Transforming Growth Factor-/? Induces Platelet-Derived Growth Factor (PDGF) Messenger RNA and PDGF Secretion while Inhibiting Growth in Normal Human Mammary Epithelial Cells
Diane A. Bronzert, Susan E. Bates, James P. Sheridan, Ralph Lindsey, Eva M. Valverius, Martha R. Stampfer, Marc E. Lippman, and Robert B. Dickson Clinical Investigations Branch (D.A.B.) and Medicine Branch (S.E.B.) National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 Lombardi Cancer Research Center (J.P.S., R.L., E.M.V., M.E.L. R.D.B.) Georgetown University Washington, D.C. 20007 Lawrence Berkeley Laboratory (M.R.S.) Berkeley, California 94720
Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-/? (TGF0) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester
12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF/? function regardless of whether TGF/? is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF/? modulation of differentiation of normal or malignant mammary gland remains to be determined. (Molecular Endocrinology 4: 981-989, 1990)
INTRODUCTION
Growth factors have been proposed to serve important functions as mediators of both paracrine and autocrine control in neoplastic transformation and tumor development. Since normal mammary epithelium and mammary carcinomas in vivo consist of proliferating epithelial and mesenchymal cells as well as vascular tissue, studies of possible interactions among these various cells are important (1). Our laboratory and others have reported that breast cancer cell lines secrete significant quantities of an insulin-like growth factor (IGF-I) (2), transforming growth factor-a (TGFa)-like activity (3, 4), and TGF/3 (5, 6). Platelet-derived growth factor (PDGF) is also secreted by breast cancer cells, but no PDGF receptors have been identified in these cells to date (79). In this paper we have expanded our studies of PDGF
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to include its regulation in normal human mammary epithelial cells (HMEC). PDGF, a potent mitogen in human serum, consists of two polypeptide chains, PDGF-A and PDGF-B, linked by disulfide bonds (10, 11). The PDGF-B chain is the normal cellular homolog to the oncogene v-sis of simian sarcoma virus (12,13) and is not usually secreted (14). DNA-mediated transfection of cultured NIH 3T3 cells with the normal human PDGF-B gene has been shown to induce malignant transformation (15,16). The PDGFA chain is synthesized independently of PDGF-B and has been identified as a product of some human tumor cells, including osteosarcoma (17), melanoma (18), and glioblastoma cells (19, 20). The PDGF-A gene is markedly less efficient in inducing transformation than the PDGF-B gene when transfected into NIH 3T3 cells (21). In contrast to the PDGF-B chain, approximately 80% of the PDGF-A chain is secreted within 3 h of synthesis (21) and possesses relatively low mitogenic activity compared to the PDGF-B chain (22, 23). In contrast to human breast cancer cell lines, which synthesize both PDGF-A and PDGF-B mRNA, we report here that HMEC synthesize mostly PDGF-A mRNA under serumfree growth conditions. TGF/3 has been proposed to have an important role in growth regulation of normal tissues. It is a multifunctional regulator of growth which stimulates the growth of mesenchymal cells while inhibiting the growth of epithelial cells (5, 6). Leof et al. (1) have proposed a model for indirect mitogenesis involving the early induction of c-s/s/PDGF-B mRNA by TGF/3 in AKR-2B fibroblasts and the subsequent autocrine stimulation of cfos, c-myc, and other PDGF-inducible genes (1). TGF/3 has been shown to inhibit both human breast cancer cell lines and normal mammary epithelial cells (5, 24) and to induce a more differentiated phenotype in the HMEC (25, 26). In contrast to the AKR-2B cell model where PDGF/c-s/s induction is coupled to growth stimulation, we report here that TGF/3 stimulates the expression of PDGF-B/c-s/s mRNA while inhibiting cell growth. We conclude that in mammary epithelial cells, PDGF secretion is more likely to serve a paracrine function than a direct autocrine function.
RESULTS Production of PDGF-Like Activity in Conditioned Medium (CM) of HMEC A RRA was used to screen CM from normal HMEC from three different individuals for the secretion of PDGF (Table 1). Serum-free media from cells were conditioned for 48 h, collected, and concentrated, then assayed for competition with [125I]PDGF for binding to the PDGF receptor on human fibroblasts. All cell strains were found to secrete PDGF-competing activity. In prior studies we have shown that human breast cancer cell lines secrete similar levels of PDGF (7).Thus, no significant difference was seen in overall PDGF secretion
Table 1. Production of PDGF Receptor-Binding Activity by Normal Human Mammary Epithelial Cells Cell Line
161 172 184 184 + TGFj8
Medium Control
PDGF Activity (ng/ml CM)
PDGF Activity (ng/mg DNA)
0.32 0.45 0.51 0.74 " oo
Diane A. Bronzert, Susan E. Bates, James P. Sheridan, Ralph Lindsey, Eva M. Valverius, Martha R. Stampfer, Marc E. Lippman, and Robert B. Dickson Clinical Investigations Branch (D.A.B.) and Medicine Branch (S.E.B.) National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 Lombardi Cancer Research Center (J.P.S., R.L., E.M.V., M.E.L. R.D.B.) Georgetown University Washington, D.C. 20007 Lawrence Berkeley Laboratory (M.R.S.) Berkeley, California 94720
Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-/? (TGF0) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester
12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF/? function regardless of whether TGF/? is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF/? modulation of differentiation of normal or malignant mammary gland remains to be determined. (Molecular Endocrinology 4: 981-989, 1990)
INTRODUCTION
Growth factors have been proposed to serve important functions as mediators of both paracrine and autocrine control in neoplastic transformation and tumor development. Since normal mammary epithelium and mammary carcinomas in vivo consist of proliferating epithelial and mesenchymal cells as well as vascular tissue, studies of possible interactions among these various cells are important (1). Our laboratory and others have reported that breast cancer cell lines secrete significant quantities of an insulin-like growth factor (IGF-I) (2), transforming growth factor-a (TGFa)-like activity (3, 4), and TGF/3 (5, 6). Platelet-derived growth factor (PDGF) is also secreted by breast cancer cells, but no PDGF receptors have been identified in these cells to date (79). In this paper we have expanded our studies of PDGF
0888-8809/90/0981 -0989$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
981
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 October 2016. at 14:12 For personal use only. No other uses without permission. . All rights reserved.
Vol 4 No. 7
MOL ENDO-1990 982
to include its regulation in normal human mammary epithelial cells (HMEC). PDGF, a potent mitogen in human serum, consists of two polypeptide chains, PDGF-A and PDGF-B, linked by disulfide bonds (10, 11). The PDGF-B chain is the normal cellular homolog to the oncogene v-sis of simian sarcoma virus (12,13) and is not usually secreted (14). DNA-mediated transfection of cultured NIH 3T3 cells with the normal human PDGF-B gene has been shown to induce malignant transformation (15,16). The PDGFA chain is synthesized independently of PDGF-B and has been identified as a product of some human tumor cells, including osteosarcoma (17), melanoma (18), and glioblastoma cells (19, 20). The PDGF-A gene is markedly less efficient in inducing transformation than the PDGF-B gene when transfected into NIH 3T3 cells (21). In contrast to the PDGF-B chain, approximately 80% of the PDGF-A chain is secreted within 3 h of synthesis (21) and possesses relatively low mitogenic activity compared to the PDGF-B chain (22, 23). In contrast to human breast cancer cell lines, which synthesize both PDGF-A and PDGF-B mRNA, we report here that HMEC synthesize mostly PDGF-A mRNA under serumfree growth conditions. TGF/3 has been proposed to have an important role in growth regulation of normal tissues. It is a multifunctional regulator of growth which stimulates the growth of mesenchymal cells while inhibiting the growth of epithelial cells (5, 6). Leof et al. (1) have proposed a model for indirect mitogenesis involving the early induction of c-s/s/PDGF-B mRNA by TGF/3 in AKR-2B fibroblasts and the subsequent autocrine stimulation of cfos, c-myc, and other PDGF-inducible genes (1). TGF/3 has been shown to inhibit both human breast cancer cell lines and normal mammary epithelial cells (5, 24) and to induce a more differentiated phenotype in the HMEC (25, 26). In contrast to the AKR-2B cell model where PDGF/c-s/s induction is coupled to growth stimulation, we report here that TGF/3 stimulates the expression of PDGF-B/c-s/s mRNA while inhibiting cell growth. We conclude that in mammary epithelial cells, PDGF secretion is more likely to serve a paracrine function than a direct autocrine function.
RESULTS Production of PDGF-Like Activity in Conditioned Medium (CM) of HMEC A RRA was used to screen CM from normal HMEC from three different individuals for the secretion of PDGF (Table 1). Serum-free media from cells were conditioned for 48 h, collected, and concentrated, then assayed for competition with [125I]PDGF for binding to the PDGF receptor on human fibroblasts. All cell strains were found to secrete PDGF-competing activity. In prior studies we have shown that human breast cancer cell lines secrete similar levels of PDGF (7).Thus, no significant difference was seen in overall PDGF secretion
Table 1. Production of PDGF Receptor-Binding Activity by Normal Human Mammary Epithelial Cells Cell Line
161 172 184 184 + TGFj8
Medium Control
PDGF Activity (ng/ml CM)
PDGF Activity (ng/mg DNA)
0.32 0.45 0.51 0.74 " oo
