Transforming Growth Factor-/? (TGF/?) Inhibits TGFa Expression in Bovine Anterior Pituitary-Derived Cells
Susan G. Mueller and Jeffrey E. Kudlow Department of Clinical Biochemistry University of Toronto (S.G.M.) Toronto, Ontario, Canada Department of Medicine Division of Endocrinology University of Alabama (J.E.K.) Birmingham, Alabama 35294
Transforming growth factor-/91 (TGF/31) is a multifunctional regulator of cell growth and differentiation. We report here that TGF01 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGFa and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF01 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGFa mRNA level. The effect of TGF/S1 on TGFa mRNA downregulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF01) and time dependent (minimum of 2-day treatment with TGF/91 was required before a decrease in TGFa mRNA was observed). Studies on TGFa mRNA stability indicated that TGF01 did not alter the TGFa mRNA half-life. Treatment of the TGF01 down-regulated cells with EGF resulted in the stimulation of TGFa mRNA levels; thus, the TGF01 -treated cells remained responsive to EGF. The decreased proliferation in response to TGF/31 could be only partially reversed by simultaneous treatment of the cells with EGF (10~9 M) and TGF/S1 (3.0 ng/ml). Qualitatively, the TGF/31-induced reduction of TGFa mRNA content was independent of cell density. TGF01 treatment of the anterior pituitary-derived cells also reduced the levels of cmyc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGFa synthesis by a polypeptide growth factor and suggest that TGF01 may be a physiological regulator of TGFa production in vivo. (Molecular Endocrinology 5: 1439-1446, 1991)
INTRODUCTION Transforming growth factor-a (TGFa), a member of the epidermal growth factor (EGF) family of mitogenic polypeptides, is generally capable of stimulating the proliferation of both epithelial and mesenchymal cells (1). Although TGFa shares only a 32% amino acid sequence homology with EGF (2), both bind to and elicit their biological effects through the same receptor, the EGF receptor (3-8). Because TGF« was initially detected in the conditioned medium of transformed cells (4) and later in fetal and embryonic tissue (9, 10, 11), it was believed to be an onco-fetal form of EGF (12). More recently, TGFa has been identified in a variety of normal adult tissues, including the pituitary (13), brain (14-16), ovary (17), keratinocytes (18), macrophages (19, 20), and vascular smooth muscle cells (21). The presence of TGFa in these tissues implies a role for TGFa in normal physiological processes. Since the growth of normal cells is under tight regulation, the expression of TGFa in normal tissues must also be regulated. To study the regulation of TGFa in normal cells, we used cells cultured from bovine anterior pituitary glands. We have previously shown that TGFa is localized in the somatotrophs and lactotrophs within the anterior pituitary gland (13), and cells derived from these glands secrete TGFa into their culture medium (8, 22). The expression of TGFa can be up-regulated in these cells by exposure to EGF and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (23). The ability of EGF to stimulate TGFa synthesis suggests that TGFa can stimulate its own synthesis by an autocrine or a paracrine route. TGFa has been shown to autostimulate its own production in other cell systems, including the human breast cancer cell line MDA 468 (24) and nontransformed primary keratinocytes (18).
0888-8809/91/1439-1446$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
The ability of a growth factor to stimulate its own production could, if left unchecked, result in over1439
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 January 2016. at 22:07 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 1440
Vol5No. 10
expression of that growth factor. Recent studies have demonstrated that TGFa overexpression, driven by a strong heterologous promoter in either transgenic mice (25-27) or transfected Rat-1 fibroblasts injected into nude mice (28) resulted in pathological hyperplasia or tumor formation in the respective models. The ability of normal tissues to control TGFa expression, despite the existence of an autostimulatory mechanism, necessitates the coexistence of a negative regulatory pathway capable of down-regulating growth factor expression. The present study was undertaken to determine whether TGF/31, a potent epithelial cell growth inhibitor (29, 30), was capable of negatively regulating TGFa expression. We report here that TGF/31 inhibited both TGFa and EGF receptor expression and decreased the growth rate of the anterior pituitary cells in culture. This is the first demonstration of the down-regulation of TGFa production by a polypeptide growth factor, and it suggests TGF/31 may be a physiological regulator of TGFa production in normal tissues.
RESULTS
We investigated the effect of TGF01 on the growth rate of bovine anterior pituitary-derived cells grown in culture. Over the first 2 days, the cells treated with either 1 or 3 ng/ml TGF/31 grew at the same rate as untreated cells, but subsequently, the TGF/31-treated cells became essentially growth arrested (Fig. 1), while the untreated cells continued to proliferate. Northern blot analysis was carried out to determine whether TGF/31 could modulate the content of TGFa:
400 • 300
NoTGFB
X 1.0 ng/ml TGFG A 3.0 ng/ml TGF8
mRNA in the pituitary cells. Cells were subcultured at a low density and treated with increasing doses of TGF/31 (0-3.0 ng/ml) for 4 days. Culture medium, in the absence or presence of TGF/31, was replaced daily. Northern blot analysis of poly(A)+ RNA indicated that TGF/31 treatment resulted in a decreased content of TGFa mRNA in these cells (Fig. 2). This decrease in TGFa mRNA was dose dependent, with a maximal decrease observed at a concentration of 1.0 ng/ml TGF/31. Densimetric analysis indicated that the decrease in TGFa mRNA resulting from treatment with 1 ng/ml TGF/31 was about 70%. The level of actin mRNA was unchanged during the course of this treatment (Fig. 2). The effect of TGF/31 on the pituitary cells was also time dependent. Cells were treated daily with 1.0 ng/ml TGF/31 for the indicated periods of time. The initiation of TGF/31 treatment was staggered, such that all treatments ended simultaneously (Fig. 3). Northern blot analysis indicated that a decrease in the level of TGFa mRNA was detected only after a 2-day treatment of the cells with TGF01 (Fig. 3). Reprobing of this Northern blot with c-myc revealed that TGF/31 treatment also resulted in a decrease in the c-myc mRNA, but this decrease was first apparent after 1 day of treatment (Fig. 3). The decrease in TGFa and c-myc mRNA was shown to be relatively specific, as the level of hexosaminidase-A mRNA was not altered during the TGF/31 treatment. Other studies have indicated that the effect of TGF/31 on smooth muscle cell proliferation is density dependent (31). To determine whether cell density had an effect on the response of the pituitary cells to TGF/31, cells were subcultured at densities to approximate 10% and 70% confluence. The sparse cells were treated with TGF/31 (1.0 ng/ml) for 4 days, while the more dense cells were treated for 3 days. Culture medium in the absence or presence of TGF/31 was changed daily. TGF/31 treatment of these cells decreased the content of TGFa mRNA at both cell densities after treatment for the indicated lengths of time (Fig. 4), although the effect was more pronounced in the sparse cells. 0 0.1 05 1.0 3.0
200
ng/ml TGF-p
TGF-a
Actin Time (Days)
Fig. 1. Effect of TGF/31 on Proliferation of Cultured Bovine Anterior Pituitary-Derived Cells The pituitary cells were subcultured at a density of 2.5 x 104 cells/35-mm culture dish in 2% calf serum-DMEM. After an overnight incubation, the medium was replaced with serumfree medium alone or with 1.0 or 3.0 ng/ml TGF/31. Cells cultured in the presence of TGF/31 were given fresh growth factor daily. Cells were counted on days 0-4, using a Coulter counter. Each point represents the mean result of triplicate determinations (average SD,
Susan G. Mueller and Jeffrey E. Kudlow Department of Clinical Biochemistry University of Toronto (S.G.M.) Toronto, Ontario, Canada Department of Medicine Division of Endocrinology University of Alabama (J.E.K.) Birmingham, Alabama 35294
Transforming growth factor-/91 (TGF/31) is a multifunctional regulator of cell growth and differentiation. We report here that TGF01 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGFa and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF01 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGFa mRNA level. The effect of TGF/S1 on TGFa mRNA downregulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF01) and time dependent (minimum of 2-day treatment with TGF/91 was required before a decrease in TGFa mRNA was observed). Studies on TGFa mRNA stability indicated that TGF01 did not alter the TGFa mRNA half-life. Treatment of the TGF01 down-regulated cells with EGF resulted in the stimulation of TGFa mRNA levels; thus, the TGF01 -treated cells remained responsive to EGF. The decreased proliferation in response to TGF/31 could be only partially reversed by simultaneous treatment of the cells with EGF (10~9 M) and TGF/S1 (3.0 ng/ml). Qualitatively, the TGF/31-induced reduction of TGFa mRNA content was independent of cell density. TGF01 treatment of the anterior pituitary-derived cells also reduced the levels of cmyc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGFa synthesis by a polypeptide growth factor and suggest that TGF01 may be a physiological regulator of TGFa production in vivo. (Molecular Endocrinology 5: 1439-1446, 1991)
INTRODUCTION Transforming growth factor-a (TGFa), a member of the epidermal growth factor (EGF) family of mitogenic polypeptides, is generally capable of stimulating the proliferation of both epithelial and mesenchymal cells (1). Although TGFa shares only a 32% amino acid sequence homology with EGF (2), both bind to and elicit their biological effects through the same receptor, the EGF receptor (3-8). Because TGF« was initially detected in the conditioned medium of transformed cells (4) and later in fetal and embryonic tissue (9, 10, 11), it was believed to be an onco-fetal form of EGF (12). More recently, TGFa has been identified in a variety of normal adult tissues, including the pituitary (13), brain (14-16), ovary (17), keratinocytes (18), macrophages (19, 20), and vascular smooth muscle cells (21). The presence of TGFa in these tissues implies a role for TGFa in normal physiological processes. Since the growth of normal cells is under tight regulation, the expression of TGFa in normal tissues must also be regulated. To study the regulation of TGFa in normal cells, we used cells cultured from bovine anterior pituitary glands. We have previously shown that TGFa is localized in the somatotrophs and lactotrophs within the anterior pituitary gland (13), and cells derived from these glands secrete TGFa into their culture medium (8, 22). The expression of TGFa can be up-regulated in these cells by exposure to EGF and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (23). The ability of EGF to stimulate TGFa synthesis suggests that TGFa can stimulate its own synthesis by an autocrine or a paracrine route. TGFa has been shown to autostimulate its own production in other cell systems, including the human breast cancer cell line MDA 468 (24) and nontransformed primary keratinocytes (18).
0888-8809/91/1439-1446$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
The ability of a growth factor to stimulate its own production could, if left unchecked, result in over1439
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 January 2016. at 22:07 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 1440
Vol5No. 10
expression of that growth factor. Recent studies have demonstrated that TGFa overexpression, driven by a strong heterologous promoter in either transgenic mice (25-27) or transfected Rat-1 fibroblasts injected into nude mice (28) resulted in pathological hyperplasia or tumor formation in the respective models. The ability of normal tissues to control TGFa expression, despite the existence of an autostimulatory mechanism, necessitates the coexistence of a negative regulatory pathway capable of down-regulating growth factor expression. The present study was undertaken to determine whether TGF/31, a potent epithelial cell growth inhibitor (29, 30), was capable of negatively regulating TGFa expression. We report here that TGF/31 inhibited both TGFa and EGF receptor expression and decreased the growth rate of the anterior pituitary cells in culture. This is the first demonstration of the down-regulation of TGFa production by a polypeptide growth factor, and it suggests TGF/31 may be a physiological regulator of TGFa production in normal tissues.
RESULTS
We investigated the effect of TGF01 on the growth rate of bovine anterior pituitary-derived cells grown in culture. Over the first 2 days, the cells treated with either 1 or 3 ng/ml TGF/31 grew at the same rate as untreated cells, but subsequently, the TGF/31-treated cells became essentially growth arrested (Fig. 1), while the untreated cells continued to proliferate. Northern blot analysis was carried out to determine whether TGF/31 could modulate the content of TGFa:
400 • 300
NoTGFB
X 1.0 ng/ml TGFG A 3.0 ng/ml TGF8
mRNA in the pituitary cells. Cells were subcultured at a low density and treated with increasing doses of TGF/31 (0-3.0 ng/ml) for 4 days. Culture medium, in the absence or presence of TGF/31, was replaced daily. Northern blot analysis of poly(A)+ RNA indicated that TGF/31 treatment resulted in a decreased content of TGFa mRNA in these cells (Fig. 2). This decrease in TGFa mRNA was dose dependent, with a maximal decrease observed at a concentration of 1.0 ng/ml TGF/31. Densimetric analysis indicated that the decrease in TGFa mRNA resulting from treatment with 1 ng/ml TGF/31 was about 70%. The level of actin mRNA was unchanged during the course of this treatment (Fig. 2). The effect of TGF/31 on the pituitary cells was also time dependent. Cells were treated daily with 1.0 ng/ml TGF/31 for the indicated periods of time. The initiation of TGF/31 treatment was staggered, such that all treatments ended simultaneously (Fig. 3). Northern blot analysis indicated that a decrease in the level of TGFa mRNA was detected only after a 2-day treatment of the cells with TGF01 (Fig. 3). Reprobing of this Northern blot with c-myc revealed that TGF/31 treatment also resulted in a decrease in the c-myc mRNA, but this decrease was first apparent after 1 day of treatment (Fig. 3). The decrease in TGFa and c-myc mRNA was shown to be relatively specific, as the level of hexosaminidase-A mRNA was not altered during the TGF/31 treatment. Other studies have indicated that the effect of TGF/31 on smooth muscle cell proliferation is density dependent (31). To determine whether cell density had an effect on the response of the pituitary cells to TGF/31, cells were subcultured at densities to approximate 10% and 70% confluence. The sparse cells were treated with TGF/31 (1.0 ng/ml) for 4 days, while the more dense cells were treated for 3 days. Culture medium in the absence or presence of TGF/31 was changed daily. TGF/31 treatment of these cells decreased the content of TGFa mRNA at both cell densities after treatment for the indicated lengths of time (Fig. 4), although the effect was more pronounced in the sparse cells. 0 0.1 05 1.0 3.0
200
ng/ml TGF-p
TGF-a
Actin Time (Days)
Fig. 1. Effect of TGF/31 on Proliferation of Cultured Bovine Anterior Pituitary-Derived Cells The pituitary cells were subcultured at a density of 2.5 x 104 cells/35-mm culture dish in 2% calf serum-DMEM. After an overnight incubation, the medium was replaced with serumfree medium alone or with 1.0 or 3.0 ng/ml TGF/31. Cells cultured in the presence of TGF/31 were given fresh growth factor daily. Cells were counted on days 0-4, using a Coulter counter. Each point represents the mean result of triplicate determinations (average SD,
