Advances in Bioscience and Biotechnology, 2012, 3, 657-664 ABB
http://dx.doi.org/10.4236/abb.2012.326085 Published Online October 2012 (http://www.SciRP.org/journal/abb/)
Increased survivin expression contributes to
apoptosis-resistance in IPF fibroblasts
Thomas H. Sisson1, Toby M. Maher2, Iyabode O. Ajayi1, Jessie E. King1, Peter D. R. Higgins3,
Adam J. Booth1, Rommel L. Sagana1, Steven K. Huang1, Eric S. White1, Bethany B. Moore1,
Jeffrey C. Horowitz1*
1Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan Medical Center, Ann Arbor,
USA
2Interstitial Lung Disease Unit, Royal Brompton Hospital, London, UK
3Division of Gastroenterology, Department of Medicine, University of Michigan Medical Center, Ann Arbor, USA
Received 30 July 2012; revised 6 September 2012; accepted 16 September 2012
ABSTRACT
Fibroblasts perform critical functions during the
normal host response to tissue injury, but the inap-
propriate accumulation and persistent activation of
these cells results in the development of tissue fibrosis.
The mechanisms accounting for the aberrant accu-
mulation of fibroblasts during fibrotic repair are
poorly understood, although evidence supports a role
for fibroblast resistance to apoptosis as a contributing
factor. We have shown that TGF-β1 and endothelin-1
(ET-1), soluble mediators implicated in fibrogenesis,
promote fibroblast resistance to apoptosis. Moreover,
we recently found that ET-1 induced apoptosis resis-
tance in normal lung fibroblasts through the upregu-
lation of survivin, a member of the Inhibitor of
Apoptosis (IAP) protein family. In the current study,
we sought to determine the role of survivin in the
apoptosis resistance of primary fibroblasts isolated
from the lungs of patients with Idiopathic Pulmonary
Fibrosis (IPF), a fibrotic lung disease of unclear eti-
ology for which there is no definitive therapy. First,
we examined survivin expression in lung tissue from
patients with IPF and found that there is robust ex-
pression in the fibroblasts residing within fibroblastic
foci (the “active” lesions in IPF which correlate with
mortality). Next, we show that survivin expression is
increased in fibroblasts isolated from IPF lung tissue
compared to cells from normal lung t issue. Co nsist ent
with a role in fibrogenesis, we demonstrate that TGF-
β1 increases survivin expression in normal lung fi-
broblasts. Finally, we show that inhibition of sur-
vivin enhances susceptibility of a subset of IPF fibro-
blasts to apoptosis. Collectively, these findings suggest
that increased survivin expression represents one
mechanism contributing to an apoptosis-resistant phe -
notype in IPF fibroblasts.
Keywords: Myofibroblast; Idiopathic Pulmonary
Fibrosis; Inhibitor of Apoptosis; Lung Fibrosis; Fas
1. INTRODUCTION
Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung
disease characterized by the progressive development of
scar tissue which can ultimately lead to respiratory fail-
ure and death. The incidence of IPF is increasing and,
with an average survival of 2 - 3 years following the dia-
gnosis, the overall prognosis is poor [1-3]. Although
several randomized clinical trials have evaluated a num-
ber of therapeutic targets in IPF over the last decade,
there continues to be a lack of clearly effective pharma-
cologic therapy [4]. Despite the generally poor prognosis,
a well-recognized clinical feature of IPF is the markedly
heterogeneous clinical course, with some patients main-
taining stable lung function for years while others ex-
perience a rapid deterioration culminating in respiratory
failure and death within months of the diagnosis [5]. It is
critical to better understand the mechanisms of biologic
heterogeneity that underlie the variable clinical course of
IPF in order to identify novel targets for intervention that
can be applied in a personalized manner [6].
Alveolar scarring in IPF is hypothesized to result from
dysfunctional wound repair which is characterized by the
accumulation of mesenchymal cells (including fibro-
blasts and their differentiated phenotype, myofibroblasts)
within pathologic lesions called fibroblastic foci [7,8].
During normal reparative responses following injury,
mesenchymal cells perform critical functions which pro-
vide healing wounds with tensile strength and facilitate
the homeostatic restoration of epithelial integrity, in-
cluding the synthesis, secretion, organization and con-
*Corresponding author.

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 658
traction of new extracellular matrix (ECM), [1,9-11].
Resolution of the normal repair process requires clear-
ance of fibroblasts via apoptosis [12,13]. However, in the
dysfunctional repair that is evident in IPF and other dis-
eases characterized by fibrosis, the persistence of repara-
tive fibroblasts leads to excessive deposition and organi-
zation of ECM [14]. Several recent studies demonstrate a
paucity of apoptosis within the mesenchymal cells that
comprise the fibroblastic foci in IPF, highlighting one
mechanism that contributes to the persistence of these
cells [15-18]. Accumulating studies also show that fi-
brotic lung fibroblasts are resistant to a variety of apop-
totic stimuli, further supporting a role for impaired fibro-
blast apoptosis in the pathobiology of IPF [18-25]. How-
ever, the mechanisms regulating resistance to apoptosis
in lung fibroblasts have not been fully elucidated [14].
Recent studies suggest that Inhibitor of Apoptosis
(IAP) family proteins can regulate susceptibility to apop-
tosis in normal lung fibroblasts [18,20,24]. The IAP
genes encode at least eight different proteins with diverse
functions, including the direct and indirect regulation of
apoptosis. The protein family is characterized by the
presence of at least one baculoviral IAP repeat (BIR)
domain which is critical for the ability of these proteins
to inhibit caspase activation in vitro [26,27]. The small-
est member of the IAP family, survivin (also known as
BIRC5) is a 16.3 kD protein with one BIR domain that is
predominantly expressed as a homodimer. Physiologi-
cally, survivin serves a critical function in cell-cycle
progression and is essential during embryonic develop-
ment. In adult cells, however, it is typically expressed
only in actively dividing cells [28]. As with other IAP
family members, survivin inhibits activation of caspase 3,
7 and 9 in-vitro. Moreover, a number of studies demon-
strate regulation of cell susceptibility to apoptosis by
survivin, although the precise in vivo mechanisms re-
main unclear [27]. In accord with its crucial function in
cell division and apoptosis regulation, inappropriate sur-
vivin expression is associated with cancers involving
multiple organs including lung, colon, breast, brain and
skin [27,29,30]. We have recently shown that induction
of survivin by the soluble fibrogenic mediator endo-
thelin-1 (ET-1) reduces the apoptotic susceptibility of
normal fetal lung fibroblasts and that the anti-fibrotic
mediator prostaglandin E2 enhances fibroblast suscepti-
bility to apoptosis while suppressing survivin expression
[20,24]. The goal of this study was to examine the ex-
pression of survivin in IPF lung fibroblasts and to deter-
mine if survivin regulates IPF fibroblast susceptibility to
Fas-mediated apoptosis.
2. MATERIALS AND METHODS
2.1. Cells and Cell Culture
Normal human adult lung fibroblasts (CCL-210) were
obtained from ATCC (Manassas, VA). Cells between
passages 8 and 16 were cultured as previously described
and growth arrested for 24 hours in serum-free media
prior to experiments [20]. Patient-derived primary human
lung fibroblasts were cultured from the lungs of patients
with IPF or from normal lung tissue from patients un-
dergoing thoracic surgery for non-fibrotic disease as we
have described previously [31]. Written informed con-
sent was obtained from all subjects in accordance with
the University of Michigan Institutional Review Board.
2.2. Reagents
Porcine TGF-β1 was from R&D Systems (Minneapolis,
MN). The activating anti-Fas antibody (clone CH11) was
from Millipore (Billerica, MA). The mouse monoclonal
antibody to survivin used for Western blotting was from
Abnova. Antibodies to glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) were from Cell Signaling (Dan-
vers, MA). The Cell Death Detection ELISA Kit detect-
ing histone-associated DNA fragments was from Roche
Applied Science (Indianapolis, IN). Horseradish peroxi-
dase conjugated secondary antibodies were from Pierce
(Rockford, IL). The survivin inhibitor CAY10625, which
disrupts the interaction between survivin and Smac/
Diablo (second mitochondrial activator of caspases/direct
IAP binding protein with low isoelectric point), thereby
allowing Smac/Diablo to carry out its pro-apoptotic func-
tion, was from Cayman Chemical Company (Ann Arbor,
MI) [32]. YM155, a functionally distinct inhibitor which
inhibits survivin transcription [33], was from Selleck
Chemicals (Houston, TX).
2.3. Immunohistochemistry
4 µm sections on polylysiene coated slides (VWR,
Leicestershire, UK) were de-waxed and rehydrated by
immersion in xylene followed by decreasing concentra-
tions of ethanol. Sections were washed with tris-buffered
saline (TBS, pH 7.6), and endogenous peroxidise activity
was blocked by immersion in 3% H2O2 (v/v) for 30 min-
utes at room temperature. Sections were washed twice in
TBS and treated with a 1 in 6 solution of goat serum
containing 4 drops/ml of Avidin block (Vector Laborato-
ries, Peterborough, UK) for 20 minutes and incubated
overnight with rabbit anti-survivin polyclonal antibodies
(4 µg/ml; Santa Cruz Biotechnology) or an isotype con-
trol in TBS with 1% bovine serum albumin and 4 drops/
ml of Biotin block. Following another wash, samples
were treated with a 1:200 dilution of biotinylated second-
dary antibody (Dako, Cambridshire, UK), washed again
and immersed in a 1:200 dilution of Streptavadin HRP
(Dako) for 30 minutes at room temperature. After wash-
ing, DAB peroxidise substrate (Vector Laboratories) was
added for ten minutes. Samples were rinsed in H2O,

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 659
counterstained with Gill’s Haematoxylin, dehydrated in
Xylene and mounted using a Sakura Coveraid system.
2.4. Quantitative Reverse Transcription
Real-Time PCR
Quantitative reverse transcription real time-PCR was
performed on an Applied Biosystems (Foster City, CA)
7300 real time PCR machine as we have reported previ-
ously [31]. Relative quantitation was based on the ΔΔCT
method and used predesigned primer-probe sets obtained
from Applied Biosystems TaqMan Gene Expression As-
says for human Survivin (BIRC5; product number
Hs00153353_m1).
2.5. SDS Page Electrophoresis and Western
Blotting
Whole cell lysates were collected and subjected to SDS-
PAGE electrophoresis and Western blotting as previ-
ously described [20]. All Western blots were stripped
and re-probed for GAPDH.
2.6. Densitometry
Western blots were analyzed using the public domain
NIH ImageJ program available at http://rsbweb.nih.gov/ij/
as previously described [20]. The relative expression of
survivin was determined by dividing the survivin band
density (adjusted for the background) by the density of
the corresponding GAPDH. This ratio of survivin to
GAPDH was then normalized by dividing these values
by the average ratio in the “normal” fibroblasts, such that
the average of all normal (or untreated) lung fibroblasts
is equal to 1.0 and the IPF samples (or the TGF-β1
treated samples) represent the fold change.
2.7. Apoptosis
Apoptosis was assessed by ELISA-based detection of
histone-associated DNA fragments using the Cell Death
Detection ELISA Kit (Roche Applied Science, Indian-
apolis, IN) as previously described [20]. To allow com-
parisons across experiments using different IPF fibro-
blast cell lines, the apoptosis data is represented as the
percentage of the absorbance of the positive control
sample (provided by the manufacturer) that was run on
each ELISA plate. Each IPF sample was run in triplicate.
2.8. Statistical Analyses
Statistical analyses were done using ANOVA with Tukey
post-test for experiments involving multiple comparisons
(apoptosis assays). An unpaired T-test with Welch’s cor-
rection was used for comparisons between normal and
IPF samples (Graphpad Prism software version 5.01 for
Windows). The relationship between apoptosis induced
by Fas-activating ligand combined with YM155 or
CAY10625 was determined using a Spearman correlation
(r).
3. RESULTS
3.1. Survivin Is Expressed in IPF Lung Tissue
To determine if survivin was increased in fibroblasts
residing in areas of active fibrosis, we used immunohis-
tochemistry to examine survivin expression in normal
and IPF lung biopsies (Figure 1). In normal lung tissue,
survivin staining was observed in subsets of alveolar
epithelial cells (Figure 1(a), arrows) and in the normal
bronchial epithelium (data not shown). In IPF tissue,
survivin expression was identified in interstitial cells
within thickened alveolar septae (Figure 1(b), arrowhead)
and in the alveolar epithelium (Figure 1(b), arrow).
Marked survivin expression was also observed in mes-
enchymal cells residing within the IPF fibroblastic foci
and in the epithelium overlying the fibroblastic foci
(Figure 1(c)).
3.2. Survivin Expression Is Increased in IPF
Lung Fibroblasts
Having shown expression of survivin within fibroblastic
foci of IPF tissue, we next sought to compare survivin
expression in primary lung fibroblasts from patients with
normal lung architecture to fibroblasts isolated from IPF
lung tissue. Using quantitative real-time PCR of banked
RNA from 12 patient-derived normal lung fibroblast cell
lines and 21 IPF fibroblast cell lines, we found a signifi-
cant (2-fold) increase in survivin expression in the IPF
fibroblasts (Figure 2). While the overall difference was
statistically significant, the distribution of survivin ex-
pression in IPF fibroblasts was heterogeneous and ap-
peared to segregate into two distinct groups, with ap-
proximately half (12/21) of the IPF cell lines showing
survivin expression that was no different than the aver-
age expression seen in the normal lung fibroblasts (Fig-
ure 2, white diamonds). In contrast, 9/21 IPF fibroblast
cell lines had a more pronounced (average 3.5-fold) in-
crease in survivin expression compared to the normal
lung fibroblasts (Figure 2, black diamonds).
We next assessed the relative survivin protein levels in
12 IPF fibroblast cell lines compared to 5 normal lung
fibroblast cell lines (Figure 3). Consistent with the RNA
findings, survivin expression was significantly (approxi-
mately 2.5-fold) higher in the IPF fibroblasts. Survivin
protein expression in IPF fibroblasts was also heteroge-
neous, with some cell lines indistinguishable from the
average expression in normal lung fibroblasts (white dia-
monds), and others demonstrating more than a 3-fold

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 660
(a) (b)
(c) (d)
Figure 1. Biopsies from architecturally normal (a) and IPF
(b)-(d) lung were immunohistochemically stained with an an-
tibody to survivin (a)-(c) or with an isotype control antibody
(d). (a) Architecturally normal lung tissue from a patient with-
out pulmonary fibrosis shows heterogeneous expression of
survivin in alveolar epithelial cells (arrows); (b) An area of
alveolar septal thickening in a patient with IPF shows survivin
expression in alveolar epithelial cells (arrow) and in cells em-
bedded within the thickened interstitium (arrowhead); (c) A
fibroblastic focus from a patient with IPF shows survivin ex-
pression in the mesenchymal cells within a fibroblastic focus
and in the alveolar epithelial cells overlying the fibroblastic
focus; (d) Isotype control staining of IPF lung tissue.
Normal IPF
Figure 2. Quantitative reverse transcription
real-time PCR for survivin expression in
RNA isolated from explanted lung fi- bro-
blasts derived from 12 normal and 21 IPF
lungs. Expression levels in the IPF fibro-
blasts appeared to segregate into a cluster
that was indistinguishable from the normal
lung fibroblasts (white diamonds) and group
that had markedly increased levels of sur-
vivin mRNA (black diamonds) p = 0.05.
(a)
Normal IPF
(b)
Figure 3. (a) Cell lysates were obtained from normal and IPF
lung fibroblasts and survivin expression assessed by Western
blotting. The representative Western blot shows two different
normal lung fibroblast cell lines (N) and five different IPF lung
fibroblast cell lines (F). The blots were stripped and re-probed
for GAPDH; (b) Relative survivin expression in the IPF fibro-
blasts in comparison to the normal lung fibroblasts was deter-
mined by densitometric analysis of indexed to the ratio of sur-
vivin to GAPDH in normal lung fibroblasts. Survivin expres-
sion in the IPF fibroblasts segregated in to low (white dia-
monds), and high (black diamonds) levels relative to the nor-
mal lung fibroblasts. p = 0.004.
increase in survivin expression (black diamonds).
3.3. TGF-β1 Increases Survivin Expression in
Normal Fibroblasts
TGF-β1 is strongly implicated in the pathogenesis of
fibrosis and has been shown to promote myofibroblast
differentiation, ECM deposition, and the induction of an
apoptosis-resistant phenotype [22,23,34]. To determine if
TGF-β1 enhanced survivin expression in normal lung
fibroblasts, we treated CCL-210 (normal adult lung) fi-
broblasts with TGF-β1 and assessed survivin expression
by Western blotting. Consistent with the induction of an
apoptosis-resistant phenotype in normal fibroblasts, TGF-
β1 induced a robust increase in survivin expression within
six hours of treatment (Figure 4). A similar response to
TGF-β1 was observed in IMR-90 fibroblasts (data not
shown). In combination with our prior study showing
induction of survivin by ET-1, these findings suggest that
survivin induction represents a conserved response of

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 661
(a)
TGF-β1 (2 ng/ml)
(b)
Figure 4. Normal adult lung fibroblasts (CCL-210)
were cultured to 60% confluence in DMEM supple-
mented with 5% fetal bovine serum and growth ar-
rested in serum-free DMEM for 24 hours prior to
treatment with TGF-β1 (2 ng/ml) for 6 hours. (a) Sur-
vivin expression was assessed by Western blotting.
The blot was stripped and re-probed for GAPDH,
and is representative of three independent experi-
ments; (b) Densitometric analysis of three independ-
ent replicates of this experiment, indexed to the ratio
of survivin to GAPDH in untreated CCL-210 cells.
normal lung fibroblasts to soluble pro-fibrotic mediators
[20].
3.4. Survivin Inhibition Enhances IPF Fibroblast
Susceptibility to Apoptosis
In normal lung fibroblasts, enhanced survivin expression
by ET-1 is associated with decreased susceptibility to
apoptosis [20]. In contrast, the suppression of survivin in
response to treatment with PGE2 is associated with en-
hanced susceptibility to apoptosis [20,24]. Finding that
survivin is expressed within the fibroblastic foci in IPF
tissue, that IPF fibroblasts demonstrate increased levels
of survivin, and that TGF-β1 induced survivin expression
in normal lung fibroblasts, we sought to determine if
survivin inhibition could modulate susceptibility to
apoptosis in IPF fibroblasts. Eight patient derived IPF
fibroblast cell lines were treated with/without Fas-acti-
vating antibody in the presence/absence of two mecha-
nistically distinct inhibitors of survivin (CAY10625 or
YM155) and apoptosis was assessed at 16 hours (Figure
5). Consistent with prior reports, treatment with Fas-
activating ligand alone did not significantly increase IPF
lung fibroblast apoptosis [18,34,35]. However, treatment
with the Fas-activating antibodies in the presence of ei-
ther survivin inhibitor was associated with a statistically
significant increase in IPF fibroblast apoptosis. As with
(a)
(b)
Figure 5. IPF fibroblasts from 8 different patients were cul-
tured to 60% confluence in DMEM supplemented with 10%
fetal bovine serum and growth-arrested for 24 hours in serum-
free DMEM prior to treatment for 16 hours with/without the
Fas-activating antibody CH11 (FasL; 250 ng/ml) in the pres-
ence/absence of the survivin inhibitors CAY10625 (5 µM) or
YM155 (10.0 µM), or with the survivin inhibitors alone. (a)
Apoptosis was assessed using ELISA-based detection of his-
tone associated DNA fragments. To allow relative comparisons
of apoptosis across experiments, the data for each cell line is
expressed as the percentage of the assay positive control that
was included on each ELISA plate. The scatter plot shows the
distribution of responses in individual cell lines along with the
mean ± standard error for each treatment group. p = 0.038
overall (ANOVA). p = 0.027 for FasL compared to FasL/
CAY10625, and p = 0.046 for FasL compared to FasL/YM155;
(b) The correlation of apoptosis between IPF fibroblasts treated
with FasL/CAY10625 and FasL/YM155. Each point represents
a different IPF cell line. The Spearman correlation (r) = 0.95.
survivin expression levels, the IPF fibroblast cell lines
demonstrated heterogeneity in their apoptotic responses
to Fas-activation in the presence of the survivin inhibit-
tors, with enhanced apoptosis observed in only half of
the IPF fibroblast cell lines. Neither inhibitor signifi-
cantly increased apoptosis in the absence of Fas-activate-

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 662
ing antibodies. Finally, there was strong correlation be-
tween apoptosis induced by Fas-activation with YM155
and Fas-activation with CAY1062 (Figure 5(b)), sug-
gesting that the increased susceptibility to apoptosis was
specifically related to survivin inhibition and not the re-
sult of an off-target effect of the inhibitors used.
4. DISCUSSION
In the current study, we examined the role of survivin in
the regulation of IPF lung fibroblast apoptosis. We show
that survivin expression is increased in the mesenchymal
cells within the fibroblastic foci of IPF lungs, supporting
a role for this IAP in the apoptosis-resistant phenotype of
these cells. Further, we demonstrate increased survivin
expression in IPF lung fibroblasts compared to normal
lung fibroblasts, although the expression of survivin in
these IPF fibroblasts is heterogeneous. Additionally, we
show that TGF-β1, like ET-1, induces survivin expres-
sion in normal lung fibroblasts [20]. Finally, we demon-
strate that pharmacologic inhibition of survivin enhances
the apoptotic susceptibility of a subset of IPF lung fibro-
blasts. Collectively, these findings indicate that increased
survivin expression represents one mechanism by which
IPF fibroblasts acquire resistance to apoptosis.
IPF lung tissue is characterized by the heterogeneous
distribution of fibroblastic foci composed of fibroblasts
and myofibroblasts which, in contrast to the overlying
epithelium, lack evidence of active apoptosis [2,14,15,
17,18]. The mechanisms leading to the lack of apoptosis
in IPF fibroblasts have not been fully elucidated. Given
the heterogeneity seen in IPF, it is possible that different
stimuli converge on overlapping signaling pathways and
effector mechanisms to promote pro-fibrotic fibroblast
phenotypes in different patients. Supporting this concept
of mechanistic heterogeneity with phenotypic conver-
gence in complex disease states such as fibrosis, we have
shown that TGF-β1 and ET-1, two soluble mediators
implicated in fibrosis in-vivo, independently promote
fibroblast resistance to apoptosis through activation of
the pro-survival tyrosine kinases, focal adhesion kinase
(FAK) and phosphatidylinositol 3-hydroxy kinase/AKT
[23,34].
We recently demonstrated a mechanistic role for sur-
vivin in the regulation of normal lung fibroblast apop-
tosis, showing that FAK and PI3K/AKT activation are
required for the upregulation of survivin by ET-1 and
that survivin inhibition attenuates ET-1 mediated resis-
tance to apoptosis in these cells [20]. In this study, we
extend these findings into primary fibrotic lung fibro-
blasts, showing that a significant subset of IPF fibroblast
cell lines have markedly increased levels of survivin.
Moreover, while all of the IPF lung fibroblast cell lines
studied were resistant to Fas-mediated apoptosis, sur-
vivin inhibition enhanced apoptosis in only one-half of
the cell lines. The implication of this observation is that
alternative mechanisms that are not dependent on sur-
vivin mediate apoptosis resistance in the other half of the
IPF fibroblast cell lines. Such segregation of IPF fibro-
blasts into similar apoptosis-resistant phenotypes via he-
terogeneous mechanisms is reminiscent of a prior study
demonstrating that diminished expression of the EP2
receptor accounted for the lack of IPF fibroblast respon-
siveness to the anti-fibrotic mediator PGE2 in 4/9 cell
lines while the remaining IPF fibroblasts had decreased
responsiveness to PGE2 despite normal levels of the EP2
receptor [36]. Interestingly, we have also shown that
PGE2 suppresses survivin expression in normal lung fi-
broblasts, suggesting a potential mechanistic link be-
tween PGE2 resistance and increased survivin expression
in the IPF fibroblasts.
Although the mechanisms by which survivin regulates
cell apoptosis are controversial, there is abundant evi-
dence demonstrating that survivin inhibition can enhance
the susceptibility of cancer cells to apoptotic stimuli,
making survivin an attractive target for anti-cancer treat-
ments [27,30,33,37-39]. We employed two mechanisti-
cally distinct inhibitors to assess the role of survivin in
IPF fibroblast resistance to Fas-mediated apoptosis [32,
33,40]. Similar to the distribution of survivin expression
in IPF fibroblasts, inhibition of survivin significantly
enhanced Fas-mediated apoptosis in approximately half
of the IPF fibroblasts studied. While all pharmacologic
inhibitors can have off target effects, we found a high
correlation between the effects of YM155 and CAY10625
on cell susceptibility to apoptosis (r = 0.95, p = 0.002).
This high correlation between the effects of two mecha-
nistically distinct inhibitors, combined with the lack of
any significant effect on apoptosis when cells were
treated with the inhibitors alone, suggests that the en-
hanced apoptosis was specific to the inhibition of sur-
vivin and did not represent an off-target effect of either
inhibitor.
Studies exploring a relationship between survivin and
fibrosis are lacking. Our previous in vitro studies support
a link between survivin and a pro-fibrotic fibroblast
phenotype [20,36]. Additionally, several studies have
linked survivin expression with PI3K/AKT activation,
findings consistent with the established roles of PI3K/
AKT signaling in pulmonary fibrosis [18,41-44]. In vivo,
only one prior study has shown a link between survivin
and fibrosis [45]. In that study, which employed a murine
model of reversible liver fibrosis, survivin expression
was significantly increased during the establishment of
fibrosis. Moreover, the resolution phase of this fibrosis
model, which is dependent on hepatic stellate cell (myo-
fibroblast) apoptosis, coincided with the reduction of
survivin expression to pre-fibrosis levels. However, a

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 663
mechanistic link between survivin and fibrosis was not
examined, and no reports have shown that survivin is
required for the establishment of fibrosis or that suppres-
sion of survivin is necessary for the reversal of fibrosis.
Together with the current demonstration that inhibition
of survivin can restore susceptibility of IPF fibroblasts to
Fas-mediated apoptosis, these findings suggest that sur-
vivin may represent a potential target for anti-fibrotic
therapies.
In conclusion, this study is the first to show that sur-
vivin is expressed in the fibroblastic foci of IPF tissue.
Moreover, it is the first study to examine survivin ex-
pression in IPF fibroblasts and to assess the impact of
survivin inhibition on fibroblast susceptibility to apop-
tosis. Our findings demonstrate that survivin expression
is increased in a subset of IPF fibroblasts and that it is
possible to enhance the susceptibility of these cells to
Fas-mediated apoptosis by inhibiting survivin. The het-
erogeneity of survivin expression in IPF fibroblasts, and
the heterogeneous responses to apoptotic stimuli seen in
the presence of survivin inhibitors, may be a reflection of
the biologic and clinical heterogeneity observed in pa-
tients with IPF. Further studies are required to elucidate
the mechanisms by which survivin regulates apoptosis
susceptibility in these cells, and to determine if survivin
inhibition can modulate fibrosis in vivo.
5. ACKNOWLEDGEMENTS
Funding for these studies was provided by NIH/NHLBI HL105489
(JCH), HL R01085083 (ESW), and HL078871 (THS).
REFERENCES
[1] Collard, H.R., et al. (2012) Burden of illness in idiopathic
pulmonary fibrosis. Journal of Medical Economics, 15,
829-835. doi:10.3111/13696998.2012.680553
[2] Raghu, G., et al. (2011) An official ATS/ERS/JRS/ALAT
statement: Idiopathic pulmonary fibrosis: Evidence-based
guidelines for diagnosis and management. American
Journal of Respiratory and Critical Care Medicine, 183,
788-824. doi:10.1164/rccm.2009-040GL
[3] Navaratnam, V., et al. (2011) The rising incidence of
idiopathic pulmonary fibrosis in the UK. Thorax, 66,
462-467. doi:10.1136/thx.2010.148031
[4] Maher, T.M. (2012) Idiopathic pulmonary fibrosis: Pa-
thobiology of novel approaches to treatment. Clinics in
Chest Medicine, 33, 69-83.
doi:10.1016/j.ccm.2011.11.002
[5] Ley, B., Collard, H.R. and King Jr., T.E. (2011) Clinical
course and prediction of survival in idiopathic pulmonary
fibrosis. American Journal of Respiratory and Critical
Care Medicine, 183, 431-440.
doi:10.1164/rccm.201006-0894CI
[6] Ding, Q., et al. (2011) New insights into the pathogenesis
and treatment of idiopathic pulmonary fibrosis. Drugs, 71,
981-1001. doi:10.2165/11591490-000000000-00000
[7] King Jr., T.E., Pardo, A. and Selman, M. (2011) Idio-
pathic pulmonary fibrosis. Lancet, 378, 1949-1961.
doi:10.1016/S0140-6736(11)60052-4
[8] Horowitz, J.C. and Thannickal, V.J. (2006) Idiopathic
pulmonary fibrosis: New concepts in pathogenesis and
implications for drug therapy. Treatments in Respiratory
Medicine, 5, 325-342.
doi:10.2165/00151829-200605050-00004
[9] Desmouliere, A., Chaponnier, C. and Gabbiani, G. (2005)
Tissue repair, contraction, and the myofibroblast. Wound
Repair and Regeneration, 13, 7-12.
doi:10.1111/j.1067-1927.2005.130102.x
[10] Hinz, B., et al. (2012) Recent developments in myofibro-
blast biology: Paradigms for connective tissue remodel-
ing. American Journal of Pathology, 180, 1340-1355.
doi:10.1016/j.ajpath.2012.02.004
[11] Laurent, G.J., et al. (2008) Escape from the matrix: Mul-
tiple mechanisms for fibroblast activation in pulmonary
fibrosis. Proceedings of the American Thoracic Society, 5,
311-315. doi:10.1513/pats.200710-159DR
[12] Desmouliere, A., et al. (1995) Apoptosis mediates the
decrease in cellularity during the transition between
granulation tissue and scar. American Journal of Pathol-
ogy, 146, 56-66.
[13] Tomasek, J.J., et al. (2002) Myofibroblasts and mech-
ano-regulation of connective tissue remodelling. Nature
Reviews Molecular Cell Biolog, 3, 349-363.
doi:10.1038/nrm809
[14] Thannickal, V.J. and Horowitz, J.C. (2006) Evolving
concepts of apoptosis in idiopathic pulmonary fibrosis.
Proceedings of the American Thoracic Society, 3, 350-
356. doi:10.1513/pats.200601-001TK
[15] Cha, S.I., et al. (2010) Compartmentalized expression of
c-FLIP in lung tissues of patients with idiopathic pulmo-
nary fibrosis. American Journal of Respiratory Cell and
Molecular Biology, 42, 140-148.
doi:10.1165/rcmb.2008-0419OC
[16] Lepparanta, O., et al. (2010) Transcription factor GATA-
6 is expressed in quiescent myofibroblasts in idiopathic
pulmonary fibrosis. American Journal of Respiratory Cell
and Molecular Biology, 42, 626-632.
doi:10.1165/rcmb.2009-0021OC
[17] Korfei, M., et al. (2008) Epithelial endoplasmic reticulum
stress and apoptosis in sporadic idiopathic pulmonary fi-
brosis. American Journal of Respiratory and Critical
Care Medicine, 178, 838-846.
doi:10.1164/rccm.200802-313OC
[18] Maher, T.M., et al. (2010) Diminished prostaglandin E2
contributes to the apoptosis paradox in idiopathic pulmo-
nary fibrosis. American Journal of Respiratory and Cri-
tical Care Medicine, 182, 73-82.
doi:10.1164/rccm.200905-0674OC
[19] Buhling, F., et al. (2005) Altered expression of mem-
brane-bound and soluble CD95/Fas contributes to the re-
sistance of fibrotic lung fibroblasts to FasL induced
apoptosis. Respiratory Research, 6, 37.
doi:10.1186/1465-9921-6-37

T. H. Sisson et al. / Advances in Bioscience and Biotechnology 3 (2012) 657-664 664
[20] Horowitz, J.C., et al. (2012) Survivin expression induced
by endothelin-1 promotes myofibroblast resistance to
apoptosis. International Journal of Biochemistry & Cell
Biology, 44, 158-169. doi:10.1016/j.biocel.2011.10.011
[21] Horowitz, J.C., et al. (2006) Constitutive activation of
prosurvival signaling in alveolar mesenchymal cells iso-
lated from patients with nonresolving acute respiratory
distress syndrome. American Journal of Physiology—
Lung Cellular and Molecular Physiology, 290, L415-
L425. doi:10.1152/ajplung.00276.2005
[22] Horowitz, J.C., et al. (2004) Activation of the pro-sur-
vival phosphatidylinositol 3-kinase/AKT pathway by
transforming growth factor-beta1 in mesenchymal cells is
mediated by p38 MAPK-dependent induction of an
autocrine growth factor. Journal of Biological Chemistry,
279, 1359-1367. doi:10.1074/jbc.M306248200
[23] Horowitz, J.C., et al. (2007) Combinatorial activation of
FAK and AKT by transforming growth factor-beta1 con-
fers an anoikis-resistant phenotype to myofibroblasts.
Cell Signal, 19, 761-771.
doi:10.1016/j.cellsig.2006.10.001
[24] Huang, S.K., et al. (2009) Prostaglandin E(2) induces
fibroblast apoptosis by modulating multiple survival
pathways. FASEB Journal, 23, 4317-4326.
doi:10.1096/fj.08-128801
[25] Xia, H., et al. (2004) Focal adhesion kinase is upstream
of phosphatidylinositol 3-kinase/Akt in regulating fibro-
blast survival in response to contraction of type I collagen
matrices via a beta 1 integrin viability signaling pathway.
Journal of Biological Chemistry, 279, 33024-33034.
doi:10.1074/jbc.M313265200
[26] Rumble, J.M. and Duckett, C.S. (2008) Diverse functions
within the IAP family. Journal of Cell Science, 121,
3505-3507. doi:10.1242/jcs.040303
[27] Sah, N.K., et al. (2006) Structural, functional and thera-
peutic biology of survivin. Cancer Letters, 244, 164-171.
doi:10.1016/j.canlet.2006.03.007
[28] Altieri, D.C. (2010) Survivin and IAP proteins in cell-
death mechanisms. Biochemical Journal, 430, 199-205.
doi:10.1042/BJ20100814
[29] Guha, M. and Altieri, D.C. (2009) Survivin as a global
target of intrinsic tumor suppression networks. Cell Cycle,
8, 2708-2710. doi:10.4161/cc.8.17.9457
[30] Altieri, D.C. (2008) New wirings in the survivin networks.
Oncogene, 27, 6276-6284. doi:10.1038/onc.2008.303
[31] White, E.S., et al. (2003) Integrin alpha4beta1 regulates
migration across basement membranes by lung fibro-
blasts: A role for phosphatase and tensin homologue de-
leted on chromosome 10. American Journal of Respira-
tory and Critical Care Medicine, 168, 436-442.
doi:10.1164/rccm.200301-041OC
[32] Oikawa, T., et al. (2010) Identification of a small-mole-
cule inhibitor of the interaction between Survivin and
Smac/DIABLO. Biochemical and Biophysical Research
Communications, 393, 253-258.
doi:10.1016/j.bbrc.2010.01.113
[33] Ryan, B.M., O’Donovan, N. and Duffy, M.J. (2009) Sur-
vivin: A new target for anti-cancer therapy. Cancer
Treatment Reviews, 35, 553-562.
doi:10.1016/j.ctrv.2009.05.003
[34] Kulasekaran, P., et al. (2009) Endothelin-1 and trans-
forming growth factor-beta1 independently induce fibro-
blast resistance to apoptosis via AKT activation. Ameri-
can Journal of Respiratory Cell and Molecular Biology,
41, 484-493. doi:10.1165/rcmb.2008-0447OC
[35] Frankel, S.K., et al. (2006) TNF-alpha sensitizes normal
and fibrotic human lung fibroblasts to Fas-induced apop-
tosis. American Journal of Respiratory Cell and Molecu-
lar Biology, 34, 293-304.
doi:10.1165/rcmb.2005-0155OC
[36] Huang, S.K., et al. (2008) Variable prostaglandin E2
resistance in fibroblasts from patients with usual intersti-
tial pneumonia. American Journal of Respiratory and
Critical Care Medicine, 177, 66-74.
doi:10.1164/rccm.200706-963OC
[37] Andersen, M.H., et al. (2007) The universal character of
the tumor-associated antigen survivin. Clinical Cancer
Research, 13, 5991-5994.
doi:10.1158/1078-0432.CCR-07-0686
[38] Kelly, R.J., et al. (2011) Impacting tumor cell-fate by
targeting the inhibitor of apoptosis protein survivin. Mo-
lecular Cancer, 10, 35. doi:10.1186/1476-4598-10-35
[39] Pavlyukov, M.S., et al. (2011) Survivin monomer plays
an essential role in apoptosis regulation. Journal of Bio-
logical Chemistry, 286, 23296-23307.
doi:10.1074/jbc.M111.237586
[40] Nakahara, T., et al. (2011) YM155, a novel survivin sup-
pressant, enhances taxane-induced apoptosis and tumor
regression in a human Calu 6 lung cancer xenograft
model. Anticancer Drugs, 22, 454-462.
doi:10.1097/CAD.0b013e328344ac68
[41] Anandharaj, A., Cinghu, S. and Park, W.Y. (2011) Ra-
pamycin-mediated mTOR inhibition attenuates survivin
and sensitizes glioblastoma cells to radiation therapy.
Acta Biochim Biophys Sin (Shanghai), 43, 292-300.
[42] Hideshima, T., et al. (2007) Inhibition of Akt induces
significant downregulation of survivin and cytotoxicity in
human multiple myeloma cells. British Journal of Hae-
matology, 138, 783-791.
doi:10.1111/j.1365-2141.2007.06714.x
[43] Kang, H.R., et al. (2007) Semaphorin 7A plays a critical
role in TGF-beta1-induced pulmonary fibrosis. Journal of
Experimental Medicine, 204, 1083-1093.
doi:10.1084/jem.20061273
[44] Le Cras, T.D., et al. (2010) Inhibition of PI3K by PX-866
prevents transforming growth factor-alpha-induced pul-
monary fibrosis. American Journal of Pathology, 176,
679-686. doi:10.2353/ajpath.2010.090123
[45] Marsillach, J., et al. (2008) Changes in the expression of
genes related to apoptosis and fibrosis pathways in
CCl4-treated rats. Molecular and Cellular Biochemistry,
308, 101-109. doi:10.1007/s11010-007-9617-0