Elevated Levels of PDGF α Receptors in Keloid Fibroblasts ... · Elevated Levels ofPDGF a Receptors in Keloid Fibroblasts Contribute to an Enhanced Response to PDGF Minoru Haisa,

Elevated Levels ofPDGF a Receptors in Keloid Fibroblasts Contribute to an Enhanced Response to PDGF

Minoru Haisa, Hitoshi 0 kochi, and Gary R. Grotendorst Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida, U.S.A.

Despite a nUIIlber of studies, the etiology of keloids reIIlains unknown. We have investigated the response of fibroblasts derived froIIl keloid tissue and norIIlal adult skin to platelet-derived growth factor (PDGF), epiderIIlal growth factor (EGF), and fibroblast growth factor (FGF). Keloid fibroblasts were IIlore responsive in both cheIIlotactic and IIlitogenic assays to all three isoforIIls ofPDGF than fibroblasts froIIl norIIlal skin.

A major connective tissue cell growth factor present in serum and platelets is platelet derived growth factor (PDGF) . PDGF is an interchain disulfide-bonded di­me ric protein, consisting of homologous polypeptide chains denoted A and B (AA, AB, and BB) that is

mitogenic and chemotactic for connective tissue cells [1-6]. PDGF also stimulates collagenase production [7], and synthesis of extracel­lular matrix components such as fibronectin [8] and hyaluronic acid [9]. The biologic and biochemical actions ofPDGF are mediated by specific cell surface receptors. Currently, two types ofPDGF recep­tors (types 0' and {J) have been identified and cloned [10-14]. Both of these receptors exhibit tyrosine kinase activity and are autophos­phorylated upon activation with PDGF. These receptors exhibit differential ligand binding specificities for the three PDGF isoforms (AA, AB, BB). The type 0' receptor binds all three isoforms and the type P receptor binds the BB isoform with high affinity [15] and the AB isoform in certain cell types [16,17]. Thus, the types and amounts of PDGF receptors present on a connective tissue cell de­termine the cell's responsivity to the various PDGF isoforms [16-19].

While various types of cells have been reported to produce PDGF, the most abundant natural source of these peptides is plate­lets. Because of the multiple biologic actions of this peptide and the role of platelets in the early response to injury it is believed that the primary biologic function of PDGF is that of a wound hormone [20]. In addition to this action in n9rmal repair PDGF is also be­lieved to playa central role in variQus pathologic states including fibrotic disorders such as atherosclerosis [21] and certain cancers [22].

To investigate the potential role of PDGF and other growth factors in keloid formation we compared the response of fibroblasts derived from keloid tissue with that of fibroblasts isolated in an identical manner from normal human skin to PDGF, epidermal growth factor (EGF), and fibroblast growth factor (FGF). We found

Manuscript received February 3, 1994; accepted for publication May 16, 1994.

Reprint requests to: Gary R. Grotendorst, Department of Cell Biology and Anatomy (R-124), University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136.

No enhanced response of the cells to either EGF or FGF was detected. The euhanced PDGF response of keloid fibroblasts appears to be IIlediated by elevated levels of PDGF O! receptors, which are 4 - 5 tiIIles higher than those in nor lIla I hUIIlan skin fibroblasts. Key words: platelet-derived growth factor/ epidermal growth factor/fi­broblastgrowthfactor.] Invest DermatoI103:560-563, 1994

that the keloid cells were more responsive than the normal cells to PDGF. No difference was detected in the response between the two cell types to EGF or FGF. The elevated response of the keloid fibroblasts to PDGF could be accollnted for by elevated levels of PDGF 0' receptor.

MATERIALS AND METHODS

Culture of Fibroblasts Eight strains of keloid fibroblasts were grown from different keloid tissue obtained from five individuals. Patients were all black, four were female, and one was male. Patient ages ranged from 16 to 48 years. Five of eight keloids were located at the ear lobe, with the others at the neck. Normal skin tissue samples were obtained from age-, race-, and sex­matched volunteer donors from forearm punch biopsy. All tissue specimellS, eight keloid and four normal adult dermis, were obtained from the Depart­ment of Dermatology, University of Miami, Miami, Florida. The lesions were diagnosed as keloids on the basis of clinical appearance, persistency, and extension beyond the original wound margins. Specimens were obtained by surgical removal under local anesthesia after informed consent was obtained. Epidermis and subepidermal fat were removed from tissue and minced speci­mens (1-2 mm3

) were placed on 100-mm tissue culture plates. Explams were cultured using Dulbecco's modified Eagle's medium (DMEM, 4.5 gil glucose) supplemented with 10% fetal bovine serum (FBS, Hyclone Labora­tories Inc., Logan, UT), 5 )lg/ ml Gentamicin (Quality Biological, Inc., Gaithersburg, MD) at 37"C, in an atmosphere of 10% CO2 and 90% air. Cells were passed at confluence by trypsin/ethylenediaminetetraacetic acid treatment and used in this investigation at passages 3 to 5. During these experiments, cultures showed no evidence of crisis or senescence.

Mitogenic, Chemotactic, and PDGF Receptor Assays Mitogenic assays were performed with confluent cultures of cells. Culture medium was changed to DMEM with 2.5% FBS 24 h before addition of growth factors . DNA synthesis was determined as described previously [23]. Chemotactic assays were performed using modified Boyden chambers on confluent cul­tures as described previously [23,24].

Tyrosine autophosphorylation of PDGF receptors was determined using confluent cultures of cells as described previously [16]. Briefly, cells were treated for 30 min at 37°C with 20 ng/ 1111 PDGF, the membranes isolated, and the receptors extracted. The receptor extract was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels and Western blots with an anti­phosphotyrosine monoclonal antibody (PY -60, ICN).

RESULTS

Mitogenic Response of Normal and Keloid Fibroblasts Ini­tially, we compared the mitogenic response of the eight keloid and

0022-202X/94 /S07 .00 Copyright © 1994 by The Society for Investigative Dermatology, Ina.

560

VOL. 103, NO. 4 OCTOBER 1994

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Figure 1. PDGF induced DNA synthesis of keloid and normal fibro­blasts. Keloid fibroblasts exhibited significantly greater stimulation of DNA synthesis in response to all forms of PDGF than normal skin fibro­blasts. The data represent the mean values of the fo ld stimulation of DNA synthesis by PDGF in the eight keloid and four normal skin fibroblast cultures. A significant (p < 0.01, two-tailed Student t test) difference in mitogenic response was obtained with 2.5-20 ngl ml ofPDGF AA and An and at 20 ngl ml PDGF BE.

four normal fibroblast cultures to the three POGF isoforms (Fig 1). The normal fibroblasts responded to POGF AB and BB, with a dose response curve of 0.5-20 ng/ml POGF and a concentration of 5 -10 ng/ml eliciting a maximal response. In comparison, POGF AA was weakly active on these cells and stimulated less than a twofold increase in DNA synthesis over non-treated control levels. This is consistent with previous studies on various human fibro­blasts [16-18] . In marked contrast, the POGF AB and BB response of keloid fibroblasts was approximately twice that of normalfibro­blasts, indicating that keloid fibroblasts are more responsive to PDGF than normal fibrob lasts. The enhanced response of keloid fibroblasts was more pronounced to PDGF AA where the keloid­derived cells exhibited a nearly sixfold stimulation of D N A synthe­sis. The elevated response to POGF by the keloid cells was not paralleled by an increased response to other growth factors. Basic FGF was equally active on both normal and keloid cells and the keloid cells were much less responsive to EGF than the normals (Fig 2). The diminished response to EGF is a property that had been observed previously in keloid-derived fibroblasts by Russell and coworkers [25]. These data demonstrate that the enhanced response to keloids is not due to a general increase in growth response to any growth factor but is limited to the action of POGF.

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Figure 2. EGF- and FGF-induced DNA synthesis of keloid and nor­mal fibroblasts. Parallel cultures to those used in Fig 1 were tested for a mitogenic response to A) EGF and B) FGF. The data arc representative of the mitogenic response of the cells to each of these factors. These studies were repeated 4 times using different cells that exhibited a similar pattern of response to EGF and FGF.

Chemotactic Response of Normal and Keloid Fibroblasts Next we investigated the chemotactic ac tivity of the PDGF iso­forms on keloid and normal fibroblasts using the Boyden chamber assay (Fig 3). Both keloid and normal fibroblasts migrated toward POGF in a dose-dependent manner. The results of these studies were similar to those of the mitogenic response to POGF. Fibro­blasts isolated from normal skin responded strongly to PDGF AB and BB but weakly to PDGF AA. In contrast, the keloid cells re­sponded well to all three isoforms of POGF and exhibited a nearly threefold greater migratory response to equivalent amounts of POGF than the normal cells.

PDGF Receptor Content and Activation in Norm.al and Ke­loid Fibroblasts POGF mediates its biologic action via specific cell surface receptors and, as discussed above, two distinct PDGF receptors (types ex and fJ) that exhibit differential li gand binding have been identified. Because of the enhanced responsiveness of the cells to POGF we wanted to determine whether there was an alter­ation in the relative levels of the POGF ex and jJ receptors present on the keloid fibroblasts compared to the cells cultured from normal skin. Cell extracts from equivalent amounts of cells were prepared and analyzed by Western blotting using either PDGF-ex-receptor­or POGF-fJ-receptor-specific antiserum (Fig 4A). Both cell types contained nearly equivalent amounts of PDGF fJ receptor based on the amount detected in the Western blot. However, the keloid fibrobl as ts contained a 4- 5 times increase in the amount ofpOGF ex receptors compared to the normal fibrob lasts .

W e next examined the functional activity of the POGF ex recep­tors present on both normal and keloid fibrobl asts using tyrosine autophosphorylation as a measure of receptor activation. Confluent cu ltures of fibroblasts were incubated with 20 ng/ ml of one of the POGF isoforms for 10 min at 37"C and the membrane fra ction

562 HAISA ET AL

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Figure 3. Chemotactic response of keloid and normal dermal fibro­blasts to PDGF. Keloid fibroblasts exhibited a greater migratory response to all th ree isoforms ofPDGF than normal fibroblasts. The data represent the mean v.al ues of the chemotactic response of the eight keloid and four normal skin fibroblast cultures to PDGF. A significant (p < 0.01, two-tailed Student t test) difference in mitogenic response was obtained at all concentrations and isoforms of PDGF tested.

extracted. The PDGF a receptor was immunoprecipitated and ana­lyzed by Western blot using a specific anti-phosphotyrosine mono­clonal antibody (Fig 4B). All three isoforms stimulated a large increase in the tyrosine autophosphorylation of the PDGF a recep­tor on the keloid fibroblasts . However, no detectable tyrosine auto­phosphorylation was observed in the normal fibroblasts at equal concentrations of PDGF and cells. These results confirm our bio­logic assays and demonstrate that the elevated response of the keloid cells to PDGF is due to an increase in the number of functional PDGF a receptors present. Because the PDGF a receptor is acti­vated equally well by all three isoforms of PDGF, it alone can account for the enhanced responsiveness of the keloid cells to all isoforms of PDGF. The PDGF P receptor is activated solely by PDGF BB and all elevation in this type of receptor would not account for the enhanced AB and AA response we have detected in the keloid cells.

DISCUSSION

These data strongly suggest that the fibroblasts present in keloid ti ssue are a different population from those present in normal dermis. Although it is possible that the cells we are studying in culture have been altered from those residing in the tissue by place-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

ment in tissue culture [26]. the keloid-derived cells maintain a phe­notype distinct from that of fibroblasts derived from normal skin cultured under identical conditions . Furthermore, this phenotype is maintained after multiple cell divisions long after removal from the in vivo environment, indicating that the differences we have de­tected are not due to a temporary change induced by some cytokille or other environmental parameter unique to the keloid site.

Past studies have reported other phenotypic differences between keloid and normal fibroblasts, including increased deposition of extracellular matrix components such as collagen, fibronectin, and chondroitin sulfate proteoglycan in keloid tissue and in keloid fi­broblast cultures [27 - 31]. In addition to enhanced matrix synthesis, hypercellularity of keloid lesions has also been reported based on quantitative histopathologic examination and chemical determina­tion of DNA content [32]. However, in contrast to the irl villo observations of hyper cellularity of keloid tissue, previous studies on the growth characteristics of the fibroblasts isolated from keloids have found them similar to those of normal fibroblasts under stan­dard culture conditions [25,32]. We could not detect any significant difference in the mean population doubling time, cell density at confluence, or cellular volume of the keloid fibroblasts used in this study compared to the normal skin fibrob lasts when grown in DMEM containing 10% fetal bovine serum. Our finding that ke­loid fibroblasts are more responsive to PDGF than normal skin fibroblasts is the first reported enhanced growth factor response by keloid fibroblasts.

Altered phenotypes of fibroblastic cells have been implicated in other fibrotic diseases including atherosclerosis . Benditt has sug­gested that the atherosclerotic plaque is similar to a benign tumor and has found that the cells present in plaques appear to be derived from a single cell based on genetic analysis of X-linked genes in atherosclerotic plaques obtained from heterozygotic females [33] . Two potential mechanisms for the generation of such a cell popula-

A .

B.

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Figure 4. Type a and P PDGF receptors on keloid and normal fibro­blasts. A) Total amounts of types a and pPDGF receptors. Immunoprecipi­tates prepared from equivalent numbers of cells were analyzed by W estern blot. La/Ie N, extracts from normal skin fibroblasts; /a/Je K, from keloid fibroblasts. Fibroblasts from kcloids contained a 4 - 5 times higher amount of PDGF a receptors than those in normal skin. The number of PDGF P receptors was nearly equal in both cell types. B) Autophosphorylation of PDGF ex receptors by PDGF. Immunoprecipitatcs were prepared from equivalent numbers of the cells from both keloid and normal fibroblast cultures. La/Je C, no PDGF added; AA, 20 ngj mI ofPDGF AA hotnodimer; AB, 20 ngj ml of PDGF AB heterodimer; BB, 20 ngj mI of PDGF BB homodimer. Keloid fibroblasts exhibited a high level of autophosphoryla­tion of the PDGF a receptor induced by all PDGF isoforms, whereas normal skin fibroblasts exhibited no detectable autophosphorylation under the same conditions.

VOL. 103, NO.4 OCTOBER 1994

tion are the transformation of a single cell that replicates to form the lesion or the selective recruitment of a subpopulation of fibroblasts that preferentially responds to an environmental factor present at the site of lesion formation. It is also possible that a combination of these two events leads to the fibrotic state.

Keloid formation occurs exclusively at sites of trauma and in areas of the body associated with increased tension and motion of the skin, such as joints [34]. Whereas no overt trauma has occurred in the areas of repeated skin stretching, microvessel occlusion has been observed at these sites and in keloids [35], which would indicate platelet aggregation and release of PDGF. These phenomena, cou­pled with an elevated response of fibroblasts in the tissue to PDGF, could lead to the formation of the keloid.

The keloid fibroblasts responded well to PDGF AA, whereas normal skin fibroblasts responded poorly to PDGF AA. This may prove to be one of the most important differences between the keloid and normal fibroblasts. We have recently reported that the predominant PDGF isoform present in human platelets and acute human wound fluid is PDGF AA [36,37]. Thus, platelet aggrega­tion and release ofPDGF at a site of trauma supplies a growth factor that is potent for the keloid-lesion-derived fibroblast but not those present in the normal skin cultures . The elevated levels of PDGF a receptor in the keloid fibroblasts present in the keloid-derived cells W"ould make them much more responsive to PDGF AA as a che­moattractant and mitogen. The presence of PDGF AA in the tissue could selectively recruit cells with elevated PDGF a receptors, forming a site with a distinct subpopulation of fibroblasts. Subse­quent incidents of trauma that release more PDGF AA at this site W"ould amplify these differences and support the overgrowth of these cells.

Studies examining the expression of PDGF receptors in wounds have found an elevation of the PDGF P receptor compared to non­injured dermis in both chronic wounds [38] and normal healing W"ounds [39]. No increases in PDGF a receptors have been reported. In addition, active lesions of systemic sclerosis [40) and psoriasis (38) are populated by connective tissue cells, expressing elevated levels of PDGF P receptors . These data suggest that the elevation of PDGF a receptors may not be a common phenomena for activation of dermal fibrotic lesions and that in normal wound healing the PDGF a receptors may not be elevated. Thus, the increased expres­sion ofPDGF a receptors may be unique to keloids. Whether these cells represent a normal subpopulation of dermal fibroblasts or are the result of transformation remains to be determined.

We thatlk Drs. A lbert Nemetil, William Eaglesteitl, atld Vitlcetlt FalallgaJor tlleir assistat.ce ill obtait.itlg keloid atld lIormallwman skitl tisslle, at.d Dr. Naoko lida Jor her advice atld assistatlCe. Tlris lVork lVas supported by NIH gra tlt GM)?223 to GRG atld by Junds from tire University oj Miami.

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40. Klareskog L, Gustafsson R, Scheynius A, Hallgren R: Increased expression of platelet-derived growth factor type B receptors in the skin of patients with systemic sclerosis. Art!'r alld R!.CII/tl 33:1534- 1541, 1990