Carcinogenesis, Vol. 21, No. 5, 1063-1066,
May 2000
© 2000 Oxford University Press
Expression of PGF2
receptor mRNA in normal, hyperplastic and neoplastic skin
Karsten Müller,
Peter Krieg,
Friedrich Marks and
Gerhard Fürstenberger1
Research Program Tumor Cell Regulation, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
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Abstract
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Reverse transcription polymerase chain reaction (RT–PCR)
and Northern blot analysis was used to determine the level of
expression of prostaglandin F
2
(FP) receptor mRNA in various
mouse tissues, including normal, hyperplastic and neoplastic
mouse epidermis. Steady-state concentrations of FP receptor
mRNA were low in normal and hyperplastic epidermis. The response
of the epidermis to the phorbol ester 12-
O-tetradecanoylphorbol-13-acetate
(TPA) was biphasic in that FP receptor mRNA was increased immediately
after treatment, followed by a long-lasting down-regulation
at later time points. FP receptor mRNA was down-regulated in
the majority of papillomas obtained by the mouse skin carcinogenesis
initiation–promotion protocol. In carcinomas, FP receptor
mRNA expression was similar to that in normal epidermis. The
steady-state concentration of FP mRNA was inversely correlated
with PGF
2 levels in normal and hyperplastic epidermis and in
papillomas, indicating that FP mRNA expression is regulated
by this eicosanoid.
Abbreviations: NSAID, non-steroidal anti-inflammatory drug; PGF2, prostaglandin F2; FP receptor, PGF2 receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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Non-steroidal anti-inflammatory drugs (NSAID), such as aspirin,
sulindac, indomethacin, ibuprofen, etc., have been shown to
inhibit experimental carcinogenesis in a variety of organs (for
review, see ref. 1). According to epidemiological and clinical
studies, NSAIDs significantly reduced colorectal tumor development
in men (for review, see ref. 2). All NSAIDs suppress prostaglandin
biosynthesis by inhibiting cyclooxygenases, i.e. enzymes catalyzing
an essential step in prostanoid formation. In fact, prostaglandins
were found to be dramatically elevated in many human and experimental
tumors, and prevention of tumor development by NSAID was attributed
to inhibition of prostaglandin production (
1,
2). Experimental
mouse skin carcinogenesis so far provides the only model in
which prostaglandins have been unequivocally shown to act as
mediators of regenerative processes and tumorigenesis (
3,
4).
Papillomas generated by the initiation–promotion protocol
[using dimethylbenz(
a)anthracene (DMBA) as an initiator and
phorbolester 12-
O-tetradecanoylphorbol-13-acetate (TPA) as a
promoter] accumulate large amounts of prostaglandins E
2 (PGE
2)
and PGF
2 (
5). Moreover, the potent anti-promoting activity of
the NSAID indomethacin could be specifically reversed by PGF
2 (
6). In addition, when applied concomitantly with TPA, only
this prostaglandin type exerted a co-promoting activity increasing
the tumor response beyond that obtained by TPA alone. No information
is yet available on the molecular mechanisms involved in the
tumor-promoting activity of PGF
2. One obvious pathway through
which PGF
2 may act is the interaction with its receptor expressed
at the cell surface. A PGF
2 (FP)-specific receptor has recently
been cloned from cDNA libraries originating from different species,
including mouse (
7). In this paper, we have analyzed the expression
of this FP receptor in normal, hyperplastic and neoplastic mouse
epidermis, as well as in various other mouse tissues.
Seven-week-old female NMRI mice (BRL, Füllinsdorf, Switzerland) were used in the animal experiments. Shaving of the back skin with electrical clippers was performed 3 days prior to treatment. For topical applications, compounds were dissolved in 0.1 ml acetone and applied onto the shaved back skin. Mice were killed at varying time-points, the back skin was dissected and snap-frozen at –70°C using a cold table. Mouse skin tumors were generated by the mouse skin carcinogenesis initiation–promotion protocol using DMBA (100 nmol/0.1 ml acetone; single epicutaneous application) and TPA (10 nmol/0.1 ml acetone; twice-weekly applications for 20 weeks). Papillomas were harvested 22 weeks after initiation, i.e. 2 weeks after the last TPA treatment, and carcinomas 40 weeks after initiation, i.e. 20 weeks after the last TPA treatment. Great care was taken during the preparation of tumors to avoid contamination by non-epithelial material. A section of each tumor sample was analyzed histologically to confirm that more than 95% of the removed biopsy material was of epithelial origin. All tumor samples were snap-frozen at –70°C immediately upon dissection.
Total RNA was isolated from frozen tissue homogenized using a dismembrator. The powdered tissue was added to a guanidinium thiocyanate solution (RNA-Clean, AGS, Heidelberg, Germany). RNA was extracted according to the manufacturer's instructions and quantified by UV absorption at 260 nm. First-strand cDNA synthesis was carried out with 1 µg total RNA in 20 µl reaction mixtures using the Gene Amp RNA PCR kit (Perkin Elmer, Weiterstadt, Germany) using a oligo(dT) primer according to the manufacturer's instructions. The reverse transcription mixture contained 1 mM of each dNTP, 2 µl 10xPCR buffer (500 mM KCl, 100 mM Tris–HCl, pH 8.3), 4 µl MgCl2 (25 mM), 1 µl oligo(dT) primer (50 µM), 1 µl RNase inhibitor (20 U/µl) and 1 µl MuLV reverse transcriptase (50 U/µl). This mixture was incubated for 10 min at room temperature and 15 min at 42°C, and then heated to 99°C for 5 min.
PCR amplification of the FP receptor DNA was performed using two specific primer pairs. Using the forward primer 5'-CTGTGTTCGTGGCTGTGCTG-3' and reverse primer 5'-TGCTTGCTGGCTCTCCTTCTC-3', a PCR product of 592 bp was obtained, and with the forward primer 5'-GCTCTTGGTGTTTCCTTCTCG-3' and reverse primer 5'-TGCTTGCTGGCTCTCCTTCTC-3', a PCR product of 446 bp was obtained. In order to discriminate the amplification products from those originating from contaminating genomic DNA, the primer sets were designed to flank intron 2, which is more than 7.5 kb in size. Amplification of a ß-actin DNA fragment (429 bp) was also performed as an internal control using the forward primer 5'-AAACTGGAACGGTGAAGGC-3' and the reverse primer 5'-GCTGCCTCAACACCTCAAC-3'. The PCR reactions were primed with 1 µl of the cDNA reactions using 20 pmol primers in 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg/ml bovine serum albumin with 0.2 mM of each dNTP and 0.5 µl Taq polymerase (5 U/µl; Appligene Oncor, Illkirch, France) in 50 µl reactions. The PCR was programmed in a PTC-200 DNA Engine (MJ Research, Watertown, USA). The amplification cycle for the FP receptor fragment consisted of an initial denaturation at 94°C for 5 min, followed by 94°C for 1 min, 60°C (fragment of 446 bp) or 55°C (fragment of 592 bp) for 1 min, 72°C for 1 min for 36 cycles and termination at 72°C for 10 min. From each PCR reaction, a 24 µl aliquot was analyzed by electrophoresis on a 1.4% agarose gel. The amplification cycle for the ß-actin fragment consisted of an initial denaturation at 95°C for 5 min, followed by 94°C for 90 s, 54°C for 90 s, 72°C for 90 s for 30 cycles, and termination at 72°C for 10 min. From each ß-actin PCR reaction, an 8 µl aliquot was analyzed by electrophoresis on a 1.4% agarose gel. The identity of the PCR products was confirmed by sequence determination using a ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit and the products were resolved on an ABI Prism 310 Genetic Analyzer (Perkin-Elmer/Applied Biosystems, Weiterstadt, Germany). The sequences were assembled and analyzed using the Heidelberg Unix Sequence Analysis Resources (HUSAR) software programs.
Northern gels loaded with 10–14 µg RNA were electrophoresed and RNA was transferred to Hybond-N+ membranes (Amersham/Pharmacia, Freiburg, Germany) by established procedures (8). Labeling was performed using the Megaprime DNA labeling kit (Amersham/Pharmacia, Freiburg, Germany) with gel electroeluted and purified cDNA fragments. The filters were washed with a final stringency of 0.1x standard saline citrate, 0.5% sodium docecyl sulfate for 20 min and exposed to film using intensifying screens at –80°C. Standardization of RNA loading was performed by rehybridization of the blots with a 18S-rRNA-specific probe.
Using murine FP receptor cDNA sequence information (7), two pairs of oligonucleotide primers were designed, yielding FP receptor-specific amplification products of 592 and 446 bp, as confirmed by DNA sequencing. RT–PCR analysis was used to measure the expression of FP receptor mRNA in murine tissues, including footsole, forestomach, trachea, lung, tongue, intestine, colon, kidney, liver and skeletal muscle, as well as brain. No FP receptor mRNA expression was detected in testis, thrombocytes or reticulocytes (Figure 1
). These results confirm previous data showing that uterine tissue and kidney exhibited the highest level of FP receptor mRNA expression (7). Expression in footsole, forestomach, tongue and trachea is reported here for the first time. In contrast to a previous study (7), FP receptor mRNA was also detected in brain, liver and intestines.
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Fig. 1. RT–PCR of FP receptor mRNA from various mouse tissues. RNA from each sample was reverse-transcribed to cDNA and PCRs were run with a specific primer set for FP receptor mRNA (upper panel) and with a primer set for ß-actin as an internal control (lower panel), as described in the text. Lanes 1, footsole; 2, trachea; 3, lung; 4, tongue; 5, forestomach; 6, brain; 7, thrombocytes; 8, reticulocytes; 9, colon; 10, intestine; 11, liver; 12, testis; 13, skeletal muscle; 14, H2O (no cDNA template); 15, kidney (positive control); 16, marker.
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Using two different specific primer sets, FP receptor mRNA was
also found in murine skin epidermis (Figure 2
). Upon epicutaneous
administration of the phorbol ester TPA, a rapid increase of
the FP receptor mRNA content was observed, which fell back to
control levels around 2–4 h after treatment, and later
on dropped below the control, to remain suppressed for up to
48 h after treatment (Figure 2
). The transient increase of FP
receptor mRNA expression induced by TPA was confirmed by northern
blot analyses (Figure 3
). As shown in Figure 4
, FP receptor
mRNA was also detected in hyperplastic epidermis obtained upon
exposure of adult mouse skin to chronic TPA treatment (
9), and
in neonatal mouse skin, which constitutively exhibits a hyperplastic
phenotype (
10). In papillomas obtained by the initiation–promotion
protocol, the steady-state concentration of FP receptor mRNA
was found to be moderately or strongly reduced (Figures 3 and
5
). Treatment of papilloma-bearing animals with TPA, however,
induced a transient increase of FP receptor mRNA. In contrast
to papillomas, carcinomas exhibited a FP receptor mRNA content
which was only slightly reduced when compared with normal skin
(Figure 5
). These data show the FP receptor mRNA to be constitutively
expressed at low levels in murine epidermis, indicating that
keratinocytes not only generate PGF
2 (
6), but are also effector
cells for this eicosanoid. This conclusion was confirmed by
the observation that keratinocytes in culture were also found
to express FP receptor mRNA (data not shown). A rapid induction
of FP receptor mRNA expression by TPA has also been reported
for corpora lutea granulosa cells, which show a similar time
course (
11). A unique feature of skin epidermis is the down-regulation
of FP receptor mRNA seen upon prolonged TPA treatment. This
drop in expression of FP receptor mRNA, which is inversely correlated
with TPA-induced synthesis of PGF
2 (
5,
6), indicates an agonist-induced
down-regulation of the receptor mRNA. Such an effect of PGF
2 has indeed been reported for ovine corpora lutea (
12). Conversely,
suppression of prostaglandin biosynthesis led to an induction
of FP receptor mRNA expression in blood vessels and brain synaptosomes
of the newborn pig. This effect was specifically counteracted
by PGF
2 or FP receptor agonists (
13,
14). Therefore, the high
level of PGF
2 in papillomas (
5) may be responsible for the down-regulation
of FP receptor mRNA in these tumors. On the other hand, FP receptor
mRNA expression was found to be at similar levels, rather than
reduced, in chronic hyperplastic skin as compared with normal
epidermis. This observation is in agreement with a low PGF
2 level both in neonatal and chronically TPA-treated hyperplastic
skin epidermis (
5). It remains to be established, however, whether
direct treatment of mouse skin with NSAID or PGF
2 leads to changes
in FP receptor mRNA expression and whether such changes are
reflected at the level of receptor protein or whether they influence
receptor activity.
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Fig. 2. RT–PCR of FP receptor mRNA from normal and TPA-treated mouse epidermis. After epicutaneous application of acetone (0.1 ml) or 10 nmol TPA (dissolved in 0.1 ml acetone), total RNA was extracted from epidermis at the times indicated and reverse transcribed to cDNA. PCRs were run with two FP-receptor-specific primer sets (upper panel) and with a primer set for ß-actin as internal control (lower panel). Lane 1, acetone-treated epidermis (30 min); 2, epidermis 30 min after TPA treatment; 3, 1 h after TPA treatment; 4, 2 h after TPA treatment; 5, 4 h after TPA treatment; 6, 6 h after TPA treatment; 7, 24 h after TPA treatment; 8, 48 h after TPA treatment; 9, H2O (no cDNA template); 10, kidney (positive control); 11, marker.
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Fig. 3. Northern blot analyses of FP receptor mRNA from normal, TPA-treated and neoplastic mouse skin. Total RNA from normal, TPA-treated and neoplastic mouse epidermis was isolated, separated on northern gels, blotted and probed with 32P-labeled FP receptor cDNA. Standardization of RNA loading was performed by rehybridization with a 18S-rRNA-specific probe. Lane 1, epidermis 30 min after TPA treatment; 2, 1 h after TPA treatment; 3, 2 h after TPA treatment; 4, 4 h after TPA treatment; 5, 6 h after TPA treatment; 6, 24 h after TPA treatment; 7, 48 h after TPA treatment; 8, kidney, positive control; 9, acetone-treated epidermis (30 min); 10, epidermis 30 min after TPA treatment; 11, carcinoma 294; 12, carcinoma 300; 13, kidney (positive control).
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Fig. 4. RT–PCR of FP receptor mRNA from normal and neoplastic mouse epidermis. RNA from samples from untreated epidermis of adult mice, of epidermis from 2- and 4-day-old mice and from mice chronically treated with TPA for 20 weeks were reverse-transcribed to cDNA and PCRs were run with a FP-receptor-specific primer set (upper panel) and a primer set for ß-actin as internal control (lower panel), as described in the text. Lane 1, hyperplastic epidermis of adult mice treated for 20 weeks with twice-weekly applications of TPA and sacrificed 2 weeks after the last TPA treatment; 2, hyperplastic mouse epidermis, 2 days after birth; 3, hyperplastic mouse epidermis, 4 days after birth; 4, normal adult epidermis; 5, H2O, no cDNA template; 6, kidney (positive control); lane 7, marker.
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Fig. 5. RT–PCR of FP receptor mRNA from normal epidermis, papillomas and carcinomas. RNA from normal epidermis, untreated papillomas, papillomas treated with TPA (10 nmol) for 1 h, and carcinomas were reverse-transcribed to cDNA and PCRs were run with an FP-receptor-specific primer set (upper panel) and a primer set for ß-actin as internal control (lower panel), as described in the text. Lanes 1 and 2, papillomas treated with TPA for 1 h; 3 and 4, individual papillomas; 5, pooled papillomas (one out of three pools); 6 and 7, carcinomas 294 and 298 (two out of six); 8, untreated skin epidermis; 9, H2O (no cDNA template); 10, kidney (positive control); 11, marker.
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As far as the co-promoting effect of PGF
2 is concerned, it is
still unclear whether a down-regulation of FP receptor expression
in papillomas provides a selective advantage for initiated cells,
e.g. through protection against cell death. In support of this,
a stimulatory effect of PGF
2 on programmed cell death might,
for instance, be postulated. Such an effect may indeed be involved
in the degradation of corpus luteum cells (
15), whereas for
keratinocytes this still remains to be shown.
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Acknowledgments
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We thank Prof. Dr G.Eisenbrand for kindly supporting this work.
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Notes
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1 To whom correspondence should be addressed Email:
g.fuerstenberger{at}dkfz-heidelberg.de 
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Received October 20, 1999;
revised January 14, 2000;
accepted January 17, 2000.
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Abstract
Introduction