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Department of Morphology, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland
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ABSTRACT |
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INTRODUCTION |
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PAI-2 is expressed in a limited number of tissues and cells, including placenta (12) , monocytes/macrophages (5) , and epidermis (13, 14, 15) . The epidermis is an interesting model system in which to study the role of PAI-2 in vivo. It comprises stem cells and transiently proliferating cells in the basal layer and differentiating cells in the suprabasal layers (16 , 17) . Epidermal differentiation culminates in the production of dead, flattened, enucleated squames consisting of keratin filaments surrounded by a proteinaceous, cross-linked cornified envelope and is thus a physiological process of apoptosis (18) . PAI-2 is preferentially synthesized in differentiated epidermal cells in vivo and in vitro (15 , 19) and can serve as a differentiation marker for keratinocytes. To investigate whether dysregulation of PAI-2 expression may disturb epidermal differentiation, we targeted overexpression of mouse PAI-2 to the proliferating population of mouse epidermis and hair follicle cells by placing a PAI-2-encoding transgene under the control of the K5 promoter (20) . Transgenic mice were highly susceptible to chemically induced papilloma formation. Furthermore, PAI-2-overexpressing papillomas did not undergo extensive apoptosis on cessation of tumor promotion and therefore continued to develop.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was released with KpnI and SalI from
the K5-PAI-2 plasmid, purified, and used to produce transgenic mice by
pronuclear injection of fertilized CBAJ/B6 F1
zygotes.
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Generation of K5-PAI-2/K5-uPA Double Transgenic Mice.
The generation and characterization of the K5-uPA transgenic line have
been described previously (21)
. Phenotypically, K5-uPA
mice are readily recognizable for having chalky white teeth because of
a defect in enamel formation due to the enzymatic activity of
transgene-encoded uPA, which is expressed in ameloblasts
(21)
. Double transgenic mice were generated by crossing
K5-PAI-2 with K5-uPA mice and identified by PCR using primers for
K5-PAI-2 as described above and primers for K5-uPA (21)
.
After having been genotyped, mice were anesthetized, and their incisors
were photographed.
In Situ Hybridization.
A PstI-EcoRI fragment of mouse PAI-2 cDNA
was cloned into pBSKS. Constructs containing sense (SP65-muk) or
antisense (SP64-muk) mouse uPA cDNA
(PstI-HindIII) were kindly provided by Dominique
Belin. The plasmids were linearized and transcribed with the
appropriate enzymes to generate digoxigenin-labeled sense or antisense
RNA probes, using a digoxigenin labeling kit (Boehringer,
Mannheim, Germany). Untreated dorsal skin or papillomas were
collected from anesthetized mice, snap-frozen in liquid nitrogen, and
cryosectioned (10 µm). Processing of cryosections, hybridization, and
detection were performed as described previously (22)
.
Binding Assay and Zymography.
Untreated adult mice were anesthetized, and their backs were shaved. A
piece (8 mm in diameter) of dorsal skin was biopsy punched and
homogenized in 1 ml of homogenization buffer [100 mM Tris
(pH 8.0) and 0.3% Triton X-100] containing 1 mM EDTA and
1 mM phenylmethylsulfonyl fluoride. The homogenates were
centrifuged (10,000 rpm, 15 min at 4°C), and the supernatants were
collected. An aliquot of 20 µl of each supernatant was incubated with
5 µl of 125I-radiolabeled human uPA for 30 min
on ice and resolved by 10% SDS-PAGE under nonreducing conditions; the
gel was dried and exposed to Kodak X-ray film. For zymographic
analysis, samples were prepared as described above, except that EDTA
and phenylmethylsulfonyl fluoride were omitted in the homogenization
procedure. After a 4-h incubation at 37°C, 10 µl of samples were
loaded on an indicator gel (23)
that contains plasminogen,
casein, and agarose. Lysis zones indicating the presence of uPA
activity were photographed under dark-background illumination.
Papilloma Induction.
Transgenic founder mice were backcrossed with mice of the CBA/J strain.
Third generation mice were used for carcinogenesis. The backs of
2-month-old mice were shaved and treated with a single dose of DMBA (25
µg in 200 µl of acetone), followed by twice weekly applications of
200 µl of TPA (10-4 M in acetone)
for 11 weeks. Alternatively, mice were treated with a single
application of DMBA or treated for 11 weeks with TPA. Development of
papillomas was verified macroscopically every week, and representative
individuals were photographed. Negative littermates were treated in
parallel as controls. Mice were individually caged as soon as
papillomas became visible.
In Situ Detection of DNA Fragmentation.
Papillomas were collected 3 weeks after TPA termination, fixed in 4%
paraformaldehyde, and processed to yield 5-µm paraffin sections. The
sections were dewaxed in xylene, rehydrated in graded ethanol
solutions, and denatured in water at 70°C for 60 min. Tailing
reactions were carried out in a mixture containing 1 µM
digoxigenin-11-dUTP (Boehringer), 0.2 µM ddATP, 0.5
unit/µl terminal transferase (Pharmacia), and 1x reaction buffer at
37°C for 60 min. Incorporated digoxigenin-11-dUTP was revealed by an
antidigoxigenin antibody conjugated to alkaline phosphatase
(Boehringer) with 5-bromo-4-chloro-3-indoly-phosphate and nitroblue
tetrazolium chloride as substrates. Results were recorded using a Zeiss
microscope.
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RESULTS |
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; the nature of the fragment was confirmed
by digestion at a unique ApaI site (Fig. 1A)
,
which yielded two fragments of the expected sizes (Fig. 1B)
.
Both individuals transmitted the transgene in a Mendelian fashion and
were used as founders to establish lines 23 and 52. Transgenic mice
were indistinguishable from their negative littermates in terms of body
size, life span, coat fur, and fertility throughout a 2-year period of
observation (data not shown). In situ hybridization
performed on a sample from line 23 revealed PAI-2 mRNA in basally
located cells of the epidermis and the outer root sheath of hair
follicles (Fig. 1C)
; the specificity of the signal was
confirmed using a corresponding sense probe, which did not hybridize to
these sites (data not shown). No signal was detected in wild-type skin
analyzed in parallel (data not shown); the signal observed in
transgenic epidermis thus did not correspond to endogenous PAI-2 mRNA,
which was under detection in our experiment. The presence of the
transgene-encoded protein was also determined. Total protein extracts
from an equal surface of skin from control, line 23, and line 52 mice
were incubated with radiolabeled human uPA, resolved by SDS-PAGE, and
autoradiographed. A uPA-PAI-2 complex of similar electrophoretic
mobility was revealed in both the wild-type and the two transgenic
lines (Fig. 1D)
, indicating that transgene-encoded PAI-2 was
qualitatively similar to endogenous PAI-2. PAI-2 levels in wild-type
and line 52 skin extracts were similar, whereas the amount of PAI-2 in
line 23 mice was much higher (Fig. 1D)
. Thus, line 23 was
selected for additional experiments, and, unless otherwise indicated,
the term "transgenic mice" used hereafter refers to this line.
PAI-2 Expression in Basal Keratinocytes Does Not Alter
Epidermal Differentiation.
To investigate the impact of PAI-2 overexpression on epidermal
structure, we compared the histology of adult back skin from wild-type
and transgenic mice (Fig. 2)
. Consistent with the normal gross appearance of the skin, the two
samples were indistinguishable in terms of epidermal thickness and hair
follicle density and structure.
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, whereas only 7 of 20 control mice bore barely
recognizable lesions (Fig. 3A)
. At the end of TPA
application (week 11), all but 1 transgenic mouse (32 of 33) and 13 of
20 control mice had developed papillomas (97% versus 65%;
P = 0.0016; Fig. 3C
). Papilloma
density (number of papillomas/papilloma-bearing mouse) was not
significantly different between the transgenic (12.03 ± 6.00) and control (14.17 ± 7.58) groups. However, on
average, papillomas in transgenic mice were of larger size, probably
because they developed earlier than papillomas in control mice.
In agreement with the low level of transgenic PAI-2 expression,
carcinogenesis in line 52 mice was similar to that in control mice
(data not shown). Treatment with DMBA or TPA alone did not result in
any detectable lesions in transgenic or control mice, indicating that
carcinogenesis in these mice follows the multistep model
(24)
and that PAI-2 overexpression does not circumvent the
requirement for either initiation or promotion.
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; in transgenic mice, in contrast, they kept growing (Fig. 4B)
. Given that transgenic PAI-2 expression persists at the
papilloma stage (Fig. 6A)
, the differential response to TPA
withdrawal suggests that PAI-2 prevents papilloma regression.
Regression may involve apoptotic cell death. To verify this hypothesis,
eight papillomas of comparable sizes were collected from four
transgenic and four control mice 3 weeks after cessation of TPA
treatment. Transgenic and control papillomas were analyzed in parallel
for the presence of apoptotic cells by in situ DNA nick
labeling. Comparable results were obtained for all transgenic and for
all control samples, and a representative experiment from one pair of
animals is shown in Fig. 4, C–F
. Whereas in control
papillomas, many cells were undergoing apoptosis (Fig. 4, C and E)
, very few cells were labeled in transgenic papillomas
(Fig. 4, D and F)
. Thus, PAI-2 overexpression may
prevent the regression of papillomas by decreasing apoptosis.
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). PAI-2 was detected in the tissue extract but not in the
conditioned medium (data not shown), suggesting that transgene-encoded
PAI-2 as well as endogenous PAI-2 remains predominantly intracellular
in keratinocytes.
Because such an in vitro assay may not faithfully represent
the situation in vivo, we designed an in vivo
strategy to evaluate the capacity of transgene-encoded PAI-2 to prevent
uPA-mediated effects. This strategy is based on the finding that
transgenic mice expressing uPA in the enamel epithelium, under the
control of the K5 promoter, have abnormal tooth development and are
easily recognized by their chalky white teeth (21)
.
Expression of PAI-2 was also targeted to the enamel epithelium in
K5-PAI-2 mice, as revealed by in situ hybridization (data
not shown). Thus, in K5-uPA/PAI-2 double transgenic mice, if PAI-2 is
secreted as is uPA, it should inhibit uPA activity and hence prevent
the occurrence of the tooth phenotype. However, like K5-uPA mice,
double transgenic mice had chalky white teeth (Fig. 5A)
, indicating that uPA activity had not been inhibited by
PAI-2. The failure of PAI-2 to prevent the tooth phenotype could be due
to insufficient PAI-2 expression. To evaluate this possibility, we
compared the expression levels of transgene-encoded PAI-2 and uPA by
measuring the net uPA activity of skin extracts from wild-type, K5-uPA,
and K5-uPA/PAI-2 mice (Fig. 5B)
. Coexpression of PAI-2 in
double transgenic mice reduced uPA activity to a level even lower than
that in wild-type mice, indicating that transgenic PAI-2 expression was
sufficient to abolish transgenic uPA activity. Taken together, these
results suggest that in PAI-2 transgenic papillomas, the repression of
apoptosis appears to be independent of inhibition of uPA but rather
involves an intracellular effect of PAI-2.
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. Whereas transgenic PAI-2 was widely expressed in the basal layer of
both normal skin and papillomas (Fig. 6A)
, very few cells
expressed uPA at discrete sites (Fig. 6B)
. No signal for
endogenous PAI-2 mRNA was detected in the 10 wild-type papillomas
analyzed (data not shown). Similar results were obtained in
hyperproliferative lesions after TPA termination (Fig. 6, C and D)
. Thus the effect of PAI-2 on transgenic papilloma
formation and skin hyperproliferation appears to be independent of uPA
expression. Interestingly, when lesions progressed to an invasive
carcinoma stage, they were characterized by switching on expression of
uPA (Fig. 6D)
and switching off expression of the K5-PAI-2
transgene (Fig. 6C)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have observed that K5-PAI-2 transgenic mice are highly susceptible to skin carcinogenesis elicited by an initiation-promotion protocol: in comparison with nontransgenic mice, papillomas arose earlier and developed on essentially all treated mice; most strikingly, they did not regress when promotion was discontinued. Importantly, expression of the transgene did not overcome the requirement for either initiation or promotion, suggesting that it did not mimic one or the other of these steps and thus acted in a manner different from H-ras mutations, which can replace the initiation step (24) . We therefore favor the hypothesis that K5-PAI-2 transgene expression facilitated another set of events relevant to carcinogenesis. In this context, our finding that papilloma regression did not occur in transgenic mice, taken together with the observation that this is accompanied in control mice by extensive apoptosis, suggests that PAI-2 may have accelerated papilloma formation by influencing the balance between cell proliferation and cell death. In accord with in vitro results that have shown effects of PAI-2 on cell death (8, 9, 10, 11) , our observations provide the first in vivo evidence that this serpin may control apoptosis and could thereby play an important role in certain hyperproliferative disorders, either nonmalignant or malignant. In this context, it is noteworthy that overexpression of PAI-2 has been reported in human hyperproliferative skin diseases such as lupus erythematosus (26) and in several human cancers (27) including squamous cell carcinoma (28 , 29) . It has recently been suggested that a possible role of PAI-2 on tumor development may depend on its cellular origin because in esophageal squamous cell carcinoma, fibroblastic PAI-2 expression was correlated with a good prognosis, whereas expression in cancer cells was associated with poor prognosis (29) . Our study extends these descriptive reports in that it suggests that PAI-2, when expressed in epithelial tumor cells, may promote cancer development and progression via an antiapoptotic effect.
Driven by the K5 promoter, PAI-2 was constitutively expressed in basal epidermis and hair follicle cells, as demonstrated by in situ hybridization. Why then did PAI-2 overexpression not influence the cellularity and differentiation of the epidermis and its appendages in the absence of exposure to the carcinogenic protocol? Both in vivo and in vitro studies have shown that endogenous PAI-2 is preferentially expressed in differentiating keratinocytes (15 , 19) ; if its role, as has been proposed, is to prevent cells from premature terminal differentiation, the pathway involved may be active only in differentiating cells, and transgene-encoded PAI-2, which is expressed in the proliferating cell population, would therefore not affect the life cycle of the intact skin. During carcinogenesis, however, rapid cell proliferation may be accompanied by rapid cell death, and the proposed antiapoptotic function of PAI-2 would become manifest. A similar phenomenon has been reported in transgenic mice overexpressing Bcl-xL in the same cell population as that which expresses the K5-PAI-2 transgene. Bcl-xL is a "classical" antiapoptotic molecule, and its expression in proliferating keratinocytes does not influence the differentiation of the skin or its appendages, but it does render the mice highly susceptible to carcinogenesis (30 , 31) . Interestingly, rapid cell proliferation accompanied by rapid cell death does occur in normal skin in vivo: it provides a means to "sculpt" the epidermis during development (32) .
Although PAI-2 can exist in two topographically different compartments (the cytosolic and extracellular compartments), PAI-2 remains mostly intracellular in keratinocytes and does not affect extracellular proteolysis. As an intracellular molecule, how can PAI-2 regulate cell death? At least two hypotheses can be envisioned. First, PAI-2 could inhibit an as yet unidentified intracellular protease because intracellular proteolysis is involved in apoptosis (33) . Other serpins, such as CrmA, SPI-1, and PI-9 have been implicated in the regulation of cell death (34) . CrmA, for instance, prevents cytokine processing by inhibiting caspase-1 and protects cells against Fas-1-, tumor necrosis factor-, and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis. A putative intracellular PAI-2 target could perhaps be identified by incubating radiolabeled PAI-2 with extracts of cells induced to commit apoptosis and searching for enzyme-inhibitor complexes. Because a putative protease may be complexed mostly with endogenous PAI-2, the recent availability of PAI-2 knockout mice (35) should be helpful in this respect. Second, given that PAI-2 is a good substrate for transglutaminase (36) , and because this enzyme is involved in the terminal differentiation of the epidermis (37) , the high levels of PAI-2 achieved in the keratinocytes overexpressing and accumulating intracellular PAI-2 may compete with the "natural" transglutaminase substrates, thereby interfering with terminal differentiation. A similar hypothesis has been proposed by O’Brien et al. (38) , who demonstrated that transgenic mice producing high levels of polyamine, an excellent substrate for transglutaminase, are highly susceptible to carcinogenesis.
Whatever the precise mechanism may be, the present study provides in vivo evidence for a role of PAI-2 in inhibiting apoptosis and for a cocarcinogenic effect of its dysregulated expression, at least in chemically induced skin carcinogenesis. Because PAI-2 is normally expressed in a limited number of cell types (39) , it is unlikely to be involved in general cell apoptosis during development and cell differentiation. This may explain why PAI-2 deficiency does not alter overall development, fertility, or survival of mice (35) . However, it will be of interest to determine whether overexpression or aberrant expression of PAI-2 accompanies the development of other cancerous lesions in the skin or other tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants 3100-039717 and 3100-055889
from the Fonds National Suisse de la Recherche Scientifique. 
2 Present address: Department of Cellular
Biochemistry, Serono Pharmaceutical Research Institute S.A., 14 Chemin
des Aulx, CH-1228 Plan-les-Ouates, Switzerland.
3 To whom requests for reprints should be
addressed, at Department of Morphology, University of Geneva Medical
School, 1 Rue Michel-Servet, CH-1211 Geneva, Switzerland. Phone:
41-22-7025223; Fax: 41-22-7025260; E-mail: Jean-Dominique.Vassalli{at}medecine.unige.ch
4 The abbreviations used are: PAI, plasminogen
activator inhibitor; DMBA,
7,12-dimethylbenz(a)anthracene; K5, bovine keratin type
5; tPA, tissue-type plasminogen activator; TPA,
12-O-tetradecanoylphorbol-13-acetate; uPA,
urokinase-type plasminogen activator.
Received 2/24/00. Accepted 11/29/00.
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