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
Abstract
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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.
Introduction
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The Lung, COPD, and HGF/c-MET signaling1: 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
Introduction
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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,
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
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
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
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.
Introduction
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BASC
Sftp-C
Scgb1a1
AEC2
Self Renewal
Differentiation
Post-Injury
Sftp-C
AEC1
Proliferation
Self Renewal
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
RAS
PI3K
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.
Specific Aims
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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 cMet 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.
Specific Aims
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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 miR199a 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.
Specific Aims
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3.1 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.
Hgf
AIM 1
HGF
c-MET
Pulmonary
Fibroblast
AEC2
Proliferation/Differentiation
Hgf
NK2
Nk2
AIM 2
Apoptosis
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 cMet 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-CreERT;c-MetΔ/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-
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-METΔ/f mice. If cMet 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].
Research Strategy Krah 15 1.2 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-CreERT;
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-
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, pMet, 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, CreERTM 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-CreERTM; Rosa26R-eYFP, will
be exposed to CS or ambient air for two months and subsequently administered recombinant Hgf, as
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
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 miR199a 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
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].
Research Strategy Krah 20 2.2 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.
Experimental Design for Aim 2.2 Procedures: Previous studies have utilized a mouse model of
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 ZnSO4 (25mM) in drinking water to drive Nk2
expression (See Figure, above). Lungs will be harvested one month after ZnSO4 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 cMet 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
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 cMet 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 [http://www.exiqon.com/microrna-in-situ-Hybridization]. 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 21-24 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 miR199a will not be highly regulated in lung following CS exposure. In this case I would propose that
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 miR199a 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
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
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 cMet, 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 cMet 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 p40Met 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.
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
Experimental Design Aim 3.2
immunoprecipitation with a Fas antibody followed by Western blot
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
Research Strategy Krah 26 Perforin/GranzymeB pathway increases cell death, I would expect to observe an increase in
GranzymeB staining within alveoli.
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.
Constitutive expression of the c-Met extracellular domain protects hepatocytes from Cd95-mediated
apoptosis in vivo [6]. However, limited tools exist to study this interaction in the lung in vivo in a
translational way. Nanobodies are derived from the Camelid genus and are antibody fragments
consisting of a single monomeric variable antibody domain. Like conventional antibodies,
nanobodies bind selectively to a specific antigen, but have a molecular weight of only 12-15 kDa
[61]. Given their small size, DNA sequences, once obtained, can be modified to enhance their
binding affinity. Importantly, previous studies indicate that nebulized antibodies are effective
experimental therapeutics in the lung [62,63]. I will utilize this tool to test the hypothesis that
stabilized c-Met/Cd95 will prevent alveolar apoptosis during and following chronic CS-induced
injury.
Procedures: To generate an effective nanobody, the cMet/Cd95 dimer will be cross-linked and digested.
Nanobodies generated against the degraded protein
fragments will be isolated. Top candidate nanobodies will
be screened in a cell-free system to determine their ability
to stabilize the c-Met/Cd95 complex. To test whether this
Experimental Design Aim 3.3
nanobody decreases alveolar apoptosis during CSinduced emphysema, mice will be divided into cohorts and subjected to CS or ambient air for two
months, as described in Aim 1. When 1 month of CS treatment remains, one room air and one CS
cohort will receive nebulized nanobodies that stabilize c-Met/Cd95, while the other cohorts will be
Research Strategy Krah 27 nebulized with control IgG. Lungs will be harvested and prepared for histology and protein. In order
to test whether c-Met/Cd95 stabilization prevents apoptosis following induction of emphysema, the
same protocol will followed, however nanobody delivery will begin upon cessation of CS exposure
(See Figure below). To determine whether c-Met/Cd95 stabilization inhibits alveolar loss, the
amount of lung epithelium will be quantified via histology and the amount of cell death will be
assayed by performing western blots for active Caspase-3.
Expected Results: Because of the accessibility of the extracellular domains of both c-Met and
Cd95, I hypothesize that generating a nanobody to link these domains together is a realistic goal. If I
am successful at generating this nanobody, I predict that stabilization of the c-Met/Cd95 interaction
will inhibit apoptosis during and after CS-induced emphysema. Inhibition of apoptosis would be
indicated by an increase in lung epithelium and a decrease in active Caspase-3 as seen via Western.
Pitfalls and Alternatives: While there are potential pitfalls with large-scale screens, such as
developing a nanobody to the crosslinking agent, the appropriate controls will be put in place to
ensure quality of the interaction. In the case of cross-linker reactivity, the nanobody will be quality
controlled against the native protein in vitro. Alternatives will be necessary if c-Met interacting with
Cd95 is not the primary mechanism of apoptotic inhibition. For example, if inflammatory cells kill
alveolar epithelium via the Perforin/GranzymeB pathway, inhaled steroids might be a necessary
addition to the treatment in this Aim. It will be interesting to test whether stabilization of cMet/Cd95 synergizes with anti-inflammatory drugs (the current standard of care) to inhibit
progression of emphysema.
Conclusions: This Aim tests the mechanism by which c-Met inhibits apoptosis and whether this
mechanism affects alveolar survival in CS-induced emphysema. If CS does cause dissociation of cMet/Cd95 and stabilization of this complex is sufficient to reduce apoptosis, then I would conclude
that the c-Met/Cd95 interaction is central to the pathophysiology of emphysema. Importantly, data
Research Strategy Krah 28 generated here could have therapeutic potential, if, for example, it is possible to stabilize the cMet/Cd95 interaction using a nanobody. Future studies could test whether c-Met mediates other
apoptotic pathways, such Perforin/GranzymeB induced cell death via controlled expression of
cytokines or other pro/anti-inflammatory molecules. Given that inflammation is a hallmark of
emphysema, Perforin mediated apoptosis may be a key process in emphysema [47]. Future studies
could also test whether stabilization of c-Met/Cd95 synergizes with steroids or other antiinflammatory drugs to halt to progression of emphysema.
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