-β in vivo, I propose to test whether PPARγ upregulates the canonical form of Hgf, but not the Nk2 variant. PPARγ is a likely regulator of Hgf in the lung, as two PPARγ binding cites reside in

23 Research Strategy Krah 23 the 5 flank of the Hgf gene, and activation of this transcription factor rescues emphysema in mice chronically exposed to CS [21,58,59]. Conclusions: Aim2 will test my hypothesis that the Hgf splice variant, Nk2, contributes to the pathogenesis of emphysema in a TGF-β and mir-199a dependent manner. Data generated here will demonstrate that Nk2 is a key regulator of c-met signaling and lung homeostasis in vivo. If mutation of the mir-199a target site on Hgf s 3 UTR expedites recovery from CS-induced injury, I would conclude that mir-199a has an important role in the pathophysiology of emphysema and represents a possible therapeutic target in this disease. Future studies will focus on how Nk2 perdurance alters downstream c-met signaling and epithelial cell function. AIM 3: Test whether c-met inhibits alveolar cell apoptosis following CS-induced injury by binding to Cd95. Apoptosis of the lung epithelium is the primary mechanism of airspace enlargement in emphysema [16,47]. While Hgf/c-Met signaling is classically a pro-survival pathway, c-met can regulate epithelial apoptosis in two distinct ways: first, an abundance of extracellular signals, such as FasL, can cause dissociation of the c-met/cd95 complex, which sensitizes epithelial cells to apoptosis [6]. Second, stress conditions mediated by Tnf-α are sufficient to cause cleavage of the c-met intracellular kinase domain. This 40kD fragment (p40-met) is sufficient to cause apoptosis in epithelial cells by localizing to the mitochondria and increasing membrane permeability [34,37]. I hypothesize that CS stimulates apoptosis of alveolar epithelial cells by dissociating c-met and Cd95. Stabilization of this protein-protein interaction could be a therapeutic target in emphysema and I will test that hypothesis in the final portion of this Aim. 3.1 Test whether CS extract disrupts the interaction between c-met and Cd95 in lung epithelial cells in vitro. Previous studies demonstrate that c-met physically interacts with the cell death receptor, CD95 [6]. This sequestration of Cd95 prevents Fas-mediated apoptosis of epithelial cells in vitro. However, certain stressors, such as increased levels of FasL, promote dissociation of c-met from Cd95, which

24 Research Strategy Krah 24 sensitizes cells to apoptosis. I hypothesize that CS extract will cause dissociation of the c-met/cd95 interaction, thus rendering cells susceptible to Fas-mediated apoptosis. Procedures: Lung epithelial (MLE12 and A549) cells will be treated with CS extract or vehicle for 72 hours after an overnight serum starvation. In order to test whether CS dissociates Cd95 from c- Met, cell membrane proteins will be cross-linked using DSS and subjected to immunoprecipitation followed by western blot probing to identify c-met/cd95 interactions. In order to test whether CS treated cells are more susceptible to apoptosis, cells will be treated with various concentrations of FasL (5-200ng/mL) and the amount of active Caspase-3 will be quantified by Western blot. Expected Results: I hypothesize that CS extract will increase the amount of isolated Cd95 and c- Met on the cell membrane, thus rendering cells more sensitive to apoptosis. In CS treated cells, I predict that there will be high levels of active Caspase-3, even at low concentrations of FasL, while control cells will activate Caspase-3 only at higher levels of Fas-L (>100ng/mL). Pitfalls and Alternatives: Because CS extract is a harsh (although widely utilized) treatment in cell culture [22], I may observe wide induction of apoptosis even in the absence of Cd95/c-Met dissociation. If this is observed, I propose to titrate the amount of CS extract to determine optimal dosing. Alternatively, cells could be treated with polycyclic aromatic hydrocarbons (PAHs), a carcinogenic component of cigarette smoke, which are sufficient to cause apoptosis of endothelial cells in vitro [51]. If CS-extract or PAHs cause wide-spread apoptosis independently of Cd95/c-Met dissociation, I will test whether p40-met is induced by CS exact and causing apoptosis via the mitochondrial intrinsic pathway [34]. This could be assayed by performing western blots for p40- Met and testing whether p40-met localizes to the mitochondria of these cells. 3.2 Chronic exposure to CS will increase the amount of unbound Cd95 in lung epithelial cells in vivo and I will test this hypothesis by assaying for c-met/cd95 interactions at different CS exposure lengths.

25 Research Strategy Krah 25 As described above, c-met interacts with Cd95 to inhibit apoptosis, however, this interaction is poorly characterized in the lung in vivo. As exposure to CS is prolonged and emphysema begins to develop, cytokines that are capable of inducing apoptosis, such as Tnf-α, become more prominent in the lung [39]. Such cytokines may be sufficient to induce c-met mediated apoptosis [6,38]. Since inflammation is increased in response to chronic CS exposure [19], I hypothesize that as emphysema progresses, there is decreased c-met/cd95 interaction. Procedures: Two cohorts of AKR/J mice will be generated and exposed to either ambient air or CS for 2 months. Lungs will be harvested for histology and protein at each of the following timepoints: 2 weeks, 1 month, and 2 months after CS induction (See Figure, below). To determine the amount of c-met/cd95 interaction, cross-linked cell lysates will be immunoprecipitation with a Fas antibody followed by Western blot Experimental Design Aim 3.2 probing with anti-met antibodies (and vice-versa). Expected Results: As temporal exposure to CS increases, I hypothesize that less Cd95 will be in a complex with c-met. This would be indicated by increased free Cd95 or c-met on western blot analysis and decreased levels of the 230kDa c-met/cd95 complex at the 1 month and 2 month time-points in CS treated animals, compared to ambient-air controls. Pitfalls and Alternatives: While the c-met/cd95 interaction represents one possible level of regulation, cell death could be promoted by other mechanisms. For example, c-met could mediate apoptosis through generation of p40-met as described in Aim 3.1 [34,37,38]. Additionally, the inflammation associated with emphysema could lead to apoptosis via the Perforin/GranzymeB pathway. To distinguish between these mechanisms, I propose to perform western blots for p40-met and immunohistochemistry for GranzymeB [60]. If p40-met is responsible for apoptosis, this protein fragment should be increased and localized to the mitochondria, while if the