Departments of Medicine and Pathology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112
Submitted 21 March 2003 ; accepted in final form 12 September 2003
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ABSTRACT |
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mitogen; mouse; platelet-derived growth factor
The newly described PDGF-C and -D chains also dimerize and bind to the PDGF- and -
receptors. In contrast to PDGF-A and -B, PDGF-C and -D require proteolytic cleavage for receptor binding (6, 9). PDGF-C and -D share substantial homology and so contain very similar protein regions. These regions include a signal sequence for secretion, a CUB (complement subcomponents Clr/Cls, Uegf, Bmp1) domain, an activation cleavage site, and a receptor-binding cysteine knot (6, 9, 14). The CUB domain may serve to bind the polypeptide to connective tissue elements, and the cysteine knot facilitates dimerization through sulfhydryl bonds.
Bleomycin is an antitumor antibiotic isolated from a strain of Streptomyces verticillus by Umezawa and coworkers in 1965 (5, 13a). Bleomycin induces pulmonary fibrosis in both mice and humans and so has been extensively used as a model to investigate the pathobiology of lung fibrosis. An extensive review of the incidence of bleomycin-induced pulmonary fibrosis was published in 1990 and reported rates of 610% in four larger studies (5). Administration of bleomycin is followed by an inflammatory lung reaction that eventuates in exuberant lung collagen deposition. Histopathology is nonspecific and demonstrates a patchy distribution of diffuse alveolar damage. An inflammatory component consists predominantly of lymphocytes and plasma cells. Histopathology demonstrates endothelial and type I epithelial cell necrosis, the presence of type II epithelial cell hyperplasia, and hyalin membranes. Fibroproliferative lesion formation and excess collagen deposition are seen as the disease progresses.
In the present study, we investigated the expression of all four PDGF ligands and both PDGF receptors in murine lung over time following administration of bleomycin compared with saline controls. The most striking changes in PDGF expression involved the two newly described PDGF isoforms, C and D. PDGF-C message was not detected by Northern analysis in saline-treated lungs but was expressed both at early and late time points following treatment with bleomycin. In contrast, PDGF-D message was constitutively expressed in murine lung but was substantially reduced in response to bleomycin. Modulation of PDGF-C and -D expression during fibrogenesis has not been described previously. Our findings demonstrate that PDGF-C and -D expression is altered during lung fibrogenesis and suggest that these two newly described isoforms may play a role in the pathogenesis of lung fibrosis.
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MATERIALS AND METHODS |
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Northern blot. Total RNA was isolated from murine lungs using the RNeasy Mini kit (Qiagen). RNA, 20 µg per lane, was separated on a 1.2% formaldehyde-agarose gel via electrophoresis. Then the RNA was transferred onto a nylon membrane (Millipore) and hybridized with an [-32P]dCTP-labeled cDNA probe in hybridization buffer [5x SSPE (sodium chloride, sodium phosphate, and EDTA), 2% sodium dodecyl sulfate (SDS), 5x Denhardt's, and 100 µg/ml single-strand DNA (ssDNA)] at 68°C overnight. The probes were labeled using the Prime-a-Gene labeling system (Promega). After hybridization, the blot was washed with 2x SSPE with 0.1% SDS twice at room temperature, then once at 68°C, and finally with 0.2x SSPE, 0.1% SDS twice at 68°C. The signal was quantified with a phosphorimager. We normalized PDGF ligand and receptor mRNA levels with 36B4 as a loading control before comparing expression from bleomycin with saline control samples.
Full-length PDGF-C cDNA was amplified with the primers 5'-CGG AAT TCT CAG CCA AAT GCT CCT CCT C-3' and 5'-CGG AAT TCT TAC AAG TCT TCT TCA GAA ATA AGC TTT TGT TCC CCT CCT GCG TTT CCT CT-3', which introduced the EcoRI site at both ends and a c-myc tag at the 3' end. Then the PDGF-C cDNA was subcloned into the pcDNA3.1 vector (Invitrogen). A 788-bp PDGF-C probe was generated from this construct by digestion with HindIII and BamHI. Full-length PDGF-D cDNA was amplified with the primers 5'-GCG GGA TCC GCC ACC ATG CAA CGG CTC GTT TTA GTC TCC ATT CTC C-3' and 5'-ACG CGT CGA CTT CTC GAG GTG GTC TTC AGC TGC AGA TAC AGT C-3', which introduced a BamHI site at the 5' end, a SalI site at the 3' end, and a mutated stop codon. Then the PCR product was subcloned into the pCMV-tagA vector (Stratagene). A 959-bp PDGF-D probe was generated from this construct by digestion with HindIII and EcoRV. The PDGF-A, PDGF-B, PDGF- receptor, and PDGF-
receptor cDNA probes were kindly donated from the laboratory of Dr. Arnold Brody (Tulane University).
RPA. A 254-bp fragment of the full-length murine PDGF-D cDNA between HaeII and EcoRI sites was designed as an RPA probe and was subcloned into the pBluescript KS (+) vector. The construct was linearized by restriction digestion with HindIII. The anti-sense RNA probe was labeled using an in vitro transcription kit (Pharmingen). One microliter (0.5 µg) of the linearized construct was incubated with 1 µl RNasin, 1 µl GAGU pool, 2 µl DTT, 4 µl 5x transcription buffer, 10 µl [-32P]UTP (Perkin Elmer), and 1 µl T7 RNA polymerase at 37°C for 1 h. Then 2 µl of DNase were added to the mixture above at 37°C for 30 min to digest the template. After digestion, the labeled probe was extracted with phenol-chloroform-isoamy alcohol (25:24:1) and precipitated with 4 M ammonium acetate (pH 5.2) and 100% ethanol. The RPA was carried out with the RiboQuant Ribonuclease Protection Assay kit (Pharmingen) according to the instruction manual. Twenty micrograms of total RNA from each sample were used in this assay. The total RNA was hybridized with 1 x 106 cpm labeled probe at 56°C for 16 h. After hybridization, 6 µl of RNase A and T1 were added at 30°C for 45 min to degrade unprotected probe, and then the samples were incubated with proteinase K at 37°C for 15 min. The hybridization product was then extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with 4 M ammonium acetate and 100% ethanol, and separated with a 5% denaturing acrylamide gel. Finally the gel was dried and exposed to film (Kodak) using an intensifying screen. A cyclophilin probe (Ambion) was used as a loading control.
Western blot. Murine lung tissue was homogenized in radioimmunoprecipitation assay buffer (1x phosphate-buffered saline, 1% Nonidet P-40, 0.5% deoxycholate, and 1% SDS) along with a protease inhibitor cocktail [4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, leupeptin, bestatin, pepstatin A, and E-64] and a phosphatase inhibitor cocktail (sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole; Sigma). Eighty micrograms of protein from lung tissue were separated by SDS-PAGE via an 816% gradient gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Amersham). The membrane was blocked with 5% bovine albumin (Sigma) in 1x phosphate-buffered saline with 0.05% Tween 20 (PBST) for 1 h at room temperature, followed by incubation with either a 1:200 dilution of anti-p-PDGFR- or a 1:200 dilution anti-p-PDGFR-
antibody (Santa Cruz) for 1 h at room temperature. After incubation, the blot was washed with PBST for 30 min and incubated with a 1:15,000 dilution of goat anti-rabbit secondary antibody (Kirkegaard and Perry Laboratories) for 1 h at room temperature. The blot was then washed with PBST for 1 h, developed using the ECL kit (Amersham), and exposed to film. After development, the blot was stripped by incubation in 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris, pH 6.8, at 50°C for 30 min. Then the blot was reprobed using either a 1:200 dilution of anti-PDGFR-
or a 1:200 dilution of anti-PDGFR-
receptor antibody (Santa Cruz).
In situ hybridization. The PDGF-C cDNA probe was labeled with the DIG DNA Labeling and Detection kit (Roche), which employs incorporation of digoxigenin-11-dUTP. Sections (5 µm) of paraffin-embedded murine lung were dewaxed with xylene and rehydrated with gradient ethanol. The sections were incubated with 0.1N HCl for 20 min and treated with proteinase K for 15 min at room temperature. Then the sections were prehybridized with 50% formamide, 5x SSC, 5x Denhardt's, and 100 µg/ml ssDNA for 2 h at room temperature. After prehybridization, the sections were hybridized with 50% formamide, 5x SSC, 5x Denhardt's, 10% dextran sulfate, 1.25 µg/µl tRNA, and 10 ng/section of probe overnight at 42°C. Finally the sections were washed, and the signals were detected with an antidigoxigenin-alkaline-phosphatase antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Control DNA (linearized pBR328), supplied with the kit, was labeled and used as a control probe. Slides were counterstained with a fast nuclear red stain (Sigma).
Statistical methods. All data are presented as means ± SE. Unpaired two-tail t-tests were performed with InStat 1.14 software. The results were considered to be statistically significant at P < 0.05. Fisher's exact test was used to evaluate the significance of the absence of PDGF-C mRNA expression in saline-treated samples with the presence of PDGF-C expression in bleomycin-treated samples.
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RESULTS |
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There were more remarkable differences in the mRNA expression of the two newly described PDGF isoforms, PDGF-C and -D, following administration of bleomycin (Fig. 2). PDGF-C mRNA was not detected by Northern analysis in the lungs of untreated mice or mice treated with saline. However, PDGF-C mRNA expression was induced as early as 4 days after bleomycin administration. PDGF-C mRNA expression after administration of bleomycin was highest at day 8 and was still elevated over saline controls at day 16. Statistical analysis was conducted by Fisher's exact test to assess the significance of a lack of PDGF-C mRNA signal in six out of six saline-treated mice with the appearance of PDGF-C mRNA in six out of six bleomycin-treated mice for each time point. Differences in PDGF-C expression between saline-treated and bleomycin-treated mice were highly significant (P < 0.005). In contrast, PDGF-D was constitutively expressed in murine lung, but its expression was diminished at 8 and 16 days following treatment with bleomycin, with the greatest decline in expression occurring at day 8 (70%, P < 0.01). We have described a short murine PDGF-D transcript with a deletion of exon 6 that renders the ligand incapable of forming into a biologically active dimer (17). The RPA shown in Fig. 3 demonstrates that the reduction in PDGF-D mRNA expression in response to bleomycin is due to a similar decrease in both the long and short PDGF-D transcripts.
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PDGF- receptor mRNA demonstrated a transient decrease (50%, P < 0.01) at day 8 following bleomycin treatment and returned to normal at day 16 (Fig. 4). There were no significant differences in PDGF-
receptor mRNA expression at any time point between bleomycin- and saline-treated mouse lung. There was, however, a reproducibly greater expression of PDGF-
receptor mRNA at the 4-day time point for both bleomycin and saline samples compared with the 8-day and 16-day time points.
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Phosphorylation of the PDGF receptors is an early event following binding of the PDGF ligand (3). As shown in Fig. 5, phosphorylation of the PDGF- receptor was greater in bleomycin-treated mouse lungs compared with saline-treated lungs at day 8 (P < 0.02), even though the protein level of the receptor in both groups was the same. At day 16, there was no difference in PDGF-
receptor phosphorylation between the saline- and bleomycin-treated animals (data not shown). PDGF-
receptor phosphorylation was not detected in the bleomycin- or saline-treated mouse lung at any time point following treatment (data not shown).
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In situ hybridization was used to localize PDGF-C mRNA expression in bleomycin-treated mouse lungs at day 8 because it was the time point showing the greatest expression (Fig. 6). PDGF-C mRNA could not be detected in saline-treated control lung but was consistently identified specifically in regions of lung injury in bleomycin-treated mice. There was no detectable PDGF-C expression by in situ hybridization in normal regions of bleomycin-treated mouse lung or in saline-treated mouse lung sections.
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PDGF-C and -D mRNA expression was compared in bleomycin-sensitive (C57BL/6) and bleomycin-resistant (BALB/c) strains (Fig. 7). Analysis was performed at the day 8 time point because earlier experiments demonstrated that this time point showed the greatest change in expression for these two PDGF isoforms in the bleomycin-sensitive strain (Fig. 2). There was no induction of PDGF-C mRNA expression in BALB/c mouse lung in response to bleomycin. PDGF-D expression was marginally reduced in bleomycin-resistant mice compared with bleomycin-sensitive mice.
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DISCUSSION |
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PDGF-C binds to the PDGF-, but not the PDGF-
, receptor (9). We found in our studies a marked increase in PDGF-
receptor phosphorylation, which is the earliest event following binding of PDGF ligands to their receptors (Fig. 5) (3). However, overall there were no significant differences in PDGF-
or -
receptor expression by Western or Northern analysis (Figs. 4 and 5). The combined observations of increased PDGF-C ligand expression with activation of the receptor that binds PDGF-C indicate that this signaling cascade is activated and suggest a role for this potent mitogen and chemoattractant in bleomycin-induced lung fibrosis. Inasmuch as PDGF-C requires proteolytic cleavage to allow receptor binding, these data also suggest that PDGF-C is activated by the proinflammatory milieu observed in response to bleomycin.
In contrast, PDGF-D mRNA expression was reduced by >50% following administration of bleomycin (Fig. 2). There was a statistically significant, but small, decrease in expression of PDGF- receptor mRNA expression 8 days following administration of bleomycin, but we did not observe any appreciable differences in PDGF-
receptor peptide expression or phosphorylation following bleomycin treatment (not shown). We recently reported (17) that murine PDGF-D has a large splicing variant involving deletion of exon 6, which causes a frame shift resulting in an early termination codon in exon 7. The large splicing variant that we have identified results in a deletion that is predicted to have significant effects on polypeptide activity, since it results in the deletion of regions within the cysteine knot domain that are important for peptide dimerization and receptor binding (6, 9). An RPA was performed to determine whether the reduction in PDGF-D mRNA expression was due to a preferential loss of either the long or short transcripts (Fig. 3), and our findings demonstrate that there is a similar reduction in both transcripts. In concert, these data suggest that PDGF-D is not a major contributor to the bleomycin-induced fibroproliferative response.
We did not find any appreciable differences in the expression of PDGF-A or -B mRNA in lungs isolated from bleomycin-treated mice compared with saline-treated animals (Fig. 1). There were small decreases in PDGF-A and -B mRNA expression as well as PDGF receptor expression at 8 days following administration of bleomycin, which could in part be due to an influx in inflammatory cells that do not express these transcripts. These data may conflict with the observations of others showing an increase in PDGF-B expression within type II cells of bleomycin-treated rats (13) and increased PDGF-A and -B expression in cells retrieved by bronchoalveolar lavage from bleomycin-treated hamsters using PCR with 40 cycles of amplification (2). Furthermore, PDGF-A and -B polypeptides were increased in the lavage fluid from bleomycin-treated rats (15). Differences in PDGF-A and -B expression in our study compared with these other studies could possibly be due to differences between species or perhaps are explained by differences between examining whole lung mRNA expression as in our study versus specific cell subtypes as performed in the studies using rats and hamsters. PDGF-A (10), but not -B, mRNA expression was reported as elevated in cells lavaged from bleomycin-treated mice, but the mice received the bleomycin by intraperitoneal injection, compared with intratracheal injection in our study.
There is also conflicting data regarding experiments that block or augment PDGF-B expression in murine models of bleomycin-induced lung fibrosis. In vivo gene transfer by the hemagglutinating virus of Japan liposome method of the extracellular portion of the PDGF- receptor, which binds PDGF-B and -D, but not PDGF-A or -C, abrogated bleomycin-induced lung fibrosis (16). In contrast, PDGF-B transgenic mice did not demonstrate an increased fibroproliferative response compared with their wild-type littermates following treatment with bleomycin (8). Our data neither support nor conclusively refute a role for the PDGF-A or -B isoforms in bleomycin-induced lung injury but instead demonstrate greater regulation of PDGF-C and -D isoform mRNA expression.
In summary, PDGF isoforms are potent fibroblast mitogens and chemoattractants that are expressed during fibroproliferative lung disease. Two PDGF isoforms that require proteolytic cleavage for activation have recently been described. We found a significant positive correlation of lung fibrosis with PDGF-C expression and a negative correlation with PDGF-D expression. This is the first study to report modulation of PDGF-C or -D expression during fibrogenesis in any organ. Our findings suggest a role for the PDGF-C isoform in the development of lung fibrosis.
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FOOTNOTES |
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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.
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REFERENCES |
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