Differential expression of platelet-derived growth factor-alpha receptor by Thy-1minus and Thy-1+ lung fibroblasts

James S. Hagood1, Patrick J. Miller2, Joseph A. Lasky3, Albert Tousson4, Benliu Guo1, Gerald M. Fuller4, and J. Clarke McIntosh5

Departments of 1 Pediatrics, 2 Microbiology, and 4 Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 Section of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, Tulane University, New Orleans, Louisiana, 70112; and 5 The Ruth and Billy Graham Children's Medical Center, Asheville, North Carolina 28801


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Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1- and Thy-1+ rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1- and Thy-1+ fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1- cells. This differential activity correlated with significantly higher expression of PDGF-alpha receptor in Thy-1- fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c-myc mRNA in Thy-1- but not in Thy-1+ fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-alpha receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-alpha receptor supports distinct roles for Thy-1- and Thy-1+ fibroblast populations in developmental and fibrotic processes in the lung.

cell surface molecules; rodent; proliferation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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FIBROSIS OFTEN FOLLOWS INJURY or inflammation in tissues and is thought to represent exaggeration or aberration of normal wound-healing events. Fibroblasts within a fibrogenic milieu clearly differ from those in normal tissues. For example, fibroblasts isolated from lungs with active fibrotic disease or from scleroderma lesions produce increased amounts of collagen (15, 29). "Fibrosis" cells have increased proliferative capacity, are capable of anchorage-independent growth, and are morphologically distinct (21, 27, 29). Evidence accumulated over the last decade suggests that the "fibrotic" phenotype arises from selective recruitment or expansion of a subset of fibroblasts with the potential for a more vigorous fibrogenic response rather than by uniform activation of all the resident mesenchymal cells within an inflamed tissue. Differences among subsets of fibroblasts have been identified on the basis of surface markers, cytoskeletal arrangement, lipid content, and cytokine profile (18). The best-characterized experimental model of fibroblast heterogeneity is based on surface expression of the Thy-1 glycoprotein. Morphological and secretory differences between Thy-1-positive (Thy-1+) and Thy-1-negative (Thy-1-) mouse and rat lung fibroblasts have shown that these subpopulations share some of the morphological criteria that distinguish normal cells from fibrotic ones. Thy-1- but not Thy-1+ fibroblasts express Ia, the rodent major histocompatibility complex class II antigen, after interferon-gamma stimulation and are capable of producing interleukin (IL)-1 (16, 19, 20). Although the exact function of Thy-1 is unknown, its structural similarity to immunoglobulin suggests an important role in cell-cell and cell-substrate interactions (7).

Cytokines and growth factors, such as transforming growth factor (TGF)-beta , tumor necrosis factor-alpha , IL-1beta , and platelet-derived growth factor (PDGF), have been demonstrated in elevated concentrations in clinical and experimental fibrogenic disorders of the lung parenchyma and airways (10, 14). The proliferative responses of fibroblasts to both TGF-beta and IL-1 are thought to be indirectly mediated by autocrine stimulation via PDGF, a powerful fibroblast mitogen and chemoattractant (13, 22). Thus the PDGF system appears to be an important regulator of fibrotic responses to multiple mediators. The TGF-beta and IL-1 responses occur largely through the interaction of the AA homodimer of PDGF with the alpha  form of the PDGF receptor (PDGFR-alpha ). PDGF A and B isoforms form homo- and heterodimers. PDGF-AA signals only through the alpha  form of the receptor, whereas AB and BB isoforms can bind to both alpha - and beta -receptor subtypes. The PDGF-AA/PDGFR-alpha pathway has been shown to mediate the mitogenic response of lung fibroblasts in both human scleroderma and rodent bleomycin injury (17, 24) as well as in other fibrotic conditions, including dermal keloid scarring (9) and in vitro asbestos-induced fibroblast mitogenesis (12). Because the alpha -receptor is capable of binding any PDGF isoform, it can process signals via all three ligands, and thus its regulation affects nearly all PDGF-mediated cellular responses. Because of the possible distinct roles of Thy-1- and Thy-1+ cells in fibrotic conditions, we examined whether these cell types differ in their responses to PDGF. As a result, we have identified differential expression of PDGFR-alpha and differential signaling in response to PDGF isoforms in these fibroblast subpopulations.


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INTRODUCTION
METHODS
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Materials. The following cDNAs were used for Northern hybridization: a rat PDGFR-alpha cDNA corresponding to the full-length coding sequence (a generous gift from Randall R. Reed, Johns Hopkins University School of Medicine, Baltimore, MD) was subcloned to generate a probe including the first 1,718 bases, which encode for the extracellular portion of the receptor; the PDGFR-beta cDNA was the generous gift from Michael Pech (Hoffman-La Roche, Basel, Switzerland); and cDNA for murine c-Myc was kindly provided by Dr. Andrew Kraft [University of Alabama at Birmingham, Birmingham, AL (UAB)]. A cDNA fragment derived from murine 18S rRNA (American Type Culture Collection, Manassas, VA) or a 700-bp fragment of rat cyclophilin cDNA, kindly provided by Dr. Etty Benveniste (UAB), were used as loading controls. The recombinant human (h) PDGF-AA and recombinant hPDGF-BB were obtained from R&D Systems (Minneapolis, MN). The following antibodies were employed in the Western blotting and immunofluoresence analyses: rabbit polyclonal anti-hPDGFR-alpha and anti-hPDGFR-beta (Santa Cruz Biotechnology, Santa Cruz, CA); FITC-conjugated mouse anti-mouse/rat Thy-1.1; and mouse IgG isotype standard (PharMingen, San Diego, CA). Secondary antibodies as indicated were obtained from Molecular Probes (Eugene, OR) and Kirkegaard and Perry Laboratories (Gaithersburg, MD). Control rabbit antiserum [anti-human epithelial sodium channel-beta (hENaC-beta )] was a kind gift from Dr. Kevin Kirk (UAB). Tissue culture plastic ware was obtained from Becton Dickinson Labware (Franklin Lakes, NJ) or Nunc (Naperville, IL). Fetal bovine serum (FBS) was purchased from Sigma (St. Louis, MO), and minimum essential medium (MEM) and Ham's F-12 medium were from Mediatech (Herndon, VA). Reagents (electrophoresis grade) used to prepare the buffers were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise stated.

Cell culture. The preparation of Lewis rat lung fibroblasts and their sorting into Thy-1- and Thy-1+ populations has been described in detail (16). We used cells that have undergone <15 culture passages from isolation, and we confirmed the presence or absence of Thy-1 staining in >95% of cells by flow cytometry (as described in Ref. 16) every second or third culture passage. Fibroblasts were screened for Mycoplasma contamination with a PCR-based kit (Stratagene, La Jolla, CA). The cells were seeded into appropriate culture dishes and grown in MEM with 10% FBS until ~90% confluent, then rendered quiescent by culturing in MEM with 0.4% FBS for 48 h. The monolayers were washed with serum-free medium (SFM), and SFM containing mediators of interest was added for the times indicated.

Proliferation assay. Fibroblasts were plated at 15,000 cells/250 µl in 24-well plates and allowed to attach overnight. The monolayers were washed twice with defined SFM (SFDM; Ham's F-12 medium and 0.25% albumin; Sigma) and rendered quiescent in SFDM plus insulin-transferrin-selenium (10 µg/ml; Life Technologies, Gaithersburg, MD) for 48 h. Growth factors were added as indicated in medium with 0.4% FBS for 8 h, after which [3H]thymidine (Amersham, Arlington Heights, IL) was added to a final concentration of 5 µCi/ml for an additional 16 h (24 h total). The wells were gently aspirated, washed three times with MEM, and placed on ice. The monolayers were treated with ice-cold 5% trichloroacetic acid (TCA) for 15 min., washed, and solubilized in prewarmed (37°C) buffer (0.2N NaOH and 0.1% SDS) for 30 min at 37°C before scintillation counting. Each condition was assayed in triplicate. The wells with medium containing either 0.4 or 10% FBS were used to determine [3H]thymidine uptake in quiescence and log-phase growth, respectively. The counts per minute obtained for cells grown in 0.4% FBS alone were averaged and subtracted from all experimental values and are expressed in arbitrary units, with the average counts per minute in wells exposed to 10% FBS alone set at 100.

Western immunoblotting. Whole cell protein lysates from Thy-1- and Thy-1+ fibroblasts (10 µg/lane) prepared in the presence of protease inhibitors were separated by SDS-PAGE with 10% acrylamide gels. The protein was electroblotted onto polyvinylidine fluoride membranes and probed with antibodies to PDGFR-alpha or -beta as indicated. The membranes were probed with an appropriate horseradish peroxidase-conjugated secondary antibody, and the labeled proteins were detected with enhanced chemiluminescence. Some membranes were stripped of the initial antibodies by incubation in 0.0625 M Tris-Cl, pH 6.8, 2% SDS, and 0.1 M beta -mercaptoethanol at 68°C for 30 min. The membranes were then washed in Tris-buffered saline and exposed to film to determine removal of the original signal before being reblocked and probed with a different antibody.

Immunofluorescence. Monolayers of Thy-1-, Thy-1+, and unsorted Lewis fibroblasts were grown to near confluence on 22 × 22-mm glass coverslips and then rendered quiescent. The coverslips were blocked with 50% normal goat serum (NGS; Sigma)-PBS and incubated with anti-Thy-1.1-FITC diluted 1:20 in 50% NGS-PBS for 1 h at 4°C. The coverslips were washed with PBS, again blocked with 50% NGS-PBS, and incubated with anti-PDGFR-alpha or anti-PDGFR-beta diluted 1:50 in blocking buffer for 1 h at 4°C. The coverslips were washed with PBS and fixed in 3% formaldehyde (transmission electron microscopy grade; Tousimis, Rockville, MD)-PBS for 45 min at room temperature. The coverslips were washed with PBS, blocked with 5% NGS-PBS, and incubated with goat anti-rabbit IgG-Texas Red-X (Molecular Probes) diluted 1:80 in 5% NGS-PBS for 40 min at 37°C. Controls for antibody specificity were mouse IgG1, kappa -FITC (anti-trinitrophenol; PharMingen) at 1:20 (control for anti-Thy-1) and rabbit antiserum (anti-hENaC-beta ) at 1:50, followed by goat anti-rabbit IgG-Texas Red-X at 1:80. All cells were also stained with Hoechst 33258 (Molecular Probes). The coverslips were mounted on glass microscope slides and examined with an Olympus IX70 inverted epifluorescence microscope. Images were acquired with a SenSys cooled charge-coupled device, high-resolution, monochromatic digital camera (Photometrics, Tucson, AZ) and analyzed with IP Lab Spectrum software (Signal Analytics, Fairfax, VA).

Northern blotting. Quiescent or proliferating fibroblast monolayers were lysed with a commercial RNA isolation buffer (Ultraspec, Biotecx, Houston, TX), and total RNA was prepared according to the manufacturer's instructions. Fifteen micrograms of total RNA in denaturation buffer were added to each well of a 1.2% agarose gel and electrophoretically separated overnight. RNA was transferred to a nylon membrane (Immobilon-N, Millipore, Bedford, MA) by capillary action. Prehybridization solution and hybridization diluent consisted of 6× saline-sodium phosphate-EDTA, 5× Denhardt's solution, 1% SDS, and 20 µg/ml of salmon sperm DNA. The membranes were prehybridized for 3-5 h at 62°C and hybridized overnight at 62°C with cDNA probes labeled with [alpha -32P]dCTP with random primers. The hybridized membranes were washed with 2× saline-sodium citrate and 0.5% SDS, then with 0.2× saline-sodium citrate and 0.5% SDS at 37 and 62°C, respectively. The resulting hybridized signal was detected by autoradiography and quantified with either a densitometric scanner (Bio-Rad, Hercules, CA) or image phosphor analysis (PhosphorImager and ImageQuant, Molecular Dynamics, Sunnyvale, CA).

Data analysis. To test for differences in signal from mediator-exposed samples and controls in Western and Northern blotting or differences in proliferation, a one-way ANOVA or paired Student's t-test was performed on the interval data generated by scanning of autoradiographs or storage phosphor scanning of hybridized membranes or on data calculated from measured counts per minute in proliferation assays. Where significant differences were noted, Dunnett's multiple comparisons procedure was employed to test for differences at particular mediator concentrations. Significance was accepted at a P value of <0.05 for all analyses.


    RESULTS
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INTRODUCTION
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Differential mitogenic response to PDGF-AA. PDGF isoforms appear to have distinct roles in fibrotic responses. We therefore determined the response of Thy-1- and Thy-1+ fibroblasts to PDGF-AA and PDGF-BB with [3H]thymidine incorporation as an indicator of proliferation. Figure 1 demonstrates that there is a concentration-dependent proliferative response of Thy-1-, but not of Thy-1+, lung fibroblasts to PDGF-AA, which is maximal at 5 ng/ml (P = 0.00006 vs. Thy-1+). In contrast, both cell populations respond to PDGF-BB; however, the Thy-1- cells have slightly higher responses than the Thy-1+ fibroblasts (42 vs. 9% compared with response to 10% FBS, P = 0.001 at 1 ng/ml of PDGF-BB; 104 vs. 69%, P = 0.01 at 5 ng/ml).


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Fig. 1.   Proliferative response to platelet-derived growth factor (PDGF) isoforms. Thy-1-negative (Thy-1-; A) and Thy-1-positive (Thy-1+; B) fibroblasts were cultured to near confluence. Quiescent monolayers were exposed to either PDGF-AA or PDGF-BB for 24 h, the last 16 of which were in presence of [3H]thymidine. Mean value for each condition was adjusted by subtracting mean value of control monolayers cultured in 0.4% fetal bovine serum (FBS) without added mediators. Data were normalized by setting rate of proliferation obtained in cells cultured in 10% FBS without added mediators at 100 arbitrary units (A and B, top, right bars). Values are means ± SD from 4 separate wells for each mediator concentration. * P < 0.05 vs. same cell type, 0.4% FBS control. + P < 0.05 vs. opposite cell type, same growth factor and/or concentration (conc). ^ P < 0.05 vs. same cell type, different PDGF isotype, same conc.

Differential expression of PDGFR-alpha protein. The contrasting responses of Thy-1- and Thy-1+ cells to PDGF-AA suggested a potential difference in PDGFR-alpha expression in these fibroblast populations. Western blotting was therefore used to establish the relative expression of PDGFR-alpha and PDGFR-beta in Thy-1- and Thy-1+ cells (Fig. 2). In quiescent monolayers (0.4% FBS), the PDGFR-alpha signal was, on average, 11 times higher in Thy-1- than in Thy-1+ cell lysates (P = 0.01; n = 3 monolayers). In overexposed Western blots, a small amount of PDGFR-alpha protein is visible in Thy-1+ lanes (data not shown). The blots were stripped and reprobed with PDGFR-beta antiserum, and the two populations were found to express nearly equivalent PDGFR-beta levels (Fig. 2B), demonstrating that differences in PDGFR-alpha are not based on unequal protein loading. The levels of either receptor subunit did not change significantly with the addition of 10% FBS, although serum withdrawal caused a transient elevation in PDGFR-alpha in Thy-1-, but not in Thy-1+, cells, which was maximal (~2-fold) at 6 h and returned to baseline at 24 h (data not shown).


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Fig. 2.   Immunodetection of PDGF receptor (PDGFR) isoforms. Confluent, quiescent monolayers were analyzed for presence of alpha  (A) and beta  (B) isoforms of PDGF receptor by immunoblotting with 10 µg whole cell lysate protein/lane. A representative enhanced chemiluminescence autoradiograph is shown, which was stripped and reprobed with PDGFR-beta antibody after detection of PDGFR-alpha . Lanes 1 and 2, 0.4 and 10% FBS-exposed cells, respectively. (-), Thy-1- cells; (+), Thy-1+ cells. Nos. in middle, relative migration of molecular-size standards (in kDa).

Differences in PDGFR-alpha mRNA. To verify the differential expression of PDGFR-alpha , total RNA was isolated from Thy-1- and Thy-1+ cells and subjected to Northern analysis. Figure 3 shows representative autoradiographs of a membrane probed with alpha - and beta -receptor cDNAs. Although both cell types have PDGFR-alpha message, the levels in Thy-1- fibroblasts are on average 3.3-fold higher than in cells expressing Thy-1. Conversely, the level of PDGFR-beta is slightly (1.4-fold) higher in Thy-1+ cells.


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Fig. 3.   Northern blot analysis of alpha -receptor RNA. Total RNA from Thy-1- and Thy-1+ populations cultured in 0.4 or 10% FBS was subjected to electrophoresis and transferred onto nylon membranes for hybridization. Shown are autoradiographs from a membrane that was initially probed with 32P-labeled PDGFR-alpha cDNA, then stripped and reprobed with cDNAs indicated at left. Identical results were seen with RNA obtained from duplicate plates.

Immunofluorescent visualization of PDGFRs. To determine whether differences in PDGFR-alpha level occur in mixed (unsorted) populations of fibroblasts and whether there is significant variation in PDGFR-alpha expression within sorted populations, monolayers were examined by digital confocal microscopy after direct immunofluorescent staining with Thy-1 antibody and indirect staining with PDGFR-alpha and PDGFR-beta antisera. Representative photomicrographs are shown in Fig. 4. Appropriate control antibodies demonstrate negligible levels of nonspecific staining (Fig. 4A). In primary, unsorted Lewis rat lung fibroblast monolayers, Thy-1 staining (FITC-labeled antibody; Fig. 4B, green) was confined to a subset of the cells. This surface-staining pattern with punctate areas of increased intensity is characteristic of many glycosylphosphatidylinositol-linked surface molecules (26). PDGFR-alpha staining (Texas Red-X-labeled secondary antibody; Fig. 4, red) is also seen only in a subset of cells in a mixed population, and there is almost no colocalization of red and green fluorescence except in areas where cells overlap. Consistent with Fig. 2, sorted Thy-1+ fibroblasts (Fig. 4, D and F) show staining with anti-Thy-1, very little staining for PDGFR-alpha , and uniform staining for PDGFR-beta . Thy-1- fibroblasts (Fig. 4, C and E) do not stain with anti-Thy-1, and most express both PDGF receptors. A combination of surface and perinuclear staining is observed for both PDGFR-alpha and PDGFR-beta , consistent with published work on PDGFR immunostaining (25). In subconfluent, sorted monolayers, cells staining positive for Thy-1 and/or PDGFR-alpha were counted in eight separate fields on two separate coverslips for each cell type. Sixty-six percent of Thy-1- cells stained positive for PDGFR-alpha vs. 0% of Thy-1+ cells (P = 0.0007; data not shown). Although not all Thy-1- cells demonstrate PDGFR-alpha immunostaining, it is clear that the differences seen in Western blotting were not due to very high levels of PDGFR-alpha expression in only a small subset of Thy-1- cells. The visualization of PDGFR-alpha staining in an unsorted population of rat lung fibroblasts (Fig. 4B) shows that differential PDGFR-alpha expression is not induced by culturing Thy-1- and Thy-1+ cells separately.


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Fig. 4.   Immunofluorescence micrographs of fibroblast monolayers. A and B: unsorted Lewis (LEW) rat lung fibroblasts. C and E: Thy-1- rat lung fibroblasts. D and F: Thy-1+ rat lung fibroblasts. alpha R, PDGFR-alpha ; beta R, PDGFR-beta . A: representative staining with appropriate control antibodies (see METHODS) for those used to stain Thy-1 and PDGFRs. B-F, representative staining with FITC-complexed antibody to Thy-1 (green). Antibodies to PDGFR-alpha (C and D) and PDGFR-beta (E and F) were visualized with a Texas Red-X-complexed secondary antibody (red). All except B were stained with Hoechst 33258 (blue nuclear stain). Bar, 30 µm.

Differences in intracellular signaling after stimulation with PDGF-AA. We measured c-myc mRNA as an indicator of intracellular signaling activated on stimulation of the cell populations with PDGF isoforms. Expression of the early-response gene c-myc is characteristic of entry into the cell cycle (11). We exposed quiescent fibroblast monolayers (n = 3) to PDGF-AA or -BB in SFM and prepared total RNA, which was then used to prepare Northern blots that were hybridized with a murine c-myc probe. Initial experiments to determine the time course demonstrated rapid induction, with elevated c-myc levels at 15 min, peaking at 30 min, and remaining elevated at 1 h (data not shown). Figure 5 demonstrates increased c-myc mRNA in Thy-1-, but not in Thy-1+, cells exposed to PDGF-AA (2 and 5 ng/ml) and -BB (1 and 2 ng/ml).


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Fig. 5.   PDGF isoform induction of c-myc RNA. Top: near-confluent monolayers of Thy-1- and Thy-1+ fibroblasts were exposed to 0.4% FBS (C) or 1, 2, or 5 ng/ml of PDGF-AA or -BB in serum-free medium for 30 min (n = 3 monolayers/condition). Total RNA was subjected to electrophoresis and transferred onto nylon membranes for hybridization to 32P-labeled cDNA probes for c-myc (myc) and cyclophilin (cyc). A representative autoradiograph is shown. Middle and bottom: mean (±SE) ratio of c-myc to cyclophilin signal relative to Thy-1- serum-free medium control (in arbitrary units).


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The data presented demonstrate a clear difference in the proliferative response of Thy-1- and Thy-1+ rat lung fibroblasts to PDGF-AA, a growth factor that is central to parenchymal remodeling in the lung. Using a combination of experimental approaches, we have shown that the preferential proliferation of lung fibroblasts lacking surface Thy-1 is based on increased expression of the PDGFR-alpha subunit. These findings demonstrate an important and relevant functional difference between these fibroblast subsets and support the independent roles of these two populations in fibrotic responses.

The divergent proliferative responses of the two subpopulations is striking in that there is no discernible response of the Thy-1+ cells to PDGF-AA. This is not based on an inability of Thy-1+ fibroblasts to respond to a proliferative stimulus because cells respond normally to PDGF-BB. The lack of any proliferative response to the AA isoform despite the presence of a small amount of alpha -receptor in Thy-1+ cells may indicate that there is a threshold of receptor expression necessary for signaling (22). The slightly stronger response of the Thy-1- cells to the BB isoform compared with that of the Thy-1+ cells at 1 and 5 ng/ml is consistent with the known ability of PDGF-BB to signal through both alpha - and beta -receptors. Interestingly, in terms of c-myc induction, limited PDGF response was seen with either isotype in Thy-1+ cells. Others (6) have demonstrated that there are differences in the sequence of intracellular signals generated through activation of the two PDGFR subtypes, suggesting that PDGF-AA and -BB initiate cell cycle traverse via separate mechanisms. Our data from immunoblotting, Northern blotting, and immunofluoresence indicate that the differences we describe in signaling are likely due to differing levels of alpha -receptor expression.

A subset of fibroblasts uniquely responsive to PDGF-AA may play an important role in pulmonary fibrogenesis. Fibroblasts isolated from lung fibrotic lesions have an increased proliferative capacity but are not transformed (4). This likely results from autocrine growth signaling in which the PDGFR-alpha plays an important role. A number of other clinical and experimental conditions have been described in which fibroblasts respond differentially to the PDGF-AA isoform, including scleroderma lung and skin lesions, keloid scars, rodent bleomycin-induced lung injury, and asbestos-induced mitogenesis (9, 12, 17, 24). Increased expression of PDGFR-alpha has been demonstrated in all of these examples, consistent with our findings in Thy-1- cells. The alpha -receptor plays an important role in regulating cellular responses to PDGF. Not only is it capable of eliciting signals in response to all three PDGF isoforms, but the PDGFR-alpha also has been shown to modulate activities mediated by the PDGFR-beta (5, 8). Notably, the latter study demonstrated functional PDGFR-alpha responses in only a subset of normal fibroblasts (8). To date, no work has correlated the expression of PDGFR-alpha to that of Thy-1.

It remains to be determined whether the presence or absence of the Thy-1 molecule influences the unique characteristics of the two populations described herein or whether Thy-1 is a "surrogate" marker for subpopulations that differ in other significant respects. In NIH/3T3 fibroblasts, loss of Thy-1 surface expression on transformation by viral oncoproteins is associated with anchorage-independent growth and increased major histocompatibility complex II expression (1, 23). Thus it appears that the presence or absence of Thy-1 impacts significantly on the ability of mesenchymal cells to interact with matrix, regulate proliferation, and participate in immune or inflammatory responses. It has been demonstrated that disruption of cell-matrix interactions may be more important than cytokine/growth factor signals in regulating PDGFR expression (2). Enhanced expression of PDGFR-alpha has been described under anchorage-independent conditions in normal rat kidney fibroblasts, conditions in which only the AA and AB isoforms (which signal through the alpha -receptor) are mitogenic (28). After injury, there is a marked disruption of normal tissue matrices. Such a change in "cellular context" may create an environment into which PDGFR-alpha -positive Thy-1- fibroblasts are recruited and induced to proliferate, thus positioning them to direct subsequent repair or scarring. The potential of PDGFR-alpha -positive fibroblasts to alter lung architecture has been underscored recently by a study (3) of the PDGF-AA null mouse, a homozygous lethal mutation that lacks normal pulmonary alveolar development related to loss of PDGFR-alpha -positive myofibroblasts.

We have demonstrated that expression of PDGFR-alpha in rat lung fibroblasts is inversely correlated with expression of Thy-1. Whether via its effect on cell cytoskeletal arrangement and matrix interaction, regulation of cytokine and growth factor profiles, or mechanisms yet to be elucidated, Thy-1 expression seems to regulate cellular properties that are central to fibrogenic responses. Further characterization of the role of fibroblast Thy-1 display will likely shed important new light on the pathophysiology of fibrosis.


    ACKNOWLEDGEMENTS

We thank Shawn Williams for invaluable assistance with digital confocal microscopy and image processing and Simon Jones and Sandra Hagood for critical reading of the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-03239 (to J. S. Hagood) and HL-03374 (to J. A. Lasky) and American Lung Association Grant RG183N (to J. A. Lasky).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. S. Hagood, 1918 University Blvd, 689 MCLM, Birmingham, AL 35294 (E-mail: jhagood{at}peds.uab.edu).

Received 15 December 1998; accepted in final form 25 February 1999.


    REFERENCES
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REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(1):L218-L224
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