T-cadherin signaling in endothelial cells

The cadherin superfamily comprises transmembrane glycoproteins that mediate calciumdependent
homophilic cell-cell adhesion. In addition to their pivotal role in mechanical adhesion
between cells, cadherins have multiple functions in tissue morphogenesis, cell recognition and
sorting, regulated cell motility and the induction and maintenance of tissue/cell polarity.
T-cadherin (T-cad) is unusual member of cadherin superfamily; while possessing the Nterminal
tandem cadherin repeat structure (EC domain), it lacks both transmembrane and
cytoplasmic domains, and is bound to the plasma membrane via a glycosylphosphatidylinositol
(GPI) anchor. T-cad has five EC domains and a propeptide in its precursor form (130kDa),
which upon cleavage gets converted to mature form (105kDa). A role for T-cad in tissue
organization was first demonstrated in the avian embryonic nervous system where the protein
influenced the pattern of neural crest cell migration and maintained somite polarity. Many cancer
cell lines (e.g. breast, colon, lung, inter alia) display allelic loss of T-cad which is correlated
with tumor progression, and hence T-cad has been described as tumor suppressor gene.
T-cad is widely expressed in the vasculature and is upregulated in proliferative vascular
disorders such as atherosclerosis and restenosis. GPI-anchored T-cad is not localized at adherent
junctions but rather distributed globally over the cell surface. T-cad is localized within lipid
rafts. In vitro data supports participation of T-cad in many cellular processes such as vascular
differentiation, migration and proliferation of smooth muscle cells (SMC) and endothelial cells
(EC) and angiogenesis. Adenoviral mediated overexpression of T-cad in EC and SMC results in
cell cycle progression and a concomitant promotion of proliferation. T-cad exhibits deadhesive
functions upon homophilic ligation with antibody against T-cad or with immobilized
recombinant protein. Homophilic ligation of T-cad induces polarization and migration of
endothelial cells in a RhoA/ROCK and Rac dependent fashion. T-cad stimulates in-gel
outgrowth of endothelial sprouts in 3-dimensional EC-spheroid and heart tissue models of
angiogenesis. In vivo, myoblast-mediated delivery of recombinant soluble T-cad to mouse
skeletal muscle facilitates VEGF-induced angiogenesis, supporting a physiological role for Tcad
as a proangiogenic protein.
The present thesis is focused on the regulation of T-cad expression and the signaling
mechanisms whereby T-cad affects vascular cell behavior. The conditions of proliferative
vascular cell disorders in which T-cad is upregulated are associated with oxidative stress and cell
survival/cell death. We observed an elevation in T-cad levels under condition of oxidative stress
induced by serum-deprivation and H2O2; this response was normalized upon inclusion of an
antioxidant, N-acetyl cysteine or NADPH oxidase inhibitor diphenyleneiodonium, suggesting T-
cad induction by reactive oxygen species is NADPH oxidase dependent. Adenoviral mediated
overexpression of T-cad in EC facilitated EC survival upon induction of apoptosis by serumdeprivation
and various apoptosis-inducing pharmacologicals. Western blot analysis of lysates
infected with Empty –adenovirus (E-EC) and T-cad adenovirus (T-cad+-EC) resulted in
hyperactivity of anti-apoptotic proteins (Akt and mTOR target p70S6 kinase) and diminished
activity of pro-apoptotic proteins (p38MAPK and active caspase3). PI3 kinase inhibitor,
wortmannin, and mTOR inhibitor, rapamycin, normalized anti-apoptotic effects of T-cad; these
data suggest that upregulation of T-cad in response to oxidative stress functions to protect EC by
concomitant induction of PI3K/Akt/mTOR pathway and suppression of p38/caspase3 pathways.
Subsequently we focused on identifying downstream targets of Akt and candidate
proximal molecular mediators for T-cad. T-cad+-EC exhibited hyperphosphorylation of glycogen
synthase kinase β (GSK3β) and concomitant nuclear accumulation of active β-catenin, a
transcription factor regulating cell cycle proteins. Using various GSK3β-carrying adenovectors
(kinase mutant, dominant negative or wild type) we demonstrated that T-cad induced nuclear
accumulation of β-catenin is GSK3β-dependent. siRNA mediated knockdown of T-cad resulted
in decreased phosphorylation of Akt and GSK3β and also in reduced nuclear accumulation of β-
catenin. T cell factor (TCF) and Leukocyte enhancer factor (LEF) are co-factors for β-catenin;
we found that luciferase (reporter) activity of TCF/LEF elements in T-cad+-EC was markedly
increased as compared to E-EC. Cyclin D1, one of the important regulators of the cell cycle is a
target of β-catenin/TCF/LEF transcription machinery; T-cad+-EC showed increased mRNA and
protein levels of cyclin D1 and increased cell proliferation. In searching for molecular mediators
of T-cad we considered Integrin linked kinase (ILK) as a putative candidate because both the
proteins are located in rafts and ILK acts upstream of Akt and GSK3β in a PI3K-dependent
fashion. T-cad+-EC exhibited increased ILK “kinase” activity in a pull-down assay. RNAimediated
knockdown of ILK abrogated effects of T-cad on both phosphorylation of Akt and
GSK3β and the nuclear accumulation β-catenin, suggesting involvement of ILK in T-cad
signaling. Confocal microscopy studies revealed colocalisation of T-cad and ILK in EC which
was most prominent within leading edges of migratory cells and at focal adhesions. Anti-ILK
immunoprecipitates contained T-cad indicating the existence of T-cad/ILK complexes, and
supporting our hypothesis that ILK can function as a proximal molecular mediator for T-cadelicited
PI3K/Akt/GSK3β signaling.
Transcriptional regulation of T-cad in endothelial cells is poorly understood. To
characterize the minimal promoter region of T-cad, we cloned serially deleted fragments of Tcad
promoter stretches into luciferase reporter vector (pGL3). Reporter gene analysis exhibited
basal levels of luciferase activity within -285bps suggesting existence of minimal promoter
region within -285bps from translational start site. Oxidative stress elevated reporter activity of
-285 bps construct, suggesting the minimal promoter region might be responsible for the redox
sensitivity of T-cad expression. To identify regulatory elements (transcription factors)
responsible for T-cad regulation gel shift assays were performed using nuclear extracts of EC
and various oligos designed from T-cad promoter region from -1 to -284 bps We identified
specific binding of regulatory protein(s) between -156 to -203 bps. Nuclear extracts from serumdeprived
EC exhibited increased binding to -156 to -203 bps oligo, suggesting that the identified
nucleoprotein complex could function to induce T-cad expression under conditions of oxidative
stress. To identify transcription factor(s) within the identified nucleoprotein complex we
performed pull-down assay using nuclear extracts of EC, biotinylated -156 to -203 bps and
streptavidin agarose beads. Proteins pulled down were subjected to microsequencing by mass
spectrometry. Interestingly thioredoxin (TRX1) was found to be present. TRX1 is a 12kDa
protein induced by NADPH oxidase under stress and it acts as an antioxidant by facilitating the
reduction of other proteins by cysteine thiol-disulfide exchange. Following its translocation to
the nucleus TRX1 reduces transcription factors, enabling their binding to regulatory elements.
Preliminary data using RNAi-mediated knockdown of TRX1 abrogates oxidative stress-induced
upregulation of T-cad in EC, suggesting that NADPH dependent-induction of T-cad involves
nuclear translocation of TRX1. These data may explain the observations of upregulation of Tcad
on vascular cells in atherosclerotic lesions where oxidative stress plays a key pathogenic
role.