Download Primary and immortalized mouse epicardial cells undergo

DEVELOPMENTAL DYNAMICS 237:366 –376, 2008
RESEARCH ARTICLE
Primary and Immortalized Mouse Epicardial
Cells Undergo Differentiation in Response to
TGF␤
Anita F. Austin,1 Leigh A. Compton,1 Joseph D. Love,2 Christopher B. Brown,3 and Joey V. Barnett1*
Cells derived from the epicardium are required for coronary vessel development. Transforming growth
factor ␤ (TGF␤) induces loss of epithelial character and smooth muscle differentiation in chick epicardial
cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with
TGF␤1 or TGF␤2 lose epithelial character and undergo smooth muscle differentiation. To further study
TGF␤ Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed
tightly packed epithelium and expressed the epicardial marker Wilm’s tumor 1 (WT1). TGF␤ induced the
loss of zonula occludens-1 (ZO-1) and the appearance of SM22␣ and calponin consistent with smooth muscle
differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the
effects of TGF␤ while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data
demonstrate that TGF␤ induces epicardial cell differentiation and that immortalized epicardial cells
provide a suitable model for differentiation. Developmental Dynamics 237:366 –376, 2008.
© 2008 Wiley-Liss, Inc.
Key words: coronary vessels; development; TGF␤; epicardium; mouse; smooth muscle
Accepted 27 November 2007
INTRODUCTION
Fifty-four percent of all cardiovascular disease in the United States effects
the coronary arteries (American
Heart Association, 2004). A detailed
understanding of the cell populations
and molecules that regulate coronary
vessel development will be required to
reveal novel drug targets and therapeutic strategies to direct the repair or
remodeling of coronary vessels in
adults. The importance of cells derived from the proepicardium (PE)
and epicardium (EP) in the formation
of the coronary vessels is well estab-
lished (reviewed in Olivey et al., 2004;
Tomanek, 2005). During development
these cells give rise to both the endothelial and smooth muscle components of the coronary vessels. In chick
embryos, the PE arises from mesothelial cells along the caudal border of the
pericardial cavity (Ho and Shimada,
1978). The PE contacts the heart at
the atrioventricular groove and migrates to the heart by means of an
extracellular matrix bridge (Nahirney
et al., 2003) between the myocardium
and the PE. These cells maintain polarity while migrating as an intact ep-
1
ithelium with the luminal surface in
contact with the myocardium. In
mammals clusters of PE cells detach
as vesicles that are transferred to the
heart by means of the pericardial fluid
(Komiyama et al., 1987; Kuhn and
Liebherr, 1988). In both avians and
mammals, after contacting the myocardium, a fraction of cells undergo
epithelial–mesenchymal transformation (EMT) and migrate into the subepicardial space. A subset of these
cells continues into the compact zone
of the myocardium (Mikawa and
Fischman, 1992; Poelmann et al.,
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee
University of Southern Indiana, Evansville, Indiana
3
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
Grant sponsor: NIH; Grant number: HL67105; Grant number: HL076133; Grant number: GM07347; Grant sponsor: American Heart
Association; Grant number: AHA0655129.
*Correspondence to: Joey V. Barnett, Department of Pharmacology, Vanderbilt University Medical Center, Room 476 RRB,
2220 Pierce Avenue, Nashville, TN 37232-6600. E-mail: [email protected]
2
DOI 10.1002/dvdy.21421
Published online 16 January 2008 in Wiley InterScience (www.interscience.wiley.com).
© 2008 Wiley-Liss, Inc.
TGF〉 AND EPICARDIAL CELLS 367
1993). Coronary vessel formation begins as angioblasts coalesce to form a
primitive vascular plexus in the subepicardial space and myocardium.
These nascent endothelial tubes form
larger vessels to become the coronary
arteries and veins that attach to the
ascending aorta and the right atrium.
Once attached, these vessels recruit
PE-derived cells to form the smooth
muscle and fibroblast components of
the vascular network. Therefore, precursor cells are delivered to the heart
by the PE and form coronary vessels
by a process of vasculogenesis (Munoz-Chapuli et al., 2002). In zebrafish,
epicardial cells are able to reinitiate
this developmental program and contribute to the genesis of new coronary
vessels in injured myocardium (Lepilina et al., 2006; Poss, 2007).
Studies of EMT in explanted PE
(Mikawa and Gourdie, 1996) and EP
(Dettman et al., 1998) have identified
factors that regulate EMT. For example, both vascular-derived endothelial
growth factor (VEGF) and fibroblast
growth factor (FGF) stimulate EMT of
epicardial cells in vitro (Morabito et
al., 2001). Recently, we showed that
transforming growth factor ␤ (TGF␤)
induces EMT and smooth muscle differentiation in chick epicardial explants (Compton et al., 2006). TGF␤
ligands are abundantly expressed in
the developing heart (Akhurst et al.,
1990; Pelton et al., 1991; Dickson et
al., 1993; Jakowlew et al., 1994; Molin
et al., 2003) and are known to play a
prominent role in stimulating EMT
(Kalluri and Neilson, 2003) and
smooth muscle differentiation (Owens
et al., 2004). Three ligands, TGF␤1,
TGF␤2, and TGF␤3 (Roberts and
Sporn, 1990; Sanford et al., 1997; Hu
et al., 1998), bind four cell surface proteins. These include two transmembrane serine/threonine kinase receptors, the type I TGF␤ receptor
(TGF␤R1) and the type II TGF␤ receptor (TGF␤R2; Lin et al., 1992; Ebner
et al., 1993; Bassing et al., 1994). In
the canonical signaling pathway (Shi
and Massague, 2003) ligand binding
to TGF␤R2 results in recruitment of
the TGF␤R1, activin receptor-like kinase (ALK) 5, to the complex. The constitutively active kinase of TGF␤R2
phosphorylates and activates the kinase domain of ALK5, which subsequently phosphorylates and activates
downstream receptor associated (R-)
Smads 2 and 3 (Kretzschmar and
Massague, 1998). These activated RSmads complex with Smad 4 and
translocate into the nucleus to alter
gene transcription. TGF␤ can activate
additional downstream effectors including RhoA (Bhowmick et al.,
2001a, 2003; Edlund et al., 2002;
Masszi et al., 2003; Deaton et al.,
2005), Ras (Ward et al., 2002), mitogen activated protein (MAP) kinases
(Bhowmick et al., 2001b; Bakin et al.,
2002; Xie et al., 2004; Deaton et al.,
2005), and PI3K/AKt (Bakin et al.,
2000), although the mechanisms by
which TGF␤ regulates these effectors
is less well described. A second class of
TGF␤ binding proteins contains two
transmembrane proteins termed the
type III TGF␤ receptor (TGF␤R3), or
betaglycan, and endoglin. Both
TGF␤R3 and endoglin contain a short,
highly conserved intracellular domains with no apparent signaling
function (Lopez-Casillas et al., 1991;
Wang et al., 1991; Cheifetz et al.,
1992). Mutations in the endoglin gene
are linked to human hereditary hemorrhagic telangiectasia (McAllister et
al., 1994), whereas targeted inactivation of the gene encoding TGF␤R3 has
been shown to result in embryonic
death at E14.5 associated with a failure of coronary vessel development
(Compton et al., 2007).
TGF␤ induced EMT in both PE and
EP explants from chick embryos
(Compton et al., 2006; Olivey et al.,
2006). Experiments using EP explants
cultured with the addition of growth
factor, specific small molecule inhibitors, and adenoviral gene transfer
demonstrated that TGF␤-stimulated
loss of epithelial character was accompanied by smooth muscle differentiation (Compton et al., 2006). These effects of TGF␤ are dependent on ALK5
kinase activity, and ALK5 kinase activity is sufficient to induce EMT and
invasion of a three-dimensional matrix by epicardial cells. Overexpression of Smad 3 is not sufficient to induce cell invasion. The loss of
epithelial character in response to
TGF␤ requires the activity of the
downstream effectors, p160 rho kinase and p38 MAP kinase. Induction
of smooth muscle differentiation requires p160 rho kinase but not p38
MAP kinase.
To determine whether TGF␤ signaling pathways play a similar role in
regulating epicardial cells in mammals, we characterized mouse epicardial cell explants. We have defined
culture conditions and demonstrated
the ability to use adenoviral gene
transfer to introduce genes of interest
into mouse explants. We determined
that, similar to the chick, the addition
of TGF␤1 or TGF␤2 causes the loss of
epithelial character and smooth muscle differentiation in mouse epicardial
cells. The type I TGF␤ receptor ALK5
is both required and sufficient to mediate EMT and smooth muscle differentiation in mouse epicardial cells.
Immortalized epicardial cells were
generated using a transgenic mouse
where the large T antigen is temperature regulated (Jat et al., 1991). Immortalized epicardial cells retain the
expression of the epicardial cell
marker WT1 and respond to TGF␤ in
a manner indistinguishable from primary epicardial cells. These data demonstrate that TGF␤ regulates epicardial cell differentiation in the mouse
and that immortalized epicardial cells
may be used as a model system to
study smooth muscle differentiation.
RESULTS
TGF␤1 or TGF␤2 Causes the
Loss of Epithelial Character
and Smooth Muscle
Differentiation
Epicardial explants from E11.5 mice
were incubated with vehicle, 250 pM
TGF␤1 or 250 pM TGF␤2 for 72 hr.
We examined responses to both
TGF␤1 and TGF␤2, because these two
ligands are known to differ in binding
affinity for TGF␤R2 (Lin and Moustakas, 1994). As soon as 24 hr after ligand addition, epicardial explants incubated with TGF␤ appeared less
epithelial when compared with vehicle. At 72 hr, cells in epicardial explants incubated with vehicle are compact and display a rounded, epithelial
phenotype (Fig. 1A). Cells in explants
incubated with TGF␤1 or TGF␤2 are
elongated and separated from one another (Fig. 1B,C). Immunostaining for
zonula occludens-1 (ZO-1), a tight
junction protein indicative of an epithelial phenotype, demonstrates abundant
staining at 72 hr in vehicle-incubated
368 AUSTIN ET AL.
explants (Fig. 1D) and a significant decrease in explants incubated with
TGF␤1 or TGF␤2 (Fig. 1E,F). Similarly,
explants incubated with vehicle show a
perinuclear staining pattern for cytokeratin and incubation with TGF␤1 or
TGF␤2 decreased cytokeratin expression (data not shown). These data demonstrate that TGF␤ causes a loss of epithelial character in epicardial cells.
Previously, we demonstrated that
chick epicardial cells lose epithelial
character and undergo differentiation
to smooth muscle in response to TGF␤
(Compton et al., 2006). Therefore, we
examined specific markers of smooth
muscle differentiation after incubation with TGF␤ (Fig. 1G–L). Explants
incubated with vehicle display little
expression of SM22␣ (Fig. 1G). Cells
in explants incubated with TGF␤1 or
TGF␤2 (Fig. 1H,I) demonstrate increased expression of SM22␣ found in
organized fibers consistent with a
smooth muscle phenotype. Similarly,
cells in explants incubated with vehicle lack calponin expression (Fig. 1J),
whereas incubation with TGF␤1 or
TGF␤2 (Fig. 1K,L) increases the expression of calponin. These data demonstrate that TGF␤ causes the loss of
epithelial character and initiates
smooth muscle differentiation of epicardial explants.
ALK5 Kinase Activity Is
Required for Epicardial Cell
Differentiation
To determine whether ALK5 activity,
a downstream molecule of TGF␤, was
required for the effects of TGF␤, we
incubated the explants on collagencoated slides with or without the
ALK5 kinase inhibitor SB431542. Explants were incubated with 2.5 ␮M
SB431542 in the presence of vehicle,
250 pM TGF␤1 or 250 pM TGF␤2 for
72 hr before fixation. Explants incubated in the presence of SB431542
and vehicle display an epithelial phenotype comparable to cells incubated
with vehicle alone (compare Fig. 2A
with 1A). In contrast, cells in explants
incubated in the presence of SB431542
and TGF␤1 or TGF␤2 did not elongate
or separate but retained an epithelial
appearance (compare Fig. 2B,C with
1B,C). Consistent with this observation
epicardial cells incubated with vehicle,
TGF␤1, or TGF␤2 in the presence of
Fig. 1. Transforming growth factor ␤ (TGF␤) induces epithelial–mesenchymal transformation
(EMT) in epicardial cells. Epicardial explants were incubated with vehicle, 250 pM TGF␤1 or 250 pM
TGF␤2 for 72 hr before fixation. A: Cells in explants incubated with vehicle are compact and display
a rounded, epithelial phenotype. B,C: Cells in explants incubated with TGF␤1 or TGF␤2 are
elongated and separated from one another. D: Cells in explants incubated with vehicle express
zonula occludens-1 (ZO-1) along the cell– cell borders consistent with an epithelial phenotype.
E,F: Cells incubated with TGF␤1 (E) or TGF␤2 (F) are elongate and have lost cell– cell contacts and
ZO-1 expression. G: Cells in explants incubated with vehicle lack distinct SM22␣ expression. H,I:
Cells incubated with TGF␤1 (H) or TGF␤2 (I) express SM22␣ in organized fibers consistent with a
smooth muscle phenotype. J: Cells in explants incubated with vehicle lack expression of calponin.
K,L: Cells incubated with TGF␤1 (K) or TGF␤2 (L) express calponin in a pattern of organized
extended fibers consistent with smooth muscle. Original magnification, ⫻100 in A–C, ⫻400 in D–L.
SB431542 retain the expression of ZO-1
(Fig. 2D–F). These data demonstrate
that inhibition of ALK5 kinase activity prevents loss of epithelial character in response to TGF␤. To determine
whether kinase activity is required for
smooth muscle differentiation, we examined the expression of the smooth
muscle markers SM22␣ and calponin.
Cells in epicardial explants incubated
with vehicle and SB431542 fail to express SM22␣ or calponin (Fig. 2G,J).
Cells incubated with TGF␤1 or TGF␤2
in the presence of SB431542 fail to express SM22␣ or calponin (compare Fig.
2H,I,K,L with 1H,I,K,L). These data
demonstrate that ALK5 kinase activity
is required for TGF␤-stimulated loss of
TGF〉 AND EPICARDIAL CELLS 369
TGF␤2 and analyzed as described
above. The addition of 10 ␮g/ml
Y27632, a specific p160 rho kinase inhibitor, had no apparent morphological effect on vehicle-incubated explants (Fig. 3A,D,G,J) and did not
completely block the loss of epithelial
character in response to TGF␤1 or
TGF␤2 (Fig. 3A–F). However, Y27632
effectively blocked the expression of
SM22␣ in response to either TGF␤1 or
TGF␤2 (Fig. 3, compare G–I with
J–L). MAP kinase is a known downstream mediator of TGF␤ signaling
and p38 MAP kinase has been implicated specifically in TGF␤-induced
EMT (Bhowmick et al., 2001b; Bakin
et al., 2002). The addition of 4 ␮M
SB202190, a p38 MAP kinase inhibitor, had no discernable effect on explants incubated with vehicle or on
the effects of TGF␤1 or TGF␤2 (Fig.
S1). These data demonstrate a requirement for p160 rho kinase activity
in mediating the expression of smooth
muscle markers in response to TGF␤.
Immortalized Epicardial
Cells Respond to TGF␤
Fig. 2. Activin receptor-like kinase 5 (ALK5) is required for the effects of transforming growth
factor ␤ (TGF␤) in epicardial explants. Epicardial explants were incubated with vehicle, 250 pM
TGF␤1 or 250 pM TGF␤2 in the presence of 2.5 ␮M SB431542, an ALK5 kinase inhibitor, for 72 hr
before fixation. A: Epicardial explants incubated with vehicle display an epithelial phenotype.
B,C: Cells incubated with TGF␤1 (B) or TGF␤2 (C) in the presence of inhibitor remain round and
compact consistent with epithelial phenotype. D–F: Cells incubated with vehicle (D), TGF␤1 (E), or
TGF␤2 (F) in the presence of inhibitor retain zonula occludens-1 (ZO-1) expression at cell– cell
borders consistent with an epithelial phenotype. G–I: Cells in explants incubated with vehicle (G),
TGF␤1 (H) or TGF␤2 (I) in the presence of inhibitor failed to express the smooth muscle marker
SM22␣. J–L: Incubation of cells with vehicle (J), TGF␤1 (K), or TGF␤2 (L) in the presence of inhibitor
failed to express the smooth muscle marker calponin. ⫻100 in A–C; ⫻400 in D–L.
epithelial character and smooth muscle
differentiation.
TGF␤ Effects on Smooth
Muscle Differentiation
Require p160 rho Kinase but
not p38 MAP Kinase Activity
RhoA is a known TGF␤ effector that
has also been shown to signal smooth
muscle differentiation in response to
platelet derived growth factor (PDGF;
Lu et al., 2001). To address the potential role of RhoA in TGF␤-stimulated
loss of epithelial character and smooth
muscle differentiation in epicardial
explants, we targeted p160 rho kinase, a downstream effector of RhoA.
Epicardial explants were harvested
and incubated with vehicle, TGF␤1, or
Immortalized epicardial cells were examined for responsiveness to TGF␤stimulated loss of epithelial character
and smooth muscle differentiation.
Cells were isolated as described and
had been in culture for at least 3
months. Before assay cells were
switched from immortomedium to
standard growth medium for 24 hr,
vehicle or ligand added, and the incubation continued for an additional 72
hr. Cells from E11.5 embryos retain
expression of the epicardial marker
WT1 (Fig. S2) and form tightly packed
epithelia (Fig. 4A,D). The addition of
either TGF␤1 or TGF␤2 resulted in
elongation and separation of cells and
the loss of ZO-1 expression (Fig.
4B,C,E,F). As in primary epicardial
explants, the addition of TGF␤1 or
TGF␤2 increased the expression of
both SM22␣ and calponin (Fig. 4G–L).
To determine whether ALK5 kinase
activity is required for the effects of
TGF␤, these same measures were performed in cells incubated with 2.5 ␮M
SB431542. The results obtained were
comparable to those obtained in primary epicardial explants. The ALK5
kinase inhibitor effectively blocked
TGF␤-stimulated loss of epithelial
370 AUSTIN ET AL.
character and the expression of both
SM22␣ and calponin (Fig. 5). Cells
incubated in the presence of
SB431542 and vehicle display an epithelial phenotype comparable to
cells incubated with vehicle alone
(compare Figs. 5A and 4A). Similar
results were obtained with cells immortalized from E10.5 and E13.5
embryos. Therefore, both cells in primary epicardial explants and immortalized cells respond similarly to
TGF␤, and this response requires
ALK5 kinase activity.
To compare the potency of TGF␤1
and TGF␤2 in inducing smooth muscle gene expression, E10.5 Sm22␣lacZ::Immorto epicardial cells were
incubated with TGF␤1 or TGF␤2
and monitored for lacZ activity (Fig.
6A,B). Incubation with concentrations from 125 to 1250 pM TGF␤1 or
TGF␤2 demonstrated a dose-dependent increase in lacZ activity with a
similar maximal response and effective concentration for 50% maximal
response (Fig. 6C). These data demonstrate similar potency for TGF␤1
or TGF␤2 in inducing SM22␣ gene
expression.
ALK5 Activity Is Sufficient
to Induce Loss of Epithelial
Character and Smooth
Muscle Differentiation
To determine whether ALK5 activity
is sufficient to induce differentiation
of epicardial cells, we infected immortalized epicardial cells from
E11.5 embryos with adenovirus encoding either constitutively active
(ca) ALK5 and green fluorescent protein (GFP) or GFP alone. The titer of
the adenovirus was adjusted so as to
infect only a fraction of the epicardial cells to allow for the scoring of
individual GFP-positive cells. Overexpression of caALK5 resulted in
loss of ZO-1 from the membrane and
a concomitant detachment of the cell
from the epithelial sheet (Fig. 7).
Overexpression of caALK5 also induced SM22␣ expression (data not
shown). Quantitation of the percent
of cells that undergo transformation
after infection with adenovirus coexpressing caALK5 and GFP or GFP
alone in immortalized cells revealed
that caALK5 expression was sufficient to cause loss of epithelial char-
Fig. 3. Inhibition of p160 rho kinase blocks transforming growth factor ␤ (TGF␤) -stimulated
SM22␣ expression in epicardial cells. Epicardial explants were incubated with vehicle, 250 pM
TGF␤1 or 250 pM TGF␤2 for 72 hr before fixation. A–C: Cells incubated with vehicle (A), 250 pM
TGF␤1 (B), or 250 pM TGF␤2 (C). TGF␤1 and TGF␤2 cause the loss of zonula occludens-1 (ZO-1)
expression at cell– cell borders. D–F: Cells were incubated with vehicle (D), 250 pM TGF␤1 (E), or
250 pM TGF␤2 (F) in the presence of 10 ␮g/ml Y27632, a p160 rho kinase inhibitor. Cells elongate
and have diminished cell– cell contacts and ZO-1 expression. G–I: Cells incubated with vehicle (G),
250 pM TGF␤1 (H), or 250 pM TGF␤2 (I). Cells incubated with vehicle did not express SM22␣ (G).
Cells incubated with TGF␤1 or TGF␤2 are elongate and display SM22␣ expression (H,I). J–L: Cells
preincubated with 10 ␮g/ml Y27632 before the addition of vehicle (J), TGF␤1 (K), or TGF␤2 (L)
lacked SM22␣ expression. ⫻400 in A–L.
acter. Only 20% of cells expressing
GFP only were scored as transformed, whereas 80% of cells expressing caALK5 were transformed
(Fig. 7). These results demonstrate
that ALK5 kinase activity is sufficient to induce both loss of epithelial
character and smooth muscle differentiation in epicardial cells.
Smooth Muscle
Differentiation Requires
p160 rho Kinase
Experiments next addressed the role
of p160 rho kinase and p38 MAPK in
mediating the effects of TGF␤ in immortalized epicardial cells. The addition of the specific p160 rho kinase
TGF〉 AND EPICARDIAL CELLS 371
markers in response to TGF␤ in both
primary and immortalized epicardial
cells.
DISCUSSION
Fig. 4. Transforming growth factor ␤ (TGF␤) induces epithelial–mesenchymal transformation
(EMT) in immortalized epicardial cells. Cells were incubated with vehicle, 250 pM TGF␤1 or 250 pM
TGF␤2 for 72 hr before fixation. A–C: Cells incubated with vehicle form a compact monolayer and
display a rounded, epithelial phenotype. A: Cells incubated with TGF␤1 (B) or TGF␤2 (C) are
elongated and separated from one another. D–F: Cells incubated with vehicle express zonula
occludens-1 (ZO-1) along the cell– cell borders consistent with an epithelial phenotype (D). Cells
incubated with TGF␤1 (E) or TGF␤2 (F) are elongate and have decreased cell– cell contacts and
ZO-1 expression. G–I: Cells incubated with vehicle lack SM22␣ expression (G). Cells incubated with
TGF␤1 (H) or TGF␤2 (I) express SM22␣ in organized fibers consistent with a smooth muscle
phenotype. J–L: Cells incubated with vehicle lack expression of calponin (J). Cells incubated with
TGF␤1 (K) or TGF␤2 (L) express calponin in a pattern of organized extended fibers in the cytoskeleton. ⫻100 in A–C; ⫻400 in D–L.
inhibitor Y27632 to immortalized epicardial cells had actions comparable
to that seen in primary explants.
Y27632 did not completely block the
loss of epithelial character in response
to TGF␤1 or TGF␤2 (Fig. S3A–F).
However, as in primary explants,
Y27632 effectively blocked the expression of SM22␣ in response to either
TGF␤1 or TGF␤2 (Fig. S3G–L). The
addition of 4 ␮M SB202190, a p38
MAP kinase inhibitor, had no discernable effect on explants incubated with
vehicle or on the effects of TGF␤1 or
TGF␤2 (Fig. S4). These data demonstrate comparable requirements for
p160 rho kinase activity in mediating
the expression of smooth muscle
Here, we demonstrate that TGF␤1
and TGF␤2 induce loss of epithelial
character and the appearance of
smooth muscle markers in mouse epicardial cells. To facilitate the study of
epicardial cell differentiation, we generated immortalized epicardial cells
and determined that immortalized
cells respond to TGF␤1 and TGF␤2 in
a manner indistinguishable from primary cells. ALK5 kinase activity is
both required and sufficient for the
effects of TGF␤. Inhibition of p160 rho
kinase activity blocks the effects of
TGF␤ on smooth muscle differentiation, whereas the inhibition of p38
MAPK activity is without effect.
These data demonstrate that the effects of TGF␤ on mouse epicardial
cells are similar to those we described
in chick epicardial cells (Compton et
al., 2006). Our observations support a
role for TGF␤ in the regulation of epicardial cell differentiation during coronary vessel development.
These data are consistent with the
well-described actions of TGF␤ in mediating EMT in several experimental
systems and cell types (Kalluri and
Neilson, 2003). However, Morabito et
al. (2001) noted that addition of
TGF␤2 or TGF␤3 to epicardial explants did not support invasion of cells
into a collagen matrix, whereas
TGF␤1 weakly stimulated invasion.
All TGF␤ isoforms inhibited FGF2
and heart conditioned media-stimulated EMT. Our experiments in chick,
and now mouse, demonstrate that
TGF␤1 and TGF␤2 cause the loss of
epithelial character as measured by
altered morphology and ZO1 expression. These effects are blocked by inhibition of ALK5 kinase activity, while
overexpression of caALK5, which activates the canonical TGF␤ signaling
pathway, results in the loss of epithelial
character. Together, these data suggest
an important role for TGF␤ in supporting epicardial cell EMT.
In both primary and immortalized
cells, the addition of TGF␤ induces
loss of epithelial character and smooth
muscle differentiation in the majority
of the cells examined, suggesting that
372 AUSTIN ET AL.
Fig. 6. Transforming growth factor (TGF) induces differentiation and Sm22␣-lacZ expression
in immortalized epicardial cells. Sm22␣-lacZ::Immorto epicardial cells were isolated from embryonic day (E) 10.5 embryos and assayed for lacZ
activity. A,B: Cells incubated with 250 pM TGF␤1
(A) or 250 pM TGF␤2 (B) for 72 hr and stained for
lacZ activity. Cells underwent transformation and
displayed a flattened smooth muscle phenotype.
Vehicle-incubated cells displayed very low levels
of single cell staining (not shown). C: To determine the dose dependence of Sm22␣-lacZ, expression cells were incubated with increasing
concentrations of TGF␤1 (triangles) or TGF␤2
(squares) for 72 hr in triplicate. Sm22␣-lacZ activity was assayed with Galacto-light Plus assay.
Luminescence readings were normalized by dividing the relative light unit readings for each
sample by the mean value for the corresponding
mock incubated cells. Data points presented are
mean fold induction of triplicate samples ⫾ SEM.
Vehicle-incubation readings were no different
from mock incubation.
Fig. 5. Activin receptor-like kinase 5 (ALK5) is required for the effects of transforming growth
factor ␤ (TGF␤) in immortalized epicardial cells. Cells were incubated with vehicle, 250 pM TGF␤1
or 250 pM TGF␤2 in the presence of 2.5 ␮M SB431542, an ALK5 kinase inhibitor, for 72 hr before
fixation. A–C: Cells incubated with vehicle form a compact monolayer and display a rounded,
epithelial phenotype (A). Cells incubated with TGF␤1 (B) or TGF␤2 (C) remain round and compact,
consistent with an epithelial phenotype. D–F: Cells incubated with vehicle express zonula occludens-1 (ZO-1) along the cell– cell borders (D). Cells incubated with TGF␤1 (E) or TGF␤2 (F) retain
ZO-1 expression. G–I: Cells incubated with vehicle did not express SM22␣, a smooth muscle
marker (G). Cells incubated with TGF␤1 (H) or TGF␤2 (I) fail to express SM22␣ in response to TGF␤.
J–L: Cells incubated with vehicle (J), TGF␤1 (K), or TGF␤2 (L) fail to express calponin. Original
magnification, ⫻100 in A–C, ⫻400 in D–L.
most, if not all, epicardial cells have
the capacity to assume a smooth muscle cell phenotype. In vivo, many epicardial cells remain epithelial and
only a fraction of cells undergo EMT,
invade the subepicardial matrix and
the myocardium, and differentiate
into smooth muscle cells. This finding
suggests that the restriction of TGF␤
ligand availability to the epicardium
may be an important mechanism to
regulate EMT and smooth muscle cell
differentiation. Although TGF␤ ligands are abundantly expressed in
the myocardium as well as the epicardium in vivo (Akhurst et al., 1990;
Pelton et al., 1991; Dickson et al.,
1993; Jakowlew et al., 1994; Molin et
al., 2003) the ligands are made as inactive precursors that can be stored in
Fig. 7.
TGF〉 AND EPICARDIAL CELLS 373
the extracellular matrix and later activated (Annes et al., 2003). Therefore,
despite the observation that mRNA
expression of the ligands is discretely
localized, the protein is found
throughout the embryo in the extracellular matrix (Ghosh and Brauer,
1996) consistent with the localized activation of ligand being an important
regulatory event.
The effects of TGF␤ in both epicardial explants and immortalized cells
require the activity of both ALK5 and
p160 rho kinase. RhoA has been implicated as a downstream mediator of
TGF␤ in multiple systems. Both pharmacological and genetic approaches in
nontransformed murine mammary
epithelial cells reveal a requirement
for RhoA/p160 rho kinase in mediating TGF␤-stimulated EMT (Bhowmick et al., 2001b). In LLC-PK1 cells,
a proximal tubule epithelial porcine
cell line, RhoA signaling downstream
of TGF␤ stimulates EMT accompanied by up-regulation of smooth muscle ␣-actin (SM␣A) gene expression
(Masszi et al., 2003). Although EMT
was not measured directly, both longand short-term actin reorganization is
stimulated by TGF␤ in a RhoA-dependent manner in human prostate carcinoma cells (Edlund et al., 2002). Our
data in mouse epicardial cells also
support a role for RhoA in signaling
loss of epithelial character downstream of TGF␤.
TGF␤ is important in recruiting undifferentiated mesenchyme into the
smooth muscle cell lineage during
blood vessel assembly and remodeling
(Grainger et al., 1998; Darland and
D’Amore, 2001; Owens et al., 2004).
TGF␤ or conditioned medium from endothelial cells up-regulates smooth
Fig. 7. Activin receptor-like kinase 5 (ALK5) is
sufficient to induce the effects of transforming
growth factor ␤ (TGF␤) in immortalized cells.
Cells were infected with adenovirus expressing
green fluorescent protein (GFP) or GFP and
constitutively active (ca) ALK5. A,B: Photomicrographs of infected cells. GFP-positive cells
express zonula occludens-1 (ZO-1) at cell– cell
borders (A). Cells overexpressing caALK5 show
absent or discontinuous ZO-1 expression (B).
C: Quantitation of the percent of cells that undergo transformation after infection with adenovirus. Data are plotted as the mean percent ⫾ SEM of three independent experiments
(P ⬍ 0.05). Original magnification, ⫻400 in A,B.
muscle myosin, SM22␣, and calponin
in embryonic 10T1/2 cells (Hirschi et
al., 1998). Up-regulation of smooth
muscle proteins by endothelial cell
conditioned medium was blocked by
preincubation with neutralizing antisera to TGF␤. Subsequent studies
showed TGF␤-mediated induction of
the late phenotypic marker smooth
muscle ␥-actin by means of serum response factor (Hirschi et al., 2002).
TGF␤ also induces smooth muscle cell
differentiation in both embryonic
stem cells (Sinha et al., 2004) and neural crest stem cells (Chen and Lechleider, 2004). Using explanted quail
hearts as an in vitro model of coronary
vasculogenesis, TGF␤ was found to inhibit endothelial cell tube formation, a
result consistent with TGF␤ inducing
smooth muscle cell differentiation at
the expense of tube formation (Holifield et al., 2004). Although targeted
deletion of the gene encoding TGF␤R3
results in a failure of coronary vessel
development, smooth muscle recruitment to the nascent vessels that do
form appears to occur normally
(Compton et al., 2007). Because the
null mice die during the time of
smooth muscle recruitment, a role for
TGF␤R3 during later stages of coronary vascular smooth muscle recruitment or differentiation cannot be excluded. However, at least the initial
stages of coronary smooth muscle differentiation and recruitment may be
independent of TGF␤R3.
Both PDGF -BB- and TGF␤-stimulated smooth muscle differentiation
requires the activity of rhoA and p160
rhokinase (Lu et al., 2001; Compton et
al., 2006). RhoA and p160 rho kinase
regulate the expression of two major
smooth muscle marker genes, SM22␣
and smooth muscle ␣ actin (SM␣A), in
rat thoracic aorta smooth muscle cells
(Mack et al., 2001). In rat pulmonary
artery smooth muscle cells, RhoA signals by means of both p160 rho kinase
dependent and independent pathways
to activate smooth muscle gene expression (Deaton et al., 2005). A chemical inhibitor of p160 rho kinase was
incubated with quail PE before isochronic transplant into chick embryos
to directly address the role of RhoA in
the developing coronary vasculature
(Lu et al., 2001). Although some quail
cells entered the subepicardial matrix, none differentiated into smooth
muscle cells or contributed to the coronary vasculature. Our data in both
chick (Compton et al., 2007) and
mouse are consistent with a RhoA and
p160 rho kinase dependent pathway
downstream of TGF␤ in epicardial
cells that regulates smooth muscle
gene expression. p38 MAP kinase has
also been implicated in TGF␤-stimulated EMT (Bhowmick et al., 2001b;
Bakin et al., 2002; Deaton et al.,
2005). TGF␤-stimulated EMT in nontransformed murine mammary epithelial cells is dependent on both
RhoA and p38 MAP kinase activity
leading to the suggestion that p38
MAP kinase might function downstream of RhoA (Bhowmick et al.,
2001a; Bakin et al., 2002; Deaton et
al., 2005). However, experiments using a dominant negative RhoA or a
p160 rho kinase inhibitor showed that
p38 MAP kinase activity is independent of RhoA activity (Bakin et al.,
2002). Studies of TGF␤ regulation of
actin cytoskeleton mobilization in human prostate carcinoma cells showed
that p38 MAPK functions in a pathway parallel to RhoA and downstream
of Cdc42 (Edlund et al., 2002). Therefore, several lines of evidence support
a model where RhoA and p38 MAP
kinase are in separate pathways
downstream of TGF␤. Our data, although implicating RhoA in smooth
muscle differentiation, fails to identify
a role for p38 MAPK in either epicardial cell EMT or smooth muscle differentiation.
Our data demonstrate that TGF␤
signaling by means of the canonical
type I receptor induces loss of epithelial character and smooth muscle cell
differentiation in both primary and
immortalized epicardial cells. ALK5
activity is both required and sufficient
for the effects of TGF␤. Smooth muscle differentiation in epicardial cells
requires p160rho kinase activity but
not p38 MAP kinase activity. These
observations suggest that TGF␤ plays
an important role in the recruitment
and differentiation of epicardial cells
into coronary smooth muscle cells.
Our characterization of immortalized
epicardial cells with properties comparable to primary cells suggests that
these cells may be an important experimental tool to probe epicardial cell
differentiation. The generation of immortalized epicardial cells from trans-
374 AUSTIN ET AL.
genic mice with epicardial or coronary
vessel defects will provide a powerful
approach to explore the role of specific
genes in epicardial cell behavior.
EXPERIMENTAL
PROCEDURES
Epicardial Explant Culture
Mouse hearts (E10.5, E11.5, and
E13.5) were harvested in Hanks buffered salt solution (HBSS) and cultured by modification of the method of
Compton (Compton et al., 2006). Embryonic hearts were placed dorsal side
down on collagen culture slides (BD
Bioscience, Bedford, MA) covered with
explant medium (M199, 5% heat inactivated fetal bovine serum (FBS) and
1:400 antibiotic/antimycotic) and cultured at 37°C, 5% CO2. After 12–15
hr, the hearts were removed to reveal
epicardial explants as monolayers attached to the collagen coated surface.
Explants were washed twice with
phosphate buffered saline (PBS), covered with explant medium, and cultured for up to 72 hr. Vehicle (TGF␤ 0.1% Bovine Serum Albumen in 4 mM
HCl; inhibitors - DMSO) or ligand was
added to the explants immediately after removal of the heart where indicated.
Immortalized Epicardial
Explant Culture
To generate inducible immortalized epicardial cell line wild type or Sm22␣lacZ mice were crossed with the ImmortoMouse line (Jat et al., 1991).
ImmortoMouse contains an interferon
inducible temperature sensitive large T
antigen that renders derived cells conditionally immortalized at 33°C in the
presence of interferon gamma. Mouse
hearts at E10.5, E11.5, and E13.5 were
placed in culture and epicardial cells
derived as above. Cells were propagated
at 33°C in DMEM 10% heat inactivated
FBS, insulin-transerrin-selenium (Biosource) and 10 units/ml mouse gamma
interferon (Peprotech). For differentiation cells were transferred to standard
M199 media without interferon as
described and cultured at 37°C for
24 hr before the 72-hr differentiation
protocol.
Immunohistochemistry
For SM22␣ (Abcam) and calponin
(Sigma) staining, explants were fixed
with 2% paraformaldehyde (PFA) for 30
min at room temperature and permeabilized with PBS and 0.1% Triton
X-100 for 5 min. Explants were fixed in
70% methanol before staining for ZO-1.
Adenovirus infected epicardial explants
for ZO-1 staining were fixed in 2% PFA
and permeabilized with 0.2% Triton
X-100 for 5 min. Explants for calponin
and ZO-1 staining were blocked with
2% bovine serum albumin in PBS for 1
hr and incubated with dilute primary
antibody (calponin, 1:400; ZO-1, 2 ␮g/
ml) overnight at 4°C. ZO-1 staining for
adenoviral-infected explants was incubated with the primary antibody for
5– 6 hr. Explants for SM22␣ staining
were blocked with 5% horse serum, and
incubated with primary antibody
(SM22␣, 1:200) overnight at 4°C. Primary antibody detection was with goat
anti-mouse cy3 (calponin), goat antirabbit cy3 (ZO-1), or donkey anti-goat
cy3 (SM22␣) secondary antibody (1:800;
Jackson ImmunoResearch). Nuclei
were stained with 4⬘,6-diamidino-2phenylinodole (DAPI; Sigma). Photomicrographs were captured with Nikon
Eclipse TE2000-E microscope and QED
imaging software or Nikon microscope
and camera with 160T film (Kodak).
Growth Factor or Inhibitor
Addition
Growth factors (TGF␤1, 250 pM;
TGF␤2, 250 pM) or small molecule inhibitors (SB431542, 2.5 ␮M; Y27632, 10
␮g/ml; SB202190, 4 ␮M) were added to
the medium immediately after removal
of the hearts. Explants incubated with
inhibitor and growth factor on collagencoated chamber slides were preincubated with medium containing inhibitor alone for 1 hr at 37°C. Afterward,
fresh medium and inhibitor were added
to explants and incubation continued.
Reagents were obtained from the following sources: TGF␤1 & TGF␤2 from
R&D Systems, SB202190 & Y27632
from Calbiochem, and SB431542 from
Sigma-Aldrich.
Adenoviral Infection of
Explants
Mouse hearts (E11.5) were harvested,
rinsed in HBSS, and incubated at
37°C for 30 min with approximately
107 PFU adenovirus (GFP alone or
caALK5 and GFP; Desgrosellier et al.,
2005). Hearts were then placed on collagen culture slides and cultured as
above. GFP-positive cells on collagen
culture slides were digitally photographed using the Nikon epifluoresence microscope and QED Imaging
software.
Scoring of Cell
Transformation
Immortalized epicardial cells were
plated on collagen-coated chamberslides at a density of 150,000 cells/well
in medium M199 with 5% fetal calf
serum (FCS). Adenovirus expressing
GFP or coexpressing caALK5 and
GFP at a titer of 107 PFU was added
and the incubation continued at 37°C
for 72 hr. Cells were fixed in 70%
methanol and immunostained for
ZO-1 as described. GFP-expressing
cells in random fields were scored as
either expressing ZO-1 in a continuous pattern at cell margins, rounded,
and in the monolayer (epithelial) or
expressing ZO-1 in a discontinuous
pattern at cell margins, elongated,
and detached from the cell monolayer
(transformed). A minimum of 100 cells
in both the GFP and GFP/caALK5
groups were scored and the percentage of epithelial and transformed cells
determined (Compton et al., 2006).
The experiment was performed three
times, the mean percentages determined, and analyzed by students ttest.
Measurement of lacZ
Staining and Activity
Immortalized E10.5 Sm22␣-lacZ::Immorto epicardial cells were plated in uncoated 24-well tissue culture dishes at a
density of 150,000 cells/well in medium
M199 with 5% FCS. After 12 hr, growth
factor (TGF␤1, TGF␤2, at 125, 250, 625,
1,250 pM) was added and cells incubated for 72 hr. One well was fixed with
2% PFA for 10 min and stained for beta
galactosidase visualization with X-gal.
Cells were incubated overnight at 37°C
in X-gal stain solution (2 mM MgCl2, 5
mM potassium ferricyanide (Sigma
P-3667), 5 mM potassium hexacyanoferrate(II)trihydrate (Sigma P-9387),
0.01% Np-40, 0.1% sodium deoxy-
TGF〉 AND EPICARDIAL CELLS 375
cholate (Fisher BP349), 0.1% X-gal(5bromo-4-chloro-3-indolyl-b-D galactopyranoside; RPI B718001) in Dulbecco’s
phosphate buffered saline (pH 8.0). Additional wells in triplicate for each concentration were assayed for beta-galactosidase activity with the Galacto-Light
Plus system (Applied Biosystems) according to company protocol. Luminescence was quantitated on a Turner
20/20 Luminometer. The quantitation
experiment was repeated twice with
comparable results.
ACKNOWLEDGMENTS
The authors thank members of the
Barnett laboratory for helpful discussions and comments. J.V.B. acknowledges the support of the VanderbiltIngram Cancer Center. J.V.B., A.F.A.,
and L.A.C. were funded by the
NIH. J.V.B. was funded by the American Heart Association.
REFERENCES
Akhurst RJ, Lehnert SA, Faissner A, Duffie E. 1990. TGF beta in murine morphogenetic processes: the early embryo and
cardiogenesis. Development 108:645–
656.
American Heart Association. 2003. Heart
disease and stroke statistics - 2004. Update. Dallas, TX: American Heart Association.
Annes JP, Munger JS, Rifkin DB. 2003.
Making sense of latent TGFbeta activation. J Cell Sci 116:217–224.
Bakin AV, Tomlinson AK, Bhowmick NA,
Moses HL, Arteaga CL. 2000. Phosphatidylinositol 3-kinase function is required for transforming growth factor
beta -mediated epithelial to mesenchymal transition and cell migration. J Biol
Chem 275:36803–36810.
Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL. 2002. p38 mitogen-activated
protein kinase is required for TGFbetamediated fibroblastic transdifferentiation and cell migration. J Cell Sci 115:
3193–3206.
Bassing CH, Yingling JM, Howe DJ, Wang
T, He WW, Gustafson ML, Shah P,
Donahoe PK, Wang XF. 1994. A transforming growth factor beta type I receptor that signals to activate gene expression. Science 263:87–89.
Bhowmick NA, Ghiassi M, Bakin A, Aakre
M, Lundquist CA, Engel ME, Arteaga
CL, Moses HL. 2001a. Transforming
growth factor-{beta}1 mediates epithelial
to mesenchymal transdifferentiation
through a Rhoa-dependent mechanism.
Mol Biol Cell 12:27–36.
Bhowmick NA, Zent R, Ghiassi M, McDonnell M, Moses HL. 2001b. Integrin beta 1
signaling is necessary for transforming
growth
factor-beta
activation
of
p38MAPK and epithelial plasticity.
J Biol Chem 276:46707–46713.
Bhowmick NA, Ghiassi M, Aakre M,
Brown K, Singh V, Moses HL. 2003.
TGF-beta-induced RhoA and p160ROCK
activation is involved in the inhibition of
Cdc25A with resultant cell-cycle arrest.
Proc Natl Acad Sci U S A 100:15548 –
15553.
Cheifetz S, Bellon T, Cales C, Vera S,
Bernabeu C, Massague J, Letarte M.
1992. Endoglin is a component of the
transforming growth factor-beta receptor system in human endothelial cells.
J Biol Chem 267:19027–19030.
Chen S, Lechleider RJ. 2004. Transforming
growth factor-beta-induced differentiation of smooth muscle from a neural
crest stem cell line. Circ Res 94:1195–
1202.
Compton LA, Potash DA, Mundell NA,
Barnett JV. 2006. Transforming growth
factor-beta induces loss of epithelial
character and smooth muscle cell differentiation in epicardial cells. Dev Dyn 235:
82–93.
Compton LA, Potash DA, Brown CB, Barnett JV. 2007. Coronary vessel development is dependent on the type III transforming growth factor beta receptor. Circ
Res 101:784 –791.
Darland DC, D’Amore PA. 2001. TGF beta
is required for the formation of capillarylike structures in three-dimensional cocultures of 10T1/2 and endothelial cells.
Angiogenesis 4:11–20.
Deaton RA, Su C, Valencia TG, Grant SR.
2005. Transforming growth factor-beta
1-induced expression of smooth muscle
marker genes involves activation of PKN
and p38 MAP kinase. J Biol Chem 280:
31172–31181.
Desgrosellier JS, Mundell NA, McDonnell
MA, Moses HL, Barnett JV. 2005. Activin receptor-like kinase 2 and Smad6
regulate epithelial-mesenchymal transformation during cardiac valve formation. Dev Biol 280:201–210.
Dettman RW, Denetclaw W Jr, Ordahl CP,
Bristow J. 1998. Common epicardial origin of coronary vascular smooth muscle,
perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart.
Dev Biol 193:169 –181.
Dickson MC, Slager HG, Duffie E, Mummery CL, Akhurst RJ. 1993. RNA and
protein localisations of TGF beta 2 in the
early mouse embryo suggest an involvement in cardiac development. Development 117:625–639.
Ebner R, Chen RH, Shum L, Lawler S,
Zioncheck TF, Lee A, Lopez AR, Derynck
R. 1993. Cloning of a type I TGF-beta
receptor and its effect on TGF-beta binding to the type II receptor. Science 260:
1344 –1348.
Edlund S, Landstrom M, Heldin CH, Aspenstrom P. 2002. Transforming growth
factor-beta-induced mobilization of actin
cytoskeleton requires signaling by small
GTPases Cdc42 and RhoA. Mol Biol Cell
13:902–914.
Ghosh S, Brauer PR. 1996. Latent transforming growth factor-beta is present in
the extracellular matrix of embryonic
hearts in situ. Dev Dyn 205:126 –134.
Grainger DJ, Metcalfe JC, Grace AA,
Mosedale DE. 1998. Transforming
growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo. J Cell Sci 111(Pt 19):2977–
2988.
Hirschi KK, Rohovsky SA, D’Amore PA.
1998. PDGF, TGF-beta, and heterotypic
cell-cell interactions mediate endothelial
cell-induced recruitment of 10T1/2 cells
and their differentiation to a smooth
muscle fate. J Cell Biol 141:805–814.
Hirschi KK, Lai L, Belaguli NS, Dean DA,
Schwartz RJ, Zimmer WE. 2002. Transforming growth factor-beta induction of
smooth muscle cell phenotype requires
transcriptional and post-transcriptional
control of serum response factor. J Biol
Chem 277:6287–6295.
Ho E, Shimada Y. 1978. Formation of the
epicardium studied with the scanning
electron microscope. Dev Biol 66:579 –
585.
Holifield JS, Arlen AM, Runyan RB, Tomanek RJ. 2004. TGF-beta1, -beta2 and
-beta3 cooperate to facilitate tubulogenesis in the explanted quail heart. J Vasc
Res 41:491–498.
Hu PP, Datto MB, Wang XF. 1998. Molecular mechanisms of transforming growth
factor-beta signaling. Endocr Rev 19:349 –
363.
Jakowlew SB, Ciment G, Tuan RS, Sporn
MB, Roberts AB. 1994. Expression of
transforming growth factor-beta 2 and
beta 3 mRNAs and proteins in the developing chicken embryo. Differentiation 55:
105–118.
Jat PS, Noble MD, Ataliotis P, Tanaka Y,
Yannoutsos N, Larsen L, Kioussis D.
1991. Direct derivation of conditionally
immortal cell lines from an H-2Kb-tsA58
transgenic mouse. Proc Natl Acad Sci
U S A 88:5096 –5100.
Kalluri R, Neilson EG. 2003. Epithelialmesenchymal transition and its implications for fibrosis. J Clin Invest 112:1776 –
1784.
Komiyama M, Ito K, Shimada Y. 1987. Origin and development of the epicardium
in the mouse embryo. Anat Embryol
(Berl) 176:183–189.
Kretzschmar M, Massague J. 1998. SMADs:
mediators and regulators of TGF-beta signaling. Curr Opin Genet Dev 8:103–111.
Kuhn HJ, Liebherr G. 1988. The early development of the epicardium in Tupaia
belangeri. Anat Embryol (Berl) 177:225–
234.
Lepilina A, Coon AN, Kikuchi K, Holdway
JE, Roberts RW, Burns CG, Poss KD.
2006. A dynamic epicardial injury response supports progenitor cell activity
during zebrafish heart regeneration. Cell
127:607–619.
Lin HY, Moustakas A. 1994. TGF-beta receptors: structure and function. Cell Mol
Biol 40:337–349.
Lin HY, Wang XF, Ng-Eaton E, Weinberg
RA, Lodish HF. 1992. Expression cloning
of the TGF-beta type II receptor, a func-
376 AUSTIN ET AL.
tional transmembrane serine/threonine
kinase. Cell 68:775–785.
Lopez-Casillas F, Cheifetz S, Doody J, Andres JL, Lane WS, Massague J. 1991.
Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.
Cell 67:785–795.
Lu J, Landerholm TE, Wei JS, Dong XR,
Wu SP, Liu X, Nagata K, Inagaki M,
Majesky MW. 2001. Coronary smooth
muscle differentiation from proepicardial cells requires rhoA-mediated actin
reorganization and p160 rho-kinase activity. Dev Biol 240:404 –418.
Mack CP, Somlyo AV, Hautmann M, Somlyo AP, Owens GK. 2001. Smooth muscle
differentiation marker gene expression
is regulated by RhoA-mediated actin polymerization. J Biol Chem 276:341–347.
Masszi A, Di Ciano C, Sirokmany G,
Arthur WT, Rotstein OD, Wang J, McCulloch CA, Rosivall L, Mucsi I, Kapus
A. 2003. Central role for Rho in TGFbeta1-induced alpha-smooth muscle actin expression during epithelial-mesenchymal transition. Am J Physiol Renal
Physiol 284:F911–F924.
McAllister KA, Grogg KM, Johnson DW,
Gallione CJ, Baldwin MA, Jackson CE,
Helmbold EA, Markel DS, McKinnon
WC, Murrell J, et al. 1994. Endoglin, a
TGF-beta binding protein of endothelial
cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet
8:345–351.
Mikawa T, Fischman DA. 1992. Retroviral
analysis of cardiac morphogenesis: discontinuous formation of coronary vessels. Proc Natl Acad Sci U S A 89:9504 –
9508.
Mikawa T, Gourdie RG. 1996. Pericardial
mesoderm generates a population of coronary smooth muscle cells migrating
into the heart along with ingrowth of the
epicardial organ. Dev Biol 174:221–232.
Molin DG, Bartram U, Van der Heiden K,
Van Iperen L, Speer CP, Hierck BP,
Poelmann RE, Gittenberger-de-Groot
AC. 2003. Expression patterns of Tgfbeta1–3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous
heart skeleton. Dev Dyn 227:431–444.
Morabito CJ, Dettman RW, Kattan J, Collier JM, Bristow J. 2001. Positive and
negative regulation of epicardial-mesenchymal transformation during avian
heart development. Dev Biol 234:204 –
215.
Munoz-Chapuli R, Gonzalez-Iriarte M,
Carmona R, Atencia G, Macias D, PerezPomares JM. 2002. Cellular precursors
of the coronary arteries. Tex Heart Inst J
29:243–249.
Nahirney PC, Mikawa T, Fischman DA.
2003. Evidence for an extracellular matrix bridge guiding proepicardial cell migration to the myocardium of chick embryos. Dev Dyn 227:511–523.
Olivey HE, Compton LA, Barnett JV. 2004.
Coronary vessel development: the epicardium delivers. Trends Cardiovasc
Med 14:247–251.
Olivey HE, Mundell NA, Austin AF, Barnett
JV. 2006. Transforming growth factorbeta stimulates epithelial-mesenchymal
transformation in the proepicardium. Dev
Dyn 235:50 –59.
Owens GK, Kumar MS, Wamhoff BR.
2004. Molecular regulation of vascular
smooth muscle cell differentiation in development and disease. Physiol Rev 84:
767–801.
Pelton RW, Johnson MD, Perkett EA, Gold
LI, Moses HL. 1991. Expression of transforming growth factor-beta 1, -beta 2,
and -beta 3 mRNA and protein in the
murine lung. Am J Respir Cell Mol Biol
5:522–530.
Poelmann RE, Gittenberger-de Groot AC,
Mentink MM, Bokenkamp R, Hogers B.
1993. Development of the cardiac coro-
nary vascular endothelium, studied with
antiendothelial antibodies, in chickenquail chimeras. Circ Res 73:559 –568.
Poss KD. 2007. Getting to the heart of regeneration in zebrafish. Semin Cell Dev
Biol 18:36 –45.
Roberts AB, Sporn MB. 1990. The transforming growth factor-betas. In: Sporn
MB, Roberts AB, editors. Peptide growth
factors and their receptors. New York:
Springer-Verlag. p 419 – 472.
Sanford LP, Ormsby I, Gittenberger-de
Groot AC, Sariola H, Friedman R, Boivin
GP, Cardell EL, Doetschman T. 1997. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout
phenotypes. Development 124:2659 –2670.
Shi Y, Massague J. 2003. Mechanisms of
TGF-beta signaling from cell membrane
to the nucleus. Cell 113:685–700.
Sinha S, Hoofnagle MH, Kingston PA, McCanna ME, Owens GK. 2004. Transforming growth factor-beta1 signaling
contributes to development of smooth
muscle cells from embryonic stem cells.
Am J Physiol Cell Physiol 287:C1560 –
C1568.
Tomanek RJ. 2005. Formation of the coronary vasculature during development.
Angiogenesis 8:273–284.
Wang XF, Lin HY, Ng-Eaton E, Downward
J, Lodish HF, Weinberg RA. 1991. Expression cloning and characterization of
the TGF-beta type III receptor. Cell 67:
797–805.
Ward SM, Gadbut AP, Tang D, Papageorge
AG, Wu L, Li G, Barnett JV, Galper JB.
2002. TGFbeta regulates the expression
of G alpha(i2) via an effect on the localization of ras. J Mol Cell Cardiol 34:1217–
1226.
Xie L, Law BK, Chytil AM, Brown KA,
Aarke ME, Moses HL. 2004. Activation
of the Erk pathway is required for TGFbeta1-induced EMT in vitro. Neoplasia
6:603– 610.