Intersectin-1s deficiency in pulmonary pathogenesis
Jeganathan et al. Respiratory Research
Intersectin-1s deficiency in pulmonary pathogenesis
Niranjan Jeganathan 0
Dan Predescu 2
Sanda Predescu 1
0 Rush University Medical Center , Chicago, IL 60612 , USA
1 Department of Pharmacology and Division of Pulmonary and Critical Care Medicine, Rush University Medical Center and Rush Medical College , 1750 W. Harrison Street, 1535 Jelke, Chicago, IL 60612 , USA
2 Department of Pharmacology and Division of Pulmonary and Critical Care Medicine, Rush University , 1750 W. Harrison Street, 1415 Jelke, Chicago, IL 60612 , USA
Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFβRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EHITSN and SH3A-EITSN fragments. The EHITSN fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EHITSN plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.
Intersectin-1s; Pulmonary arterial hypertension; Lung cancer; Acute lung injury; Eps8; mSos1; MAPK; Rac1
Intersectins (ITSNs) belong to the family of adaptor
]. Adaptor proteins, also known as scaffold proteins,
mediate the interaction between receptors and signal
transduction pathways by functioning as platforms for the
assembly of multiple protein signaling complexes [
Adaptor proteins lead to specificity in signaling via their
sequence of protein domains/motifs, subcellular localization
and their proximity to binding proteins [
adaptor proteins play a crucial role in cell signaling in a
spatial and temporal fashion, and regulate many important
cellular processes including proliferation, differentiation,
cell cycle control, cell survival and migration [
Aberrant expression of adaptor proteins is implicated in
numerous diseases .
ITSNs have a unique multi-domain structure each with
a distinct ligand preference [
]. ITSNs were initially only
associated with the regulation of endocytosis [
Subsequent studies have revealed a more complex role for these
proteins in the regulation of cell signaling and
cytoskeleton rearrangement [
]. Recent studies implicate
ITSNs, especially the transcript intersectin-1 short
(ITSN1s), in the pathogenesis of several pulmonary diseases
]. Given these novel findings, this review article will
provide a comprehensive overview of ITSN-1s’ regulation
of biochemical pathways and its clinical implications in
The sequence and initial characterization of ITSN was
first reported in Xenopus in 1998 by Yamabhai et al. [
They reported a 1270-amino acid long protein containing
two Eps15 homology (EH) domains, a central
coiledcoiled region, and five Src homology 3 (SH3) domains
(Fig. 1). The protein was named ITSN because of its
potential to bring together EH and SH3 domain binding
proteins into a macromolecular complex [
]. ITSN was the
first protein reported with both EH and SH3 domains
which interacts with asparagine-proline-phenylalanine
(NPF) and proline-rich (PXXP) motifs of the binding
proteins, respectively [
Shortly thereafter, human ITSN was identified by
Guipponi et al. [
]. ITSN exists with a high degree of
similarity in a number of higher eukaryotes [
]. There are two
ITSN genes in humans, ITSN-1 and ITSN-2, located on
chromosome 21 and 2 respectively [
]. ITSN-1 and
ITSN-2 share identical domain structure and greater than
50% sequence identity [
]. The human ITSN gene
produces two main ITSN transcript mRNAs, short (ITSN-s)
and long (ITSN-l), due to alternative splicing; human
ITSN-s is 5.3-kilobases and ITSN-l is 15-kilobases [
Both ITSN-s and ITSN-l contain two EH and five SH3
]. In addition, ITSN-l encodes for a Dbl
homology (DH), a Pleckstrin (PH) and a C2 domain ,
Fig. 1. The EH domains bind to proteins associated with
]. SH3 domains interact with proteins
implicated in cell signaling and cytoskeleton organization
6, 7, 11, 13, 24, 25
]. The DH domains promote
guaninenucleotide exchange on Rho, PH domains mediate
interactions with inositol phospholipid and C2 domains
mediate Ca2-dependent phospholipid binding [
ITSNs are widely distributed in human tissues, however
the expression of ITSN isoforms is tissue-dependent;
ITSN-1 l is specific to the brain and is absent in lung
tissue whereas ITSN-1s, ITSN-2 s and ITSN-2 l are
expressed ubiquitously [
19, 21, 23
]. Alternative splicing
plays a major role in the regulation of ITSN gene
expression, function and tissue specificity [
]. In addition to
the major splice variants there is a number of minor splice
variants of ITSN-1s protein which facilitate tissue specific
4, 19, 21
]. This is illustrated in the fact that
ITSN-1s preferentially interacts with mSos1 and Cbl in
most tissues including lung [
6, 11, 13, 28
]. However, in
brain tissue, a splicing of microexon 20 within the SH3A
domain of ITSN-1s (resulting in inclusion of 5 additional
amino acids) leads to reduced binding to mSos1 and Cbl,
and enhanced interaction with CdGAP [
transcript was conserved in numerous other eukaryotes
examined. Additional splicing events have also been reported
with ITSN-1s [
ITSN-1s is present in all subcellular compartments [
At the plasma membrane it is present in caveolae and
clathrin-coated pits [
8, 13, 26
]. Activation of receptor
tyrosine kinases (RTK) relocates ITSN-1s to the plasma
membrane where it forms a complex with important cell
signaling proteins [
]. Throughout the cytoplasm,
ITSN1s is associated vesicles, cytoskeleton elements and
Golgilike structures, in the perinuclear region [
8, 13, 26
findings in our lab also show that ITSN-1s is present in
the nucleus and interacts with important nuclear proteins
(unpublished). The wide subcellular distribution of
ITSN1s is consistent with its involvement of multiple important
signaling pathways involved in pulmonary pathogenesis.
Role of ITSN-1s in Endocytosis and cell signaling
ITSN-1s plays an important role in endocytosis and vesicle
trafficking; ITSN-1s binds to a number of important
endocytic proteins and localizes to clathrin-coated pits and
caveolae at the plasma membrane [
5, 8, 30
]. ITSN-1s binds
several dynamin-2 molecules simultaneously and clusters
them at endocytic sites creating a high concentration of
dynamin-2, which is required for collar formation around
the necks of endocytic vesicles, leading to membrane fission
and endocytosis [
5, 8, 31
]. This interaction is via the SH3
domains of ITSN-1s [
5, 8, 32
]. The SH3A domain has the
highest affinity for dynamin-2, and regulates its
assemblydisassembly and the GTPase activity in the process of
caveolae-dependent endocytosis [
]. However, ITSN-1s
effects in regulating endocytosis are
concentrationdependent as studies have shown that both silencing
ITSN1 gene (siRNA) and overexpressing ITSN-1s inhibit
]. Overexpression of SH3A in
endothelial cells (EC) as well as in mouse lung endothelium
stimulates dynamin-2 assembly and stabilizes dynamin-2
oligomers preventing detachment of caveolae from the
plasma membrane and as result, acting as a potent inhibitor
of endocytosis [
]. Similarly, acute depletion of
ITSN1s in mouse lungs results in inefficient dynamin-2
recruitment to the endocytic site leading to a decreased number
of free caveolae. This impairs trans-endothelial transport
which in turn opens interendothelial junctions and activates
paracellular transport. Prolonged inhibition of ITSN-1s
upregulates alternative endocytic structures/pathways
which partially restore the junctional integrity [
Studies have also demonstrated that ITSN-1s regulates a
number of important cell signaling pathways [
Highthroughput yeast two-hybrid screening identified more
than 100 interacting proteins with ITSNs (55 binding
proteins for ITSN-1, 62 binding proteins for ITSN-2). In
addition, ITSN-1 and ITSN-2 can dimerize with itself or
each other [
]. Given the wide subcellular distribution of
ITSN-1s, it has unique interactions and regulates different
signaling pathways depending on the intracellular
compartment and its spatial orientation.
Although ITSN-1s does not have a guanine nucleotide
exchange factor (GEF) domain like ITSN-1 l, [C-terminal
DH-PH domains act as a GEF [
]], it plays an
important role in the regulation of multiple Ras family GTPases
9, 10, 13
]. Ras family proteins regulate many processes in
the cell, including MAPK signaling, cell proliferation,
cytoskeleton organization and cell migration . Ras
GTPases cycle between an active GTP-bound form and an
inactive GDP-bound form [
]. This cycling is
regulated by GEFs that promote release of GDP and
subsequent binding of GTP to activate Ras. GTPase activating
or accelerating proteins (GAPs) enhance the intrinsic
GTPase activity to promote GTP hydrolysis to terminate
Ras GTPase activity [
]. ITSN-1s interacts with
CdGAP, mSos1 and Eps8 which regulate the activity of
GTPase proteins Cdc42, Ras and Rac1 respectively [
10, 13, 24, 43
]. ITSN-1s’ interaction with CdGAP inhibits
its GAP activity leading to activation of Cdc42 and Rac1
]. This interaction is tissue/organ dependent;
splicing of five additional amino acids in microexon 20 within
the SH3A domain of ITSN-1s leads to enhanced
interaction with CdGAP in brain tissue, and reduced binding
in lung tissue .
ITSN-1s’ interaction with mSos1 is more complex. The
SH3 domains of ITSN-1s form a stable complex with
mSos1 and compete with Grb2 for binding to the same site
on mSos1 [
]. Activation of RTK recruits mSos1-Grb2
complex to activate Ras and MAPK pathway. Consistent
with this, overexpression of the SH3 domains of ITSN alone
impairs mSos1-Grb2 complex and prevents Ras-mediated
MAPK activation [
]. However, overexpression of
fulllength ITSN-1s activates Ras in the perinuclear vesicles
without downstream activation of the MAPK pathway [
ITSN-1s mediated Ras activation in the perinuclear vesicles
was initially thought to be via mSos1, but recent studies
have disproved this observation and have implicated a novel
PI3K isoform in this interaction [
]. The novel PI3K
isoform, PI3KC2β, preferentially binds to inactive Ras and
inhibits its activation. In the presence of ITSN-1s, the
preferential interaction of PI3KC2β and ITSN-1s results in
dissociation from inactive Ras which makes it available for
immediate activation by GTP loading [
Eps8 interacts with mSos1 in the presence of RTK
activation to convert the Rac1 GTPase from its inactive
GDPbound state to the active GTP-bound state [
impairs mSos1-Eps8 interaction and favors Cbl-Eps8
interaction leading to impaired Rac1 activation and Eps8
ubiquitination, respectively. Cbl is an E3 ubiquitin ligase and plays
an important role in regulating the levels of numerous
]. These interactions are most likely between the
SH3 domain(s) of ITSN-1s and the proline-rich regions of
Eps8 and Cbl, similar to ITSN’s interactions with most
signaling proteins [
]. The multiple domains of ITSN-1s are
known to simultaneously interact with the same protein as
well as different proteins [
]. Given the presence of five
SH3 domains, ITSN-1s is able to simultaneously interact
with multiple proline-rich domains of proteins to
coordinate different cellular signaling processes [
]. Rac1 has a
complex reciprocal relationship with RhoA, and the balance
between the two determines cell migration by reorganizing
cytoskeleton elements and focal adhesions [
Consistent with this, impaired Rac1 activation in the presence of
ITSN-1s leads to increased RhoA activation [
ITSN-1s also plays an important role in RTK’s
regulation of numerous signaling pathways [
]. RTK function is
regulated by endocytosis and ubiquitination. Although
endocytosis terminates RTK signaling, it has emerged as a
prerequisite step in the activation of signaling pathways
]. Internalization and trafficking of the epidermal
growth factor receptor (EGFR) to endosomes is necessary
for maximal activation of the MAPK pathway by EGFR
. Consistent with this, silencing ITSN-1s gene
attenuates the extent and duration of Erk1/2 activation after
epithelial growth factor stimulation, an effect that is due to
decreased EGFR internalization [
]. ITSN-1s also
decreases EGFR activity by promoting ubiquitination of
EGFR by the ubiquitin E3 ligase Cbl [
]. In addition,
ITSN-1s interacts with a number of other proteins
involved in the regulation of Cbl such as sprouty2 and
1, 50, 51
ITSN-1s deficiency in lung cancer
ITSNs’ participation in the activation of numerous
mitogenic kinases provided strong suspicion for a potential
involvement in cell proliferation and cancer. Earlier
studies have shown that overexpression of ITSN-1s
induces oncogenic transformation of rodent fibroblasts
]. Both low and high levels of ITSN-1s have been
reported in several cancers. The first clinical evidence for
the role of ITSN-1s in cancer came from patients with
Down syndrome [
]. The human ITSN1 maps proximal
to the Down syndrome critical region (21q22.1-q22.2
between markers D21S319 and D21S65). Triplication
(trisomy) of this region is associated with many phenotypes
of Down syndrome [
]. As a consequence of ITSN-1s’
location in the down syndrome critical region, these
patients have an elevated ITSN-1 mRNA and protein level
]. In a cohort prospective registry study, these
patients had a significantly lower incidence of solid organ
cancers, especially lung cancer, compared to the age
matched general population [
]. In addition, the
Human Protein Atlas reports significantly lower levels of
ITSN-1s protein level in lung cancer and a number of
other solid organ malignancies [
]. However, studies in
neuroblastoma and glioblastoma have shown
upregulation of ITSN-1s and reduced tumorigenesis and cell
migration with silencing of ITSN-1s gene [
44, 56, 57
demonstrates that ITSN-1s mediated interactions and
regulation of signaling pathways is specific to the tissue
and disease. The numerous minor splice variants that
occur throughout the ITSN-1s protein, results in
changes in the EH, SH3, and DH domains and facilitate
these tissue and disease-specific interactions [
Alternative splicing generates protein isoforms with different
biological properties that differ in protein:protein
interactions, subcellular localization, protein stability and
posttranslational modifications [
]. ITSN isoforms have
also been demonstrated to play a causative role in other
diseases such as. glioblastoma, neuroblastoma,
Alzheimer’s disease, Down syndrome, retinitis pigmentosa and
44, 56, 57, 59–61
]. It is increasingly
recognized that the large number of alternative splicing
events of ITSN-1s mRNA, the alternative
polyadenylation, the tissue-specific expression level of different
transcripts and their differential binding to interacting
partners, play an important role in regulation of
ITSN1s in health and disease [
27, 62, 63
]. The detailed
characterization of isoform specific-functions of
ITSN1s, and how specific changes in splicing impact the
disease process and risk require further investigation.
The level of both ITSN-1s protein and mRNA is
significantly decreased in human lung cancer likely due to
suppression of transcription [
]. Pilot studies using
immunohistochemistry staining in lung cancer tissue
samples demonstrate a negative correlation between the
ITSN-1s protein level and the aggressiveness of lung
cancer. The subcellular distribution of ITSN-1s is not
altered in lung cancer [
]. The impact of ITSN-1s
deficiency in lung cancer progression was studied by
comparing control lung cancer cells (A549) to those
with ITSN-1s protein level restored to normal levels by
stable transfection using a myc-ITSN-1s plasmid
(A549 + ITSN-1s). ITSN-1s expression impairs lung
cancer cell proliferation, anchorage-independent growth
and tumorigenesis [
]. We have shown that ITSN-1s
expression in lung cancer cells impairs Eps8 interaction
with mSos1 and facilitates Eps8-Cbl interaction leading
to ubiquitination and downregulation of Eps8. Eps8
interacts with mSos1 in the presence of RTK activation to
convert the Rac1 GTPase from its inactive GDP-bound
state to the active GTP-bound state [
Eps8mSos1 interaction in the presence of ITSN-1s decreases
Rac1 activation. Rac1 activation is a required for
Rasmediated tumor progression [
]. Rac1 activation also
directly activates the JNK pathway leading to tumor
]. As an oncoprotein, Eps8 also translocates
to the nucleus and upregulates numerous cell cycle
proteins such as transcription factor Foxm1 [
ITSN-1s mediated inhibition of tumorigenesis is likely
due to a combined inhibitory effect on these pathways.
In addition, ITSN-1s enhances ubiquitination and
downregulation of EGFR with a potential negative impact on
In the presence of ITSN-1s, decreased Rac1 activation
and reciprocal upregulation of RhoA shift the balance in
favor of decreased lung cancer cell migration and
]. This results in a number of significant changes
in the cytoskeleton. In the presence of ITSN-1s, lung
cancer cells show phenotypic changes favoring transition
from mesenchymal-to-epithelial cells: increased
spreading, lack of elongated and polarized morphology, and
prominent actin bundles towards peripheral attachment
]. In addition, the number of vinculin focal
adhesions at the cell surface is increased and the vimentin
filament network is collapsed. In a scratch closure assay
(in vitro cell migration model) ITSN-1 mediated
cytoskeleton changes lead to significantly decreased cell
migration and scratch closure. In vivo mouse metastasis
assay shows significantly more tumor metastasis to the
lungs as well as larger size tumors in A549 cancer cells
compared to A549 + ITSN-1s cells. Altogether, these
studies demonstrate that ITSN-1s deficiency is a crucial
event in lung cancer progression. ITSN-1s’ ability to
reverse the malignant features demonstrates the capability
of this protein to regulate multiple pathways
simultaneously which makes it an attractive therapeutic target.
Further validation of ITSN-1s protein level in a large
cohort of patients at different stages of lung cancer could
establish ITSN-1s as a predictor of prognosis and
indicator of response to therapy. Given the ubiquitous
distribution of ITSN-1s and evidence that loss of ITSN-1s is a
characteristic feature of many cancers [
findings may be applicable to other types of cancer.
ITSN-1s deficiency in acute lung injury
Acute lung injury is associated with excessive apoptosis
of pulmonary endothelial and epithelial cells, which
induce endothelial and epithelial barrier dysfunction
leading to pulmonary edema [
]. Acute lung injury is
triggered by direct injury to the lung and can be
secondary to other inflammatory conditions such as sepsis,
pancreatitis or transfusion rejection. Studies have shown
that apoptotic pulmonary cells or macrophages engulfing
apoptotic cells release substances such as growth factors,
and create conditions that favor the emergence of
apoptosis-resistant cells [
In acute lung injury, as in many pro-inflammatory states,
full-length ITSN-1s expression is decreased [
11, 12, 68
ITSN-1s is important for the pro-survival signaling pathway
mSos1/Ras/Erk1/2 MAPK which regulates cell survival,
proliferation and vascular remodeling [
6, 9, 10
Downregulation of ITSN-1s, via siRNA, inhibits the Erk1/2 MAPK
pathway leading to mitochondrial apoptosis of EC [
However, EC with LPS-induced ITSN-1s deficiency do not
undergo apoptosis since ITSN-1s deficiency is countered
by upregulation of anti-apoptotic proteins leading to
protection of EC [
The use of ITSN-1s knockdown (KDITSN) mouse model
has significantly advanced our understanding of the
mechanisms involved in lung injury as well as the role of
ITSN1s in the pathogenesis of acute lung injury. KDITSN mouse
model was generated by intravenous delivery of the
cationic liposomes/siRNAITSN complex in repeated doses to
maintain continuous downregulation of ITSN-1s protein
expression up to 24 days. The amount injected (100 μg
siRNA/mouse) was efficient and specific in inhibiting
ITSN-1s mRNA and protein levels without significant
adverse effects [
Acute KDITSN (assessed 72 h post-siRNA delivery) leads
to decreased Erk1/2 MAPK signaling and causes
significant EC apoptosis, microvascular loss and alveolar
destruction. The lung injury is patchy and mostly involves
the alveolar capillary units centered on small vessels and
mid-sized vessels [
]. The morphology of ECs nuclei,
mitochondria, and Golgi are affected as well. As a
consequence, the alveolar-capillary unit becomes increasingly
permeable leading to leakage of protein-rich fluid from
the vascular to the interstitial space causing distension of
the interstitial space [
]. These findings are identical to
the pathophysiological processes that are hallmarks of
acute lung injury [
]. This pattern of lung injury
triggered by ITSN-1s deficiency persists up to about 10 days.
ITSN-1s interaction with endocytic proteins is crucial for
normal endocytosis [
]. The pulmonary EC of KDITSN mice
demonstrate impaired normal endocytosis which occurs via
caveolin and clathrin-coated vesicles [
deficiency leads to upregulation of alternative endocytic
pathways (ECs display enlarged endocytic structures,
membranous rings and tubules) to compensate for
deficiency in normal endocytosis and vesicular [
endocytosis and vesicular trafficking are crucial for growth
factor receptor signaling and activity of their downstream
]. TGFβ is a ubiquitous and multifunctional
cytokine which in the setting of lung injury expresses
antiinflammatory properties confined to the extent of septal
injury and facilitates recovery [
]. Alk5 is a widely expressed
transforming growth factor β receptor I (TGFβRI) which
complexes with TGFβRII when activated by growth factor
TGFβ, and activates Smad2 and Smad3 proteins via the
canonical TGFβ signaling pathway [
]. In normal EC,
internalization of Alk5 via clathrin-coated vesicles leads to
TGFβ-induced activation of Smad2/3 pathway which is
then recycled to the plasma membrane. In contrast
internalization of Alk5 by caveolae is directed to ubiquitin
proteasome pathway [
]. The alternative pathways
upregulated by ITSN-1s deficiency express predominantly
caveolin-1 and therefore alters the endocytic trafficking of
Alk5 in favor of enhanced degradation [
]. Consistent with
this, in acute lung injury Alk5 expression is significantly
decreased up to 10 days; however after 10 days Alk5
expression starts to gradually recover and reaches normal levels
by 24 days [
]. The recovery of Alk5 expression is via the
effects of microparticles. Microparticles are 0.5-1.0 μm
diameter in size and play a crucial role in the
communication between different cell types in normal and pathological
settings. They have double membrane morphology and
store important bio-effectors which play a crucial role in
the recovery of lung injury by inducing endothelial
modifications via membrane fusion and paracrine effects [
At day 10 of ITSN-1s deficiency when EC apoptosis and
lung injury are at their peak, there is a significant increase
in microparticles containing Alk5. In the setting of
prolonged ITSN-1s deficiency these microparticles are able to
interact and transfer Alk5/TGFβRII complexes to
dysfunctional EC . The expression of TGFβ is also significantly
increased at 10 days when lung injury is at its peak. As a
result of increased TGFβ and restoration of its receptor Alk5/
TGFβRI, the Erk1/2 MAPK pathway is restored, and
remaining non-apoptotic quiescent EC exhibit phenotypic
changes toward hyperproliferation and apoptosis resistance
leading to increased microvessel density, repair and
remodeling of the lungs [
]. Within 2 weeks after severe injury,
mouse lung function returned to normal state with little
evidence of prior damage. A lung repair process
characterized by EC proliferation and increased microvascular
density was critical for the remarkable recovery. However, the
typical TGFβ/ALK5 signaling is shifted from Smad2/3
activation towards a less common Ras/Erk1/2 MAPK pathway
]. Since ITSN-1s associates with mSos1 in a complex
that excludes Grb2, ITSN-1s deficiency increases mSos1
availability for Grb2 interaction and results in preferential
formation of ALK5/mSos1/Grb2 signaling complex. This
shifts the balance from Smad2/3-Erk1/2 towards Ras/
MEK/Erk1/2 activation [
]. Despite continuous and
efficient KDITSN, apoptosis of EC starts to decrease after
10 days, and by 24 days has reached almost normal levels.
A similar proliferative pattern was also seen with epithelial
As the timeline of acute lung injury/acute respiratory
distress syndrome (ARDS) related pathology is shared by
both mice and humans, and since ITSN-1s deficiency is
also a characteristic of human lung tissue of acute lung
injury/ARDS patients [
], we applied a translational
approach to study microparticles from the blood of ARDS
patients. Similar to the mouse studies, we have identified a
population of Alk5/TGFβRI-immunoreactive
]. Flow cytometry and calibrated/counted beads
used to quantify the microparticles indicated that the
Alk5-positive microparticle population is more numerous
in the ARDS patients compared to healthy controls,
consistent with reports of elevated levels of microparticles in
disease settings [
]. Flow cytometry and magnetic bead
separation via biotin-conjugated Alk5 Ab and streptavidin
magnetic beads demonstrated that these Alk5-positive
microparticles are immunoreactive to CD73 and CD105 and
negative to CD34 CD45, suggesting a mesenchymal stem
cell origin [
]. Additional studies demonstrate that these
particles interact with LPS-treated lung ECs, in culture
and in vivo, leading to improved permeability and
decrease in lung histological severity, consistent with
longterm follow-up studies of ARDS survivors, suggesting that
lung repair is in fact a hallmark of the normal course of
recovery from acute lung injury [
]. Altogether, these
observations demonstrate that extensive pulmonary EC
death due to ITSN-1s deficiency stimulates mesenchymal
stem cell paracrine signaling via microparticles leading to
generation of hyperproliferative and apoptotic-resistant
endothelial cells and subsequent recovery of lung injury.
ITSN-1s deficiency in pulmonary arterial hypertension
Pulmonary arterial hypertension (PAH) is a disease in
which there is persistent elevation of pulmonary artery
pressure. The pathological findings in this disease are
medial hypertrophy, intimal proliferation and fibrosis of
pulmonary arteries and arterioles [
]. The intimal
changes lead to progressive obstruction of the vessels
which, in the presence of high pressure, dilate and
evolve into microaneurysms. Areas of microaneurysms
lead to endothelial proliferation and formation of in situ
thrombosis which leads to the characteristic plexiform
lesions. The obliteration of vessels and plexiform lesions
lead to an increase in pulmonary vascular resistance and
ultimately right heart failure. Enhanced proliferation of
EC, smooth muscle cells and fibroblasts are central to
the pathogenesis of PAH [
]. Emergence of proliferative
ECs in PAH is a consequence of initial EC dysfunction
and apoptosis and subsequent selection of
apoptoticresistant proliferative EC [
Inflammatory mechanisms play a significant role in the
initiation of the pathogenesis of PAH. Inflammation
associated with PAH attracts inflammatory cells to release
granzyme B [
]. Interestingly, ITSN-1s is a substrate for
granzyme B with a cleavage site at the IDQD271GK
sequence, a well-conserved sequence among mammals [
The cleavage results in decreased expression of full-length
ITSN-1s protein and in two biologically active protein
fragments, N-terminal fragment (EHITSN) and C-terminal
fragment (SH3A-EITSN) [
]. Evidence demonstrates that
ITSN-1s is a substrate for granzyme B and its cleavage
leading to deficiency of ITSN-1s which is associated with the
pathogenesis of PAH [
]. Mice treated with
lipopolysaccharide (LPS; bacterial endotoxin which induces a strong
immune response and leads to increase in granzyme B) and
monocrotaline-induced PAH mouse and rat models results
in loss of full-length ITSN-1s expression and the presence
of a 28-kDa fragment corresponding to the molecular
weight of EHITSN [
]. Biochemical analyses of lung ECs of
PAH patients demonstrate low levels of full-length ITSN-1s
protein and mRNA expression. Immunohistochemistry
staining of human PAH specimens show lower ITSN-1s
staining of pulmonary arteries with proliferative ECs and
plexiform lesions whereas the presence of granzyme B is
increased in the milieu of these lesions [
We have reported that both fragments formed by
cleavage of full-length ITSN-1s are biologically active and impact
EC proliferation [
]. EHITSN enhances EC proliferation
via activation of p38 MAPK pathway. In the presence of
increased EHITSN expression, p38 MAPK is distributed
predominantly in the cytosol and is activated. P38 MAPK
activation leads to activation of Elk-1 transcription factor
which facilitates Elk-1 binding to the c-Fos promoter
leading to increased expression of the growth related protein
cFos. Treatment with a selective p38 MAPK inhibitor
significantly inhibits EC proliferation confirming the role of
EHITSN mediated activation of p38 MAPK in EC
proliferation. EHITSN has no effect on JNK or the PI3K/Akt
signaling pathway, and impairs Erk1/2 MAPK activation via
negative cross talk from p38 to Erk1/2. The SH3A-EITSN
fragment, however, impairs proliferation by sequestering
mSos1 and inhibiting activation of Ras/Erk1/2 MAPK
signaling. The concurrent expression of both EHITSN and
SH3A-EITSN results in a high p38/Erk1/2 MAPK activity
ratio favoring EC proliferation .
ITSN-deficient mice transduced with EHITSN developed
pathological findings similar to PAH patients. Wild-type
mice and ITSN-deficient mice [KDITSN and ITSN
knockout/heterozygous (K0ITSN+/−)] were treated with
mycEHITSN cationic lipoplexes delivered repeatedly by
retroorbital injection every 48 h for 20 days. No hypoxia or
chemical/synthetic compounds known to induce PAH were
used. EHITSN transduced ITSN-deficient mice developed
numerous and widespread clusters of proliferating
pulmonary ECs and pathological findings consistent with plexiform
lesions; hypercellular and stalk-like lesions arising from the
vessel walls protruded into the lumen of the pulmonary
artery and caused severe obliteration of the vessel. These
lesions displayed a rich matrix of collagen consistent with
vascular medial thickening seen in human PAH [
Moreover, proliferation of pulmonary artery smooth muscle cells
was also observed in the lung vessels of EHITSN-transduced
ITSN-1s deficient mice, suggesting significant cross-talk
between ECs and smooth muscle cells leading to the
pathogenesis of PAH which involves proliferation and
hypertrophy of both intimal and medial layers. Only 20 days
of EHITSN treatment, independent of any other insults,
resulted in modest increase in the RVSP values (from 21 to
25.5 mmHg) and right ventricular hypertrophy.
Similar to the findings in cultured ECs, the lungs of
EHITSN-transduced ITSN-deficient mice showed increased
activation of p38 MAPK, Elk-1 transcription factor and
increased expression of the c-Fos gene, consistent with
activation of p38 MAPK pathway [
]. Prior studies have also
implicated p38 MAPK in cell proliferation and vascular
obliteration leading to the pathogenesis of PAH [
Given that the motif NPF is an essential target of EH
], the proliferative potential of ECs expressing
EHITSN is impaired by treatment with a membrane
permeable peptide containing the NPF motif [
EHITSN is a highly specific molecular target, this could be
a very effective treatment option to ameliorate and
perhaps reverse the EC plexiform phenotype already
established in severe human PAH.
ITSN-1s is a multi-domain protein with numerous binding
partners capable of regulating many important signaling
pathways. Studies to date implicate ITSN-1s deficiency in
the pathogenesis of several pulmonary diseases (Fig. 2). In
lung cancer, ITSN-1s deficiency shifts the balance in favor
of greater Eps8-Sos1 interaction and less Eps8-Cbl
interaction leading to activation of Rac1 and increased
expression of Eps8 respectively. As a result of this and other
potential interactions, ITSN-1s plays a crucial role in lung
cancer development and progression: proliferation,
anchorage-independent growth, cytoskeleton modification,
migration and metastasis. As a pro-survival protein,
ITSN1s deficiency is a crucial early event in development of
acute lung injury. ITSN-1s deficiency impairs normal
endothelial structures and their function which leads to
endocytosis of Alk5 receptors via alternative endocytic pathways
resulting in Alk5 ubiquitination and degradation.
Downregulation of Alk5 expression in acute lung injury is
counterbalanced by circulating microparticles which, via paracrine
effects, interact and transfer Alk5/TGFβRII complex to
remaining non-apoptotic cells. These receptors, in a cellular
context characterized by ITSN-1s deficiency and aberrant
endocytosis, signal via Erk1/2 MAPK pathway, instead of
the usual Smad 2/3 pathway, regardless leading to
restoration and repair of lung architecture. In the setting of PAH,
full-length ITSN-1s is cleaved by granzyme B released by
inflammatory cells. The cleavage results in EHITSN and
SH3A-EITSN fragments. The EHITSN fragment leads to EC
proliferation via activation of p38 MAPK and Elk-1/c-Fos
signaling. The SH3A-EITSN fragment impairs Ras/Erk1/2
MAPK signaling. However, the concurrent expression of
both fragments results in high p38/Erk1/2 MAPK activity
favoring pulmonary cell proliferation. In vivo, ITSN-1s
deficient mouse transduced with EHITSN plasmid leads to EC
and smooth muscle proliferation resulting in pulmonary
vascular obliteration and plexiform lesions consistent with
pathological findings seen in severe PAH. As shown in
these studies related to pulmonary diseases and others,
ITSN-1s regulates multiple signaling pathways
simultaneously in a tissue, concentration and subcellular
distribution specific manner. Given the numerous protein-protein
interactions between the multiple domains of ITSN-1s, it is
highly likely that additional regulatory pathways remain to
be identified. Further studies will shed light into novel
mechanisms of regulation of protein, genetics and
epigenetics by ITSN-1s in pulmonary diseases. Future studies
should also explore the role of other ITSN transcripts and
potential compensatory role in pulmonary pathogenesis.
Studies of ITSN-1s involvement in acute lung injury and
PAH are limited and its role in the pathophysiology of these
diseases is less established. The KDITSN and K0ITSN+/−
mouse models of lung injury as well as the
EHITSN-transduced K0ITSN+/− mouse model of plexogenic PAH that
recapitulate many of the pathological events associated with
the human disease are valuable tools to further advance our
understanding of ITSN-1s involvement in pulmonary
pathology, and provide novel targets for treating these severe
ARDS: Acute respiratory distress syndrome; DH: Dbl homology;
EC: Endothelial cells; EGFR: Epidermal growth factor receptor; EH: Eps15
homology; EHITSN and SH3A-EITSN: N-terminal fragment and C-terminal
fragment; GAPs: GTPase activating or accelerating proteins; GEF: Guanine
nucleotide exchange factor; ITSN-1s: Intersectin-1s; ITSNs: Intersectins; K0ITSN
+/−: ITSN knockout/heterozygous; KDITSN: ITSN-1s knockdown;
NPF: Asparagine-proline-phenylalanine; PAH: Pulmonary arterial hypertension;
PH: a Pleckstrin; SH3: Src homology 3
Supported by Rush Executive Committee of Research Grant (to NJ) and
R01HL089462 (to SP).
Availability of data and materials
Not applicable as no data were generated or analyzed.
NJ drafted the article and prepared for submission. DP and SP edited the
final article. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interest.
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