From the Cutaneous Biology Research Center,
Massachusetts General Hospital and Harvard Medical School, Charlestown,
Massachusetts 02129 and¶ Department of Molecular Genetics
Biochemistry and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267-0524
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ABSTRACT |
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We have examined the role that individual TGF- Homeostasis of self renewing epithelia such as the epidermis is
determined by a complex interplay of signals, resulting from cell-cell
and cell-matrix interactions as well as diffusible factors acting on
cells of either the same or different type. The TGF- In the skin, elevated TGF- Expression of TGF- Skin homeostasis results from a delicate balance between keratinocyte
growth, differentiation, and apoptosis. Such a balance is perturbed
upon exposure to tumor promoting and/or inflammatory agents, UV light,
and wounding. The phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA)1 is a potent skin tumor
promoter. In vivo, acute treatment with TPA leads to
epidermal hyperplasia, possibly through mechanisms such as blockage of
gap-junctional intercellular communication (15) and/or differential
modulation of specific TGF- The present study was undertaken to establish the role that individual
TGF- Skin Grafting Experiments--
Homozygous TGF- Cell Culture and Viability Assays--
Primary keratinocytes
were prepared from wild type newborn mice (SENCAR) and grown at
34 °C and 8% CO2 in medium at low calcium concentrations as described previously (18). Confluent cultures were
tested 5 days after plating. Cells were either untreated or treated
with the TGF- TUNEL Assays--
Primary keratinocytes that were either
untreated or treated with specific TGF- Immunofluorescence Analysis of Focal Adhesions and Actin
Cables--
Mouse primary keratinocytes were plated onto
collagen-coated glass coverslips and grown to confluence. Cells that
were either untreated or treated with TGF- In Vitro Kinase Assays--
Primary keratinocytes in low calcium
medium were either untreated or treated with TGF 1) Normal Maturation of the Skin of TGF-
The lack of the TGF- 2) Widespread Epidermal Cell Death in the Skin of
TGF-
Apoptotic cells as well as cells undergoing necrosis are characterized
by increased nuclear DNA fragmentation, which can be detected by TUNEL
assays (21). Most of the pyknotic nuclei in the TPA-treated TGF-
In addition to cell death in the epidermis, the TPA-treated TGF- 3) Direct Protective Effects of the TGF-
Staining of cells with calcein-AM/propidium iodide provides an
alternative method to TUNEL assays to measure cell death (19). This
method relies on the ability of viable cells to cleave calcein/AM into
calcein, generating a green fluorescent signal, whereas dying cells
take up propidium iodide into the nucleus and become identifiable by
red fluorescence. Experiments using this method confirmed that the
death-inducing action of TPA was prevented by pretreatment of
keratinocytes with the TGF- 4) Disruption of Focal Adhesions and Actin Cables in TPA-treated
Keratinocytes and Counteracting Effects of TGF- 5) Specific Inhibitory Effects of TGF- Previous data from a number of laboratories have shown that the
TGF- Apoptosis is typically associated with a multistage process of DNA
fragmentation, cell shrinking, and lack of an inflammatory response
(21, 28, 29). A necrosis reaction can also be associated with random
DNA fragmentation, but it involves cell swelling and a significant
inflammatory reaction (29, 30). The histological findings of the
TGF- Under basal conditions, grafted epidermis of TGF- The mature TGF- The protective function of TGF-
isoforms, and in particular TGF-
3, play in control of epidermal
homeostasis. Mice with a knockout mutation of the TGF-
3 gene die a
few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the
skin of these mice. Grafted skin of mice with a disruption of the
TGF-
3 gene developed similarly to grafts of wild type and TGF-
1
knockout animals. However, a strikingly different response was observed
after acute treatment with the tumor promoter
12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to
TPA, the grafted skin of wild type and TGF-
1 knockout mice underwent
a hyperplastic response similar to that of normal mouse skin. In marked
contrast, TPA treatment of TGF-
3 knockout grafts induced widespread
areas of keratinocyte cell death. Analysis of cultured keratinocytes
treated with purified TGF-
isoforms revealed that TGF-
3 plays a
direct and specific function in protecting keratinocytes against
TPA-induced cell death. The protective function of TGF-
3 on
TPA-induced cell death was not because of general suppression of the
signaling pathways triggered by this agent, as ERK1/2 activation
occurred to a similar if not greater extent in TGF-
3-treated
versus control keratinocytes. Instead, TGF-
3 treatment
led to a significant reduction in TPA-induced c-Jun N-terminal kinase
activity, which was associated and possibly explained by specific
counteracting effects of TGF-
3 on TPA-induced disruption of
keratinocyte focal adhesions.
INTRODUCTION
Top
Abstract
Introduction
References
family of
diffusible factors is thought to play a key role in control of
epithelial homeostasis. Some well known functions of TGF-
s in
epithelial tissues include growth inhibition, promotion of cell
adhesion and tissue reorganization, suppression and, at later stages,
promotion of tumor development (1). Three TGF-
isoforms exist that
share a high degree of homology (~80%) in their processed active
regions. All three isoforms bind to the same cellular receptors,
although with different affinities, and elicit similar biological
responses on most cultured cells. However, expression of the three
TGF-
s is differentially regulated in vivo in different
cell types and at various developmental stages, suggesting that the
three isoforms play distinct biological roles (2). This conclusion is
consistent with the fact that mice with disruptions of each of the
three TGF-
genes have little or no overlapping phenotype. TGF-
1
knockout mice die a few weeks after birth of a wasting syndrome
accompanied by a multifocal inflammatory reaction (3, 4). Mice with a
knockout mutation of the TGF-
3 gene die a few hours after birth
because of cleft palate (5, 6). Mice with a disruption of the TGF-
2
gene die also at birth, because of a relatively large number of
developmental abnormalities affecting the cardiovascular and
musculoskeletal systems as well as other organs (7).
1 expression has been found in
keratinocytes of the basal epidermal layer and in cultured
keratinocytes under basal proliferating conditions, suggesting that
this factor is involved in a negative feedback growth regulatory loop
(8, 9). TGF-
2 expression is increased in keratinocytes upon
differentiation (10), implicating this isoform as a possible mediator
of differentiation-induced growth arrest.
3 mRNA is limited, at least in culture, to
cells of mesenchymal origin, including dermal fibroblasts, whereas it
is not found in keratinocytes (11, 12). This factor is likely to
diffuse from the dermis to the overlying epidermis (9) and may be an
important mediator of dermal-epidermal interactions, including those
involved in the wound-healing reaction (13, 14) as well as in negative
control of keratinocyte tumor development (12).
isoforms (9, 16). In vitro,
acute TPA treatment has the opposite effect of inducing keratinocyte
detachment, growth arrest, and differentiation (Ref. 17 and references
therein). The discrepancy between the in vivo and in
vitro effects has never been explained.
s, and in particular TGF-
3, play in control of epidermal
homeostasis. As mentioned, mice with a disruption of the TGF-
3 gene
die a few hours after birth (5, 6). A full-thickness skin grafting
approach was used to investigate the postnatal development and
homeostatic control of the skin of these mice, in comparison with
similarly grafted skin from wild type and TGF-
1 knockout animals.
These studies suggest that TGF-
3 may not be essential for normal
skin maturation. Instead, the in vivo findings, together with analysis of cultured keratinocytes treated with purified TGF-
isoforms, indicate that TGF-
3 plays a specific and direct function
in protecting keratinocytes against the death-inducing effects of
agents such as TPA.
EXPERIMENTAL PROCEDURES
3 knockout mice
die at birth and can be rapidly cannibalized by the mothers. For this
reason, experiments were synchronized so as to have sufficient numbers
of time-mated heterozygous pregnant females to use as source of pups
immediately before delivery (18.5 days of pregnancy). The genotype of
pups was determined by polymerase chain reaction analysis of tail DNA with oligonucleotide primers specific for the wild-type and disrupted TGF-
alleles (6). Skins from wild type and homozygous knockout littermates were used for full-thickness grafting onto nude mice. The
procedure involved a 10 × 10-mm excision of the host skin, up to
but not including the panniculus carnosus and placement of a similar
size fragment of newborn mouse skin onto the area of excision. The
grafted skin was covered with a Vaseline gauze and a silicon
transplantation chamber on top for protection. Gauze and chamber were
removed 6 days after grafting. Grafts were analyzed at 1 month after
grafting, under basal conditions and after treatment with TPA
(10
4 M in acetone, Sigma) or acetone vehicle
alone. In each case, the grafted tissue was excised, half was frozen,
and the other half was fixed in 10% neutral-buffered formalin before
histological and immunohistochemical analysis. Terminal
deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling
(TUNEL) assays of frozen sections were performed as described below for
cultured cells. Immunohistochemistry for TGF-
3 expression was
performed by incubating frozen sections of wild type and knockout
grafted skins with antibodies against TGF-
3 (Santa Cruz) in the
presence or the absence of a specific neutralizing peptide followed by
incubation with biotinylated anti-rabbit secondary antibodies (Vector,
Burlingame). Fast RED (Sigma) was used as substrate for the alkaline
phosphatase reaction.
1 or TGF-
3 isoforms (R&D Systems) at 3 ng/ml for the
indicated amounts of time followed by exposure to TPA (in
Me2SO stock solutions; 100 ng/ml final concentration) or
Me2SO vehicle alone. Cell viability was determined by the
calcein-AM/propidium iodide staining method (19). Briefly, culture
medium was replaced with 1 µM calcein-AM (Molecular
Probes) and 5 µg/ml propidium iodide in serum-free low calcium medium
for 20 min at 34 °C. Cultures were examined immediately without
fixation using a Zeiss Axiophot fluorescence microscope. Green and red
fluorescence were simultaneously recorded using a computer-digitalized
equipment. In each case, 4-5 different fields were examined, and a
minimum of 800 cells were counted to determine the fraction of
calcein-AM (green) versus propidium iodide (red) positive cells.
isoforms and/or TPA were
trypsinized, centrifuged, and brought into suspension into
serum-supplemented medium, counted, and recentrifuged in a cytospin
apparatus onto gelatin-coated glass slides. In other experiments, cells
were directly grown on tissue culture slides (Labtec) and treated under
these conditions. Cytospin preparations or keratinocytes directly grown
on slides were fixed in 10% neutral-buffered formalin for 10 h at
room temperature, air dried for 1 h, and then permeabilized with
20 µg/ml proteinase K for 5-10 h at room temperature. The terminal
deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling
assay (TUNEL) was performed using the TACS terminal
deoxynucleotidyltransferase in situ apoptosis detection kit
(Trevigen) using 3,3'-diaminobenzidine tetrahydrochloride or TUNEL blue
labeling for detection according to the manufacturer's instructions.
1, TGF-
3, and/or TPA
were fixed for 10 h at room temperature with 2% paraformaldehyde
in phosphate-buffered saline followed by permeabilization with 0.1%
Triton X-100 in phosphate-buffered saline and blocking in 5% goat
serum in phosphate-buffered saline. Cells were incubated with
anti-vinculin rabbit antiserum (Sigma) for 1 h at room
temperature, followed by incubation with fluorescein
isothiocyanate-conjugated secondary antibodies (Southern Biotech.
Associates) and BODIPY(581-591)-conjugated phalloidin (Molecular
Probes). Samples were analyzed by confocal microscopy using a LEICA TCS
4D scanner connected to an inverted LEITZ DM IRB microscope. Images
were processed using a TCS-NT software package.
1 or TGF
3 (3 ng/ml) for 18 h followed by exposure to TPA (100 ng/ml) for
different lengths of time. In vitro kinase assays were
performed using the stress-activated protein kinase/c-Jun N-terminal
kinase (JNK) or p44/42 mitogen-activated protein kinase assay kits (New
England BioLabs Inc.), according to the manufacturer's instructions.
Briefly, cells were lysed in cell lysis buffer and normalized for
protein amounts by the Bradford assay (Bio-Rad). Same amounts of total
cell extracts (250 µg of proteins) were incubated with c-Jun (1-89)
fusion protein beads to immunoprecipitate the Jun N-terminal kinase or
with phospho-specific antibody to p44/42 mitogen-activated protein
kinase to selectively immunoprecipitate active mitogen-activated
protein kinase (ERK1/2). Kinase reactions were carried out in the
presence of cold ATP using either recombinant c-Jun or Elk1 proteins as
substrates for the JNK and ERK1/2 kinases, respectively. Samples were
analyzed by 12% SDS-polyacrylamide gel electrophoresis and
immunoblotting with either phospho-specific c-Jun antibodies to detect
c-Jun phosphorylated at Ser-63 or phospho-specific Elk1 antibodies to detect the phosphorylated form of Elk1 at Ser-383. For JNK and ERK1/2
protein level determinations, total cell extracts were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting with rabbit
polyclonal antibodies against JNK and ERK1/2, respectively (Santa Cruz
Biotech., Inc.). In all cases, blots were developed with the ECL system
(Amersham Pharmacia Biotech), and results were quantified by
densitometric scanning.
RESULTS
3-deficient
Mice--
The neonatal mortality of TGF-
3-deficient mice prevents a
direct assessment of how this mutation can affect the maturation and
homeostasis of skin after birth. To overcome this problem, we used a
full-thickness grafting approach of newborn mouse skin onto nude mice
in such a way that it was possible to compare the effects of the
TGF-
3 and -
1 mutations under identical experimental settings and
for a prolonged period of time. Newborn mouse skin grafted in this
manner develops normally as if in the intact animal, with good hair
growth becoming detectable by 9-10 days after grafting and complete
fur formation by 18-20 days. Within a similar time frame, the multiple
epidermal layers characteristic of newborn skin are reduced to the two
to three layers of the adult animal. The grafts can be maintained up to
1 year with normal hair production.
3 isoform did not lead to gross alterations in
skin maturation and hair formation. Compared with control grafts, the
grafted skin of mice with specific TGF-
3 or -
1 knockout mutations
showed good hair growth by 2-3 weeks after grafting (Fig.
1 and data not shown). Within a similar
time frame, the stratified multiple squamous epidermal layers
characteristic of normal newborn mouse skin were reduced to 2-3 layers
of the adult mouse epidermis with normal structure and morphology.
Immunohistochemical analysis with antibodies against TGF-
3 produced
a significantly lower level of positive staining in the grafts of
TGF-
3 knockout skin relative to surrounding skin and/or grafts of
wild type animals (Fig. 1, lower panels). However, even in
the TGF-
3 knockout grafts, residual specific staining for TGF-
3
was detectable, especially at the periphery, consistent with some
diffusion of this factor from the neighboring skin.
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Fig. 1.
Full-thickness grafting of
TGF- 3 knockout skin onto nude mice.
A, normal maturation of a skin graft derived from a newborn
TGF-
3 knockout mouse at 1 month after grafting. B, levels
of TGF-
3 protein in grafts of TGF-
3 knockout versus
wild type skin at 1 month after grafting as determined by
immunohistochemical analysis. Skin grafts from wild type
(w.t.) and TGF-
3 knockout mice (
3
/
) were stained
with anti-TGF-
3 antibodies as described under "Experimental
Procedures." The wild type skin graft was also stained with the
antibodies in the presence of a neutralizing peptide. Bar,
250 µm.
3-deficient Mice after Phorbol Ester Treatment--
Previous
analysis of knockout mice for other potentially redundant genes has
indicated that essential functions of these genes can be unmasked under
conditions of stress, where normal homeostatic mechanisms are perturbed
or partially abrogated (18, 20). One such condition is acute treatment
of mouse skin with the phorbol ester tumor promoter TPA, which results
in marked epidermal hyperplasia as well as mild inflammatory reaction
by 24-48 h of exposure (Ref. 9 and references therein). When treated
with TPA, the grafted epidermis of wild type and TGF-
1 knockout mice
underwent a hyperplastic response similar to that of normal mouse skin
(Fig. 2A). Interestingly, a
more pronounced inflammatory infiltrate was detectable in the grafts of
TGF-
1 knockout skins, consistent with the immunosuppressive function
of this specific isoform (3). In marked contrast, TPA treatment of
TGF-
3 knockout grafts induced widespread areas of keratinocyte cell
death (Fig. 2A). Keratinocytes in these areas exhibited
mixed features of an apoptotic and necrotic process, with highly
pyknotic nuclei and cell swelling (Fig. 2A).
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Fig. 2.
Epidermal hyperplasia of wild type and
TGF- 1 knockout grafts and widespread cell
death in TGF-
3 knockout grafts in response to
acute TPA treatment. A, full-thickness grafts derived
from wild type (w.t.), TGF-
1 (
1
/
), and TGF-
3
(
3
/
) mice at 1 month after grafting were treated with TPA
(10
4 M in acetone, 200 ml) for 24 or 48 h or with control acetone alone for 48 h (Ctrl). The
integrity of the grafted skin was verified in each case at the
beginning of the experiment, after shaving. Each condition was tested
on duplicate mice with similar results. These findings were confirmed
in two other independent experiments. Bar, 180 µm.
B, grafts of TGF-
3 knockout skin treated with acetone
alone or TPA for 48 h and of wild type skin treated with TPA were
analyzed by TUNEL assay using immunoperoxidase for detection.
Bar, 90 µm.
3
knockout grafts stained positive by this assay, whereas no TUNEL
positivity was detected in the TPA-treated wild type grafts nor in the
acetone-treated controls (Fig. 2B).
3
knockout grafts exhibited signs of dermal necrosis. Many venules were
also affected by granulomatous vasculitis with segmented fibrinoid
necrosis of the cell walls, angiocentric histiocytosis, and endothelial
swelling (data not shown). Capillaries showed evidence of
microangiopathy with some vessels occluded by fibrin thrombi. None of
these changes were found in similarly treated grafts of wild type or
TGF-
1 knockout skins.
3 Isoform on TPA-induced
Cell Death of Cultured Keratinocytes--
The alterations of epidermal
cells observed in the TGF-
3 knockout grafts after TPA treatment may
be explained by a direct protective function of TGF-
3 on
keratinocytes or, alternatively, to an indirect effect on the dermis.
These possibilities were further tested in culture by examining whether
exogenous addition of either TGF-
3 or TGF-
1 can protect primary
keratinocytes from the effects of TPA. A significant fraction of
growing keratinocytes in culture undergoes nuclear DNA fragmentation,
which is likely because of the spontaneous continuous shedding of cells
into the medium. TPA treatment of control keratinocytes significantly
increased the fraction of cells exhibiting nuclear DNA fragmentation as early as 6-8 h after treatment (Fig.
3A). As previously reported for human keratinocytes (22, 23), TGF-
1 treatment was also by itself
sufficient to increase the number of TUNEL-positive cells.
Interestingly, no such effect was observed with TGF-
3 (Fig.
3A). More importantly, pretreatment of keratinocytes with this latter isoform suppressed the increase in TUNEL positivity induced
by TPA, whereas no such protective effects were observed after
pretreatment with TGF-
1 (Fig. 3A).
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Fig. 3.
TPA-induced cell death of cultured
keratinocytes and protective effects of the
TGF- 3 isoform. Primary mouse
keratinocytes were either left untreated (Ctl) or treated
with TGF-
1 or TGF-
3 for 18 h before exposure to
Me2SO vector alone or TPA (100 ng/ml) for an additional
8 h. A, nuclear DNA fragmentation as assessed by TUNEL
assay. Left panel, representative fields of control and
TGF-
3-treated keratinocytes plus/minus TPA exposure. Right
panels, composite result of four independent experiments.
B, cell viability as assessed by combined staining with
calcein-AM (green, viable) and propidium iodide
(red, dead). Right panel, composite result of
three independent experiments. In all cases, results were quantified by
examining at least 4 independent fields, counting a minimum of 800 cells. Values are expressed as fraction of TUNEL or calcein A-positive
(i.e. viable) cells.
3 but not TGF-
1 isoforms (Fig. 3B).
3--
One of the
most important consequences of TPA treatment is detachment of
keratinocytes from their substratum (Ref. 18 and references therein),
which in turn can trigger programmed cell death (24). The protective
function of TGF-
3 on TPA-induced cell death may be connected with
specific effects on keratinocyte cell attachment. Focal adhesions serve
as a bridge between integrin receptors and the actin cytoskeleton and
provide an essential adhesive structure of keratinocytes in culture
(25). Keratinocytes were analyzed by double immunofluorescence with
antibodies against vinculin, a specific focal adhesion component
(detected with fluorescein isothiocyanate-coupled secondary antibodies;
green) and phalloidin, for actin cable visualization
(rhodamine-coupled; red). Samples were analyzed by confocal
microscopy, and green and red images were superimposed. Focal adhesions
and actin cables were readily detectable in control keratinocytes,
whereas they were totally disrupted as early as 30 h after TPA
exposure (Fig. 4, upper
panels). By contrast, little or no disruption of actin cables and
focal adhesions were induced by TPA in cells that had been pretreated with TGF-
3 for 18 h (Fig. 4, lower panels). Relative
to TGF-
3, pretreatment with TGF-
1 had more limited counteracting
effects on TPA-induced disruption of focal adhesions and disassembly of actin cables (Fig. 4, middle panels). In parallel with these
findings, TPA treatment induced rounding up of keratinocytes by 6 h (Fig. 5A) and detachment
from the dish by 24 h (Fig. 5B). These effects were
almost totally prevented by pretreatment with TGF-
3, whereas TGF-
1 exerted a more partial protection (Fig. 5).
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Fig. 4.
TPA-induced disruption of focal adhesions and
actin cables and counteracting effects of
TGF- 3. Primary mouse keratinocytes were
either left untreated (Ctrl) or treated with TGF-
1 or
TGF-
3 for 18 h before exposure to Me2SO vector
alone or TPA (100 ng/ml) for an additional 30 h. Cells were
analyzed by double immunofluorescence with antibodies against vinculin
and fluorescein isothiocyanate-conjugated secondaries
(green) and Texas red-conjugated phalloidin
(red). Samples were analyzed by confocal microscopy, and
green and red images were superimposed so that
sites of overlapping staining are visualized as yellow.
Bar, 4 µm. Photographs were exposed for identical amounts
of time. Similar results were observed at 2 h of TPA treatment and
were confirmed in a second independent experiment.
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Fig. 5.
TPA-induced cell detachment and counteracting
effects of TGF- 3. Primary mouse
keratinocytes were either left untreated (Ctrl) or treated
with TGF-
1 or TGF-
3 for 18 h before exposure to TPA (100 ng/ml) for either 6 h (A) or 24 h (B).
Panel A, contrast phase microscopy (10×) illustrating the
differential rounding up of cells after 6 h of TPA treatment,
depending on whether or not cells were pretreated with TGF-
1 or
TGF-
3. Panel B, keratinocyte cell detachment at 24 h
of TPA treatment. Cells that had remained attached to the dish were
trypsinized and counted, in parallel with those that had detached in
the culture medium. Each condition was tested on duplicate dishes, and
values are expressed as ratios of detached versus detached
cells recovered from each dish.
3 Versus TGF-
1 on
TPA-induced JNK Activation--
Previous work has demonstrated that
loss of keratinocyte cell adhesion can trigger JNK kinase activation
(26), and preferential activation of the JNK versus ERK
kinase can be a critical determinant of apoptosis (27). Differential
modulation of these pathways could provide a mechanism for the specific
protective effects of the TGF-
3 versus TGF-
1 isoforms
on TPA-induced cell death. To test this possibility, primary
keratinocytes were either untreated or treated with TGF-
1 or
TGF-
3 (3 ng/ml) for 18 h followed by exposure to TPA for
different lengths of time. Total cell extracts were incubated with
beads coupled to the c-Jun protein to immunoprecipitate JNK or with
phospho-specific antibody to the ERK1/2 kinase to immunoprecipitate
the active form of this kinase. In vitro kinase assays were
carried out using recombinant c-Jun or Elk1 proteins as substrates for
the JNK and ERK1/2 kinases, respectively. In vitro kinase
activity was normalized by the total amounts of JNK and ERK proteins
present inside the cells, as determined by immunoblotting of the same
cell extracts with antibodies against these proteins. A representative
experiment is shown in Fig.
6A, whereas Fig. 6B
illustrates the composite quantification of three independent experiments. As reported for other cell types, treatment of mouse primary keratinocytes with TPA resulted in the early induction of JNK
kinase activity, which returned to basal level or even below by 4-8 h
of treatment. Activity of the related ERK kinase was also induced in
the TPA-treated keratinocytes, with a similar time course of
activation. Keratinocytes pretreated with TGF-
1 showed the same
pattern of JNK activation in response to TPA as the control, with the
exception that the basal level of JNK kinase activity appeared elevated
in these cells even before TPA treatment. ERK kinase was induced in the
TGF-
1-pretreated keratinocytes after exposure to TPA, but ERK
activity levels remained consistently lower than in controls. In
contrast, elevation of JNK activity in response to TPA was
significantly delayed and/or suppressed in the TGF-
3-treated
keratinocytes, whereas ERK activity was induced by TPA to a similar if
not greater extent than in the controls. Thus, the relative ratio of
JNK versus ERK activation was substantially lower in the
TGF-
3 versus TGF-
1-treated keratinocytes (Fig. 6B),
consistent with the specific counteracting effects of the first isoform
on TPA-induced cell death.
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Fig. 6.
JNK and ERK1/2 activation in TPA-treated
keratinocytes and differential modulatory effects of the
TGF- 3 versus
TGF-
1 isoforms. Panel
A, primary mouse keratinocytes were either left untreated
(Control, Ctl) or treated with TGF-
1 or TGF-
3 for
18 h before exposure to TPA (100 ng/ml) for the indicated amounts
of time. Total cell extracts were normalized for protein amounts and
assayed for JNK and ERK1/2 in vitro kinase activity using
c-Jun and Elk1 proteins as substrates as described under
"Experimental Procedures." The same extracts were analyzed for
total JNK and ERK protein levels by immunoblotting with the
corresponding specific antibodies. Panel B, composite result
of three independent experiments performed as described in panel
A. Quantitation was achieved by densitometric scanning of the
autoradiographs and normalization for total JNK and ERK protein levels.
In the first two panels, values are expressed as -fold of induction
relative to basal kinase activity in the untreated controls. In the
right panel, the relative ratio of JNK versus ERK
kinase activity was calculated.
DISCUSSION
3 isoform is preferentially produced by dermal fibroblasts and
not keratinocytes, and that in the skin, the presence of this factor in
the epidermis is likely because of diffusion from the underlying dermis
(9, 11, 12, 14). The present findings establish a unique function of
TGF-
3 not shared by TGF-
1 as a specific isoform able to protect
keratinocytes from the death-inducing effects of an agent such as TPA.
These findings help to explain a long-standing discrepancy between the
in vivo effects of TPA (induction of epidermal hyperplasia)
and the effects of this agent on cultured keratinocytes (induction of
growth arrest, differentiation, and as we have shown here, increased
cell death). Because keratinocytes produce very little if any TGF-
3,
these cells are intrinsically sensitive to the death-inducing effects
of TPA but could be protected from these effects in vivo by
the dermally produced TGF-
3.
3 knockout epidermis treated with TPA exhibited mixed features
of apoptosis and necrosis. An increasing number of variant cell deaths
have been described to which the dichotomy apoptosis/necrosis cannot be
applied, and even biochemically, the distinction is less clear than
expected (31). For instance, it has recently been shown that well
established inhibitors of apoptosis such as Bcl-2 and ICE inhibitors
can also suppress classical necrosis (32). In addition to cell death in
the epidermis, the TPA-treated TGF-
3 knockout grafts showed evidence
of dermal necrosis as well as microangiopathy. Our in vitro
data are consistent with the alterations of the epidermis being a
primary consequence of keratinocyte exposure to TPA in the absence of
the TGF-
3-protective function. However, alterations in the dermis
and blood vessels could also contribute to keratinocyte cell death in
the overlying epidermis.
3 knockout mice was
histologically normal, and no increased apoptosis was observed.
However, levels of TGF-
3 were reduced but not totally absent in
these grafts, consistent with this factor diffusing to some extent from
the neighboring skin in amounts that might be sufficient for normal
skin maturation
1, -
2, and -
3 isoforms share a >70% amino
acid sequence identity, including conservation of the 8 cysteine residues that contribute to the correct folding of the monomers and the
additional cysteine residue involved in subunit dimerization (33). The
sequence diversity among individual isoforms has been maintained
throughout evolution, suggesting that they may have intrinsically
distinct functions (34). In fact, although all three isoforms bind to
type I and type II TGF-
receptors and exert similar effects on many
cell types, in certain cases they behave selectively. For instance,
TGF-
2 exerts little if any growth inhibitory effects on endothelial
cells (35), and this may be because of the lack of binding of TGF-
2
to endoglin, a major surface TGF-
-binding protein that coexists with
the type I and type II TGF-
receptors on endothelial cells (36).
Similarly, TGF-
3, unlike TGF-
1 and -
2, fails to bind to
heparin and to liver heparan sulfate (37). These polysaccharides
potentiate the biological activity of TGF-
1 by antagonizing the
binding of this isoform and its inactivation by
2
macroglobulin, a major TGF-
-binding protein found in serum and the
intercellular space (38). The differential binding of the TGF-
isoforms to heparin has been related to two specific basic residues at
positions 26 and 60 in TGF-
1 and -
2, which are replaced by
neutral ones in TGF-
3 (37). In primary mouse keratinocytes grown
under our conditions, TGF-
1 and TGF-
3 were found to exert similar
growth inhibitory effects and to induce a TGF-
-responsive promoter
(3TP-luc) to a similar
extent.2 This is consistent
with the fact that these factors bind type I and type II TGF-
receptors with similar affinity (1), and are therefore expected to
trigger similar downstream reponses. However, TGF-
1 treatment was by
itself sufficient to increase the fraction of TUNEL positive
keratinocytes, whereas no such effect was elicited by TGF-
3. More
importantly, there was a substantial difference in the way pretreatment
with TGF-
3 but not TGF-
1, protected keratinocytes from
TPA-induced cell death. An attractive hypothesis is that TGF-
3,
besides triggering type I and type II receptors to a similar extent as
TGF-
1, binds specifically to other proteins on the keratinocyte cell
surface, thus affecting behavior of these cells in additional manners.
3 on TPA-induced cell death is not
because of general suppression of TPA signaling, as ERK1/2 activation
occurred to a similar if not greater extent in TGF-
3 treated
versus control keratinocytes. Instead, TGF-
3 treatment led to a significant reduction in TPA-induced JNK activity, whereas increased rather than decreased JNK activity was observed after treatment of the same cells with TGF-
1. Activation of ERK has been
connected with growth stimulation of cells in response to a variety of
stimuli, whereas induction of JNK has been closely related to apoptosis
(39). In epithelial cells, one important inducer of JNK activity (and
apoptosis) is loss of cell attachment to the substrate (40). The rapid
disruption of stress fibers and focal adhesions caused by TPA treatment
was found to be effectively counteracted by TGF-
3, thus providing an
attractive explanation for the protective effects of this isoform
against TPA-induced cell death. A specific role of TGF-
3 in
promoting epithelial cell adhesion is consistent with the major
phenotype of TGF-
3 knockout mice in which cleft palate has been
related to reduced adhesion of the epithelial medial edge (6). Future
studies will have to further investigate the consequences of this
specific function of the TGF-
3 isoform in epidermal homeostasis.
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ACKNOWLEDGEMENTS |
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We thank to Dr. Cythia Magro for reviewing the histological slides and Drs. Caterina Missero, Cathrin Brisken, and Jerry Gross for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR39190, CA16038, and CA73796 (to G. P. D.) and in part by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Dermatology, Stanford University Medical School, Stanford, CA 94305.
To whom correspondence should be addressed: CBRC, MGH-East,
13th St., Charlestown MA 02129. Tel.: 617-724-9538; Fax: 617-724-9572; E-mail: Paolo.Dotto{at}cbrc2.mgh.harvard.edu.
The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling; JNK, c-Jun N-terminal kinase.
2 J. Li, K. Foitzik, E. Calautti, H. Baden, T. Doetschman, and G. Paolo Dotto, unpublished observations.
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REFERENCES |
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