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5 and 2 Integrin Gene Transfers Mimic the PDGF-B–Induced Transformed

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5 and 2 Integrin Gene Transfers Mimic the PDGF-B–Induced Transformed 0023-6837/01/8109-1263$03.00/0 LABORATORY INVESTIGATION Vol. 81, No. 9, p. 1263, 2001 Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. ␣5 and ␣2 Integrin Gene Transfers Mimic the PDGF-B–Induced Transformed Phenotype of Fibroblasts in Human Skin Mark Nesbit, Helmut Schaider, Carola Berking, Daw-Tsun Shih, Mei-Yu Hsu, Michelle McBrian, Timothy M. Crombleholme, Rosalie Elenitsas, Clayton Buck, and Meenhard Herlyn The Wistar Institute (MN, HS, CB, D-TS, M-YH, MM, CB, MH), Philadelphia; Department of Surgery (TMC), The Children’s Hospital of Philadelphia, Philadelphia; and Department of Dermatology (RE), University of Pennsylvania, Philadelphia, Pennsylvania SUMMARY: Platelet-derived growth factor (PDGF)-B is a proto-oncogene capable of transforming fibroblasts. Using adenoviral vectors, we tested whether endogenous PDGF-B expression in human skin xenotransplants leads to changes in the expression of ␣5 and ␣2 integrin subunits and whether integrin overexpression leads to PDGF-related changes in the skin. In vitro, transduction of fibroblasts with PDGF-B or the integrin ␣5 subunit stimulated multilayered growth and spindle-type morphology, both markers of mesenchymal cell transformation. In vivo, PDGF-B transduction of the human dermis was associated with up-regulation of collagen and fibronectin synthesis, increases in ␣5 and ␣2 integrin subunit expression, vessel formation, and proliferation of fibroblasts, keratinocytes, and pericytes. A similar stromal response was induced when ␣5 and ␣2 integrin subunits were overexpressed in the human dermis, suggesting that integrins play a major role in the induction of a transformed phenotype of fibroblasts by PDGF-B. (Lab Invest 2001, 81:1263–1274). latelet-derived growth factor (PDGF) is a normal 1995). Malignant cells often express PDGF-B consti- P constituent of platelets from which it is released tutively (Fahrer et al, 1989; Ito et al, 1990; Leveen et al, by degranulation of ␣ granules at sites of injury (Ross 1990; Mauro and Bulfone, 1990), and in glioma, et al, 1986). PDGF-A and PDGF-B can function as PDGF-B is an autocrine growth factor (Mauro and homo- or heterodimers of 28 to 35 kDa (Kohler and Bulfone, 1990). Other tumors, such as melanomas, Lipton, 1974; Ross et al, 1974; Westermark and lack the receptor and produce the factor for stimulat- Wasteson, 1976). PDGF-BB and PDGF-AB bind to the ing normal cells in the tumor stroma (Forsberg et al, PDGF␤ receptor and, to a lesser extent, the PDGF␣ 1993). receptor, both present predominantly on mesenchy- In fibroblasts, activation of the PDGF␤ receptor mal cells. However, PDGF-AA homodimers bind only leads to transformation (Gazit et al, 1984; Fleming et to the PDGF␣ receptor (Hart et al, 1988; Heldin et al, al, 1992), defined as loss of contact inhibition in 1988). PDGF-B exerts a mitogenic effect on fibro- monolayer cultures and anchorage-independent blasts (Kohler and Lipton, 1974) and smooth muscle growth in soft agar (Fry et al, 1990). Immortalized, but cells (Ross et al, 1974), which express the PDGF␤ nontumorigenic murine fibroblasts can form tumors in receptor and normally do not secrete PDGF-B. Secre- mice when transduced with the proto-oncogene c-sis tion of PDGF-B can be induced with peptide regula- (Kim et al, 1994); the PDGF-B gene shares 94% tory factors, such as transforming growth factor-␤ sequence homology with the avian viral oncogene, (Soma and Grotendorst, 1989). Binding of PDGF-B to v-sis (Waterfield et al, 1983), further underscoring the PDGF␤ induces receptor dimerization and is associ- potential for fibroblast transformation by PDGF-B (Be- ated with autophosphorylation and binding of down- nito and Lorenzo, 1993; Huang et al, 1984; Leal et al, stream modulators to the cytoplasmic domain of the 1985; Valgeirsdottir et al, 1995). receptor (Benito and Lorenzo, 1993; Bornfeldt et al, Besides its growth-stimulating activity, PDGF-B up- regulates expression of ␣5 and ␣2 integrin subunits, which heterodimerize with the ␤1 integrin subunit (Ahlen and Rubin, 1994; Gailit et al, 1996). The ␣5␤1 Received June 6, 2001. integrin is the major fibronectin receptor present on MN and HS contributed equally to the study. This study was supported in most cells; it mediates adhesion, migration, assembly part by National Institutes of Health (NIH) Grants CA25874, CA76674, CA74294, DK50306, and CA10815. HS was supported by the Austrian of extracellular matrix, and signal transduction (Greil- Science Foundation (FWF-1652-Med) and the Max Kade Foundation. ing and Clark, 1997; Wu et al, 1993), which can induce Address reprint requests to: Dr. Meenhard Herlyn, The Wistar Institute, 3601 gene expression and affect transit through the cell Spruce Street, Philadelphia, PA 19104. E-mail: [email protected] cycle (Juliano and Varner, 1993). The induction of ␣5 Laboratory Investigation • September 2001 • Volume 81 • Number 9 1263 Nesbit et al integrin monomer expression may be involved in the Ad5) revealed a 36 kDa band representing the regulation of tumor cell growth and differentiation, PDGF-BB homodimer and several bands of precursor provided that ␤1 integrin subunit is available. In colon proteins, whereas LacZ transduced or nontransduced carcinomas, overexpression of ␣5 integrin subunit fibroblasts showed neither the mature nor precursor leads to increased tumorigenicity (Gong et al, 1997) bands (Fig. 1A). Western analysis of lysates of fibro- when fibronectin is present. In the absence of fi- blasts transduced with PDGF, ␣5, or LacZ revealed bronectin, ␣5␤1 integrin expression induces a nega- high levels of ␣5 integrin in ␣5-transduced cells, lower tive growth signal that can only be overcome when levels in PDGF-B–transduced cells, and very little ␣5in cells are provided with fibronectin (Allen-Hoffmann et LacZ- or nontransduced cells (data not shown). Beta1 al, 1990; Juliano and Varner, 1993; Varner et al, 1995). integrin was present at high levels independent of PDGF-B increases production of fibronectin in cells adenoviral transduction. Mitogenic activity in the cul- (Allen-Hoffmann et al, 1990) and enhances collagen ture supernatant of mouse fibroblasts was abrogated synthesis as part of the wound-healing response by addition of anti-PDGF-BB antibodies, suggesting (Pierce et al, 1995). We hypothesized that the pheno- that most of the activity in the supernatant was due to type induced in vivo by PDGF-B in fibroblasts is due to PDGF-BB. Overexpression of PDGF-B in human fibro- the up-regulation of integrin ␣5 and ␣2 subunits, blasts was mitogenic and stimulated [3H]-thymidine which, in turn, increase fibronectin and collagen pro- incorporation significantly, demonstrating the biologi- duction. We further questioned whether the induced cal activity of adenovirus-induced PDGF-BB secretion changes by PDGF-B in human skin can be partially (data not shown). Extractions of human skin injected 3 mimicked by infection with integrin ␣5or␣2 subunits. days earlier with PDGF-B/Ad5 revealed that two iso- To study the effects of locally overproduced human forms of PDGF, PDGF-AA and PDGF-AB/BB, were PDGF-B and integrins ␣5 and ␣2, we transfected more highly expressed compared with skin injected human fibroblasts with adenoviral vectors carrying the with PBS or LacZ/Ad5 (Fig. 1B). Series of bands of PDGF-B, ␣5, or ␣2 genes (PDGF-B/Ad5, ␣5/Ad5, and precursor proteins, down to 30 kDa, were detected for ␣2/Ad5, respectively). Replication-defective adenovi- both PDGF-AA and PDGF-AB/BB in the lysates. The ruses infect proliferating and nonproliferating cells and lowest bands for both isoforms are shown in Figure induce gene expression with high efficiency (Kozarsky 1B, accounting for about 32 kDa for PDGF-AA and 34 and Wilson, 1993). The biological effects of PDGF-B, kDa for PDGF-AB/BB. Seven days after injection there ␣5, and ␣2 integrin subunit overexpression were ana- was still a higher production of PDGF-AA and PDGF- lyzed in human skin grafted to immunodeficient mice. AB/BB after PDGF-B/Ad5 injection compared with The grafted skin maintains its human-specific archi- LacZ/Ad5. The increase in expression of PDGF-BB tecture throughout the lifetime of the animals and is was confirmed by ELISA, in which a significant in- well-suited for studies of human skin cell transforma- crease after 3 and 7 days was observed (p Ͻ 0.05; Fig. tion (Juhasz et al, 1993a; Soballe et al, 1996). Our 1C). results demonstrate that PDGF-B transduction of fi- broblasts induces an increase in fibronectin and col- Human Skin Remodeling by Intradermal lagen, leads to up-regulation of ␣5 and ␣2 integrin PDGF-B Transduction subunits, increases vessel formation and the numbers Human skin xenografts injected once with PDGF-B/Ad5 of undifferentiated keratinocytes in the epidermis as became raised, with increased soft tissue growth after 3 well as pericytes. A transformed phenotype, ie, loss of days (Fig. 2A, top), compared with their appearance after contact inhibited growth, was also observed in fibro- injection with LacZ/Ad5 (Fig. 2A, bottom) or before blasts overexpressing ␣5 in culture. Those cells injection (Fig. 2B). Histological analysis of hematoxylin showed up-regulated production of fibronectin, which and eosin (H&E)-stained sections revealed dermal hy- was mostly pronounced in human skin grafts com- percellularity and irregular epidermal hyperplasia 3 days pared to cultured cells. Infection of fibroblasts in after PDGF-B/Ad5 injection (Fig. 2C), which was minimal human skin xenotransplants with either integrin ␣5or in sections from the LacZ/Ad5 control group (Fig. 2D). ␣2 subunits also enhanced fibronectin and collagen After 3 days, PDGF-B/Ad5-injected grafts showed in- production. These changes resembled the trans- tense expression of PDGF-B in the dermis, with no formed phenotype after PDGF-B infection, except that expression in the epidermis (Fig. 2E).
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