Growth Factor-regulated Pathways in Epithelial Cell Proliferation 1 •2 STUART A. AARONSON, JEFFREY S. RUBIN, PAUL W. FINCH, JANE WONG,3 CINZIA MARCHESE, JOSEPH FALCO, WILLIAM G. TAYLOR, and MATTHIAS H. KRAUS
Introduction Efforts to investigate growth regulation of epithelial cellsin tissue culture have led to the development of medium and/or substrates for propagation of primary epithelial cellcultures (1-3). Despite these advances, it has been difficult to obtain long-term homogenous epithelial cell populations with normal growth properties. The development of continuous lines with normal growth properties has been extremely useful in studies of growth regulation, as well as alterations associated with malignant transformation by a variety of agents (4-6). The potential advantages of continuous clonal cell lines of epithelial origin led us to establish lines of clonally derived BALB/c mouse epidermal keratinocyte (7). Despite their aneuploid nature, the lines are nontumorigenic and retain in vitro properties similar to those of primary diploid keratinocytes. These include the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0mM in the medium. The cells also demonstrate an absolute requirement for nanomolar concentrations of epidermal growth factor (EOF) for their proliferation in the presence of serum (7). We have utilized these cellsas a model system to study growthfactor-activated proliferation pathways and the effects ofoncogenes on proliferation and differentiation of this important target cell for genetic alterations that lead to cancer. Growth Factor Requirements for Proliferation of Epithelial Cells in Culture To investigate growth factor requirements of BALB/MK cells, wedevised a chemically defined medium, taking advantage of previous reports on serum-free growth of other cell types. In this system, EOF and insulin at supraphysiologic concentration werethe only growth factors required. The fact that IOF-l was effective at much lower concentrations than was insulin supports the conclusion that the insulin effect was likely mediated by its interaction with the IOF-l signal transduction pathway. In contrast, IOF-2 induced little, if any, DNA synthesis either on its own or when used to complement EOF or insulin. Under optimal serum-free conditions, the sustained growth of BALB/MK cells was comparable to that in serum-containing medium supplemented with EOF (8). Acidic and basic fibroblast growth factors (FOFs) have previously been shown to be mitogenic for a wide variety of cell types, including fibroblasts and endothelial cells as well as other cells of mesodermal derivation (reviewed in 9). The activity of these growth
SUMMARY IlMIstlgstlons of the psthw8yll reguletlng normel growth of eplthellel cells heve l'1lV8eled the existence of two meJorgrowth-f8etor slgnellng cescedes required for prollferetlon. One pethway Is ectlveted by IGF-1 or high Insulin concentration. The other Is triggered by EGF,TOFa, or members of the FGF f8mlly, Including the recently dlecovered eplthellel-cell·speclflc growth f8ctor, dellgnet· ed keretlnocyte growth f8etor (KGF).III expresllon plttern In luggelll thet KGFpleye en lmpertent normel physiologic role es e Itromel effector of eplthellel cell prollf8retlon. Oncogenel, which represent constitutively eetlveted forme of genel crltlcelly Involved In growth·f8etor Ilgnellng path· weys, apeclflcelly ebrogete the requirement for mltogenl of the EGF pethway. Exemplel of sueh genes Include the erbBlEGF receptor end erbB·2, which encode Itructurally releted receptor pro· telns end ere often empllfled end/or overexpreeeed In eplthellel mellgnenclee. Employing reduced Itrlngency hybridization with y·erbB es a probe, we recently Identified e third member of thll receptor f8mlly, dellgnsted erbB-3. eDNA cloning l'1lV8eled a predicted 148·kD trenlmembrene polypep· tide with Itructurel f8aturel Ilmllar to thOle of the EGFreceptor. NormelerbB·3 exprellion In kere· tlnocytee Ind gllnduler epithelium luggeeta III physiologic role In these cell typel. Moreover,merked· Iy eleveted erbB·3 mRNA levell In certeln memmery tumor cellllnel luggelt thet Increased erbB·3 exprellion may ello play a role In lOme human epithelial mellgnenclel.
"'''0
AM REV RESPIR DIS 1990; 142:81-S10
factors for cells of epithelial derivation is much less welldocumented, but a few reports suggest that they are mitogenic for certain epithelial cell types (10-12). As shown in table 1, both recombinant acidic and basic FOFs were potent inducers of DNA synthesis in BALB/MK cells (8). Insulin demonstrated synergy with either acidic or basic FOF in promoting DNA synthesis. The interaction between the FGFs and EGF was weaker, with only an approximately additive stimulation of DNA synthesis. In a parallelS-day growth assay, neither acidic nor basic FGF could alone support the sustained growth of BALB/MK cells. Similarly, either of the FOFs in combination with EOF supported little or no growth. However, each of the FGFs acted in concert with insulin to stimulate growth approaching that achieved with the combination of insulin and EOF. Thus, although acidic and basic FOF acted through receptors distinct from those of EGF, they were also able to complement IOF-l signal transduction to support the growth of BALB/MK.
chymal origin in human fetal tissues (15),but they exert paracrine effects on multiple cell types. Whereas acidic-FOF and basic-FOF possess activity for epithelial cellsin addition to their known endothelial and fibroblast targets (16), neither are apparently synthesized with signal peptides (17, 18). Thus, although they may have a role in epithelial cellrenewal, their mechanisms of release are not known and may only be associated with cell damage. Thus, we have sought to detect and isolate epithelial cell mitogens from stromal cells derived from epithelial tissues. A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinoeyte growth factor (KOF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C. reversed-phase HPLC column. KOF was both acid- and heat-labile and consisted
A New FGF-related Growth Factor with Properties of a Major Paracrlne Effector of Epithelial Cell Growth Interactions between epithelial and mesenchymal tissues are important during normal development (13). However, the specific factors responsible for such interactions have not been well established. Although EOF and TGFa are structurally related growth factors with epithelial as well as fibroblast specificity, neither shows a specific pattern of expression in stromal cells from epithelial tissues (14). The insulinlike growth factors are synthesized predominantly in cells of mesen-
1 From the Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland, the Department of Neurosurgery, Rhode Island Hospital, Providence, Rhode Island, t9-e Howard Hughes Medical Institute, Bethesda, Maryland, the Universita degli Studi Catania, Catania, Italy, and The Johns Hopkins Oncology Center, Baltimore, Maryland. 2 Correspondence and requests for reprints should be addressed to Stuart A. Aaronson, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Building 37, Room lE24, Bethesda, MD 20892. 3 Research Scholar of the National Institutes of Health.
58
AARONSON, RUBIN, FINCH, WONG, MARCHESE, FALCO, TAYLOR, AND KRAUS
TABLE 1 FOLD STIMULATION OF DNA SYNTHESIS AND FOLD INCREASE IN CELL NUMBER
Factor None Acidic FGF Basic FGF EGF
Fold Stimulation of DNA Synthesis'
Fold Increase in Cell Nurnber'l
Concentration (ngml-')
Alone
+1
+E
Alone
+1
+E
100 100 20
1.0 42 30 71
6.6 170 160 500
71 110 90
< 1 < 1
Introduction Efforts to investigate growth regulation of epithelial cellsin tissue culture have led to the development of medium and/or substrates for propagation of primary epithelial cellcultures (1-3). Despite these advances, it has been difficult to obtain long-term homogenous epithelial cell populations with normal growth properties. The development of continuous lines with normal growth properties has been extremely useful in studies of growth regulation, as well as alterations associated with malignant transformation by a variety of agents (4-6). The potential advantages of continuous clonal cell lines of epithelial origin led us to establish lines of clonally derived BALB/c mouse epidermal keratinocyte (7). Despite their aneuploid nature, the lines are nontumorigenic and retain in vitro properties similar to those of primary diploid keratinocytes. These include the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0mM in the medium. The cells also demonstrate an absolute requirement for nanomolar concentrations of epidermal growth factor (EOF) for their proliferation in the presence of serum (7). We have utilized these cellsas a model system to study growthfactor-activated proliferation pathways and the effects ofoncogenes on proliferation and differentiation of this important target cell for genetic alterations that lead to cancer. Growth Factor Requirements for Proliferation of Epithelial Cells in Culture To investigate growth factor requirements of BALB/MK cells, wedevised a chemically defined medium, taking advantage of previous reports on serum-free growth of other cell types. In this system, EOF and insulin at supraphysiologic concentration werethe only growth factors required. The fact that IOF-l was effective at much lower concentrations than was insulin supports the conclusion that the insulin effect was likely mediated by its interaction with the IOF-l signal transduction pathway. In contrast, IOF-2 induced little, if any, DNA synthesis either on its own or when used to complement EOF or insulin. Under optimal serum-free conditions, the sustained growth of BALB/MK cells was comparable to that in serum-containing medium supplemented with EOF (8). Acidic and basic fibroblast growth factors (FOFs) have previously been shown to be mitogenic for a wide variety of cell types, including fibroblasts and endothelial cells as well as other cells of mesodermal derivation (reviewed in 9). The activity of these growth
SUMMARY IlMIstlgstlons of the psthw8yll reguletlng normel growth of eplthellel cells heve l'1lV8eled the existence of two meJorgrowth-f8etor slgnellng cescedes required for prollferetlon. One pethway Is ectlveted by IGF-1 or high Insulin concentration. The other Is triggered by EGF,TOFa, or members of the FGF f8mlly, Including the recently dlecovered eplthellel-cell·speclflc growth f8ctor, dellgnet· ed keretlnocyte growth f8etor (KGF).III expresllon plttern In luggelll thet KGFpleye en lmpertent normel physiologic role es e Itromel effector of eplthellel cell prollf8retlon. Oncogenel, which represent constitutively eetlveted forme of genel crltlcelly Involved In growth·f8etor Ilgnellng path· weys, apeclflcelly ebrogete the requirement for mltogenl of the EGF pethway. Exemplel of sueh genes Include the erbBlEGF receptor end erbB·2, which encode Itructurally releted receptor pro· telns end ere often empllfled end/or overexpreeeed In eplthellel mellgnenclee. Employing reduced Itrlngency hybridization with y·erbB es a probe, we recently Identified e third member of thll receptor f8mlly, dellgnsted erbB-3. eDNA cloning l'1lV8eled a predicted 148·kD trenlmembrene polypep· tide with Itructurel f8aturel Ilmllar to thOle of the EGFreceptor. NormelerbB·3 exprellion In kere· tlnocytee Ind gllnduler epithelium luggeeta III physiologic role In these cell typel. Moreover,merked· Iy eleveted erbB·3 mRNA levell In certeln memmery tumor cellllnel luggelt thet Increased erbB·3 exprellion may ello play a role In lOme human epithelial mellgnenclel.
"'''0
AM REV RESPIR DIS 1990; 142:81-S10
factors for cells of epithelial derivation is much less welldocumented, but a few reports suggest that they are mitogenic for certain epithelial cell types (10-12). As shown in table 1, both recombinant acidic and basic FOFs were potent inducers of DNA synthesis in BALB/MK cells (8). Insulin demonstrated synergy with either acidic or basic FOF in promoting DNA synthesis. The interaction between the FGFs and EGF was weaker, with only an approximately additive stimulation of DNA synthesis. In a parallelS-day growth assay, neither acidic nor basic FGF could alone support the sustained growth of BALB/MK cells. Similarly, either of the FOFs in combination with EOF supported little or no growth. However, each of the FGFs acted in concert with insulin to stimulate growth approaching that achieved with the combination of insulin and EOF. Thus, although acidic and basic FOF acted through receptors distinct from those of EGF, they were also able to complement IOF-l signal transduction to support the growth of BALB/MK.
chymal origin in human fetal tissues (15),but they exert paracrine effects on multiple cell types. Whereas acidic-FOF and basic-FOF possess activity for epithelial cellsin addition to their known endothelial and fibroblast targets (16), neither are apparently synthesized with signal peptides (17, 18). Thus, although they may have a role in epithelial cellrenewal, their mechanisms of release are not known and may only be associated with cell damage. Thus, we have sought to detect and isolate epithelial cell mitogens from stromal cells derived from epithelial tissues. A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinoeyte growth factor (KOF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C. reversed-phase HPLC column. KOF was both acid- and heat-labile and consisted
A New FGF-related Growth Factor with Properties of a Major Paracrlne Effector of Epithelial Cell Growth Interactions between epithelial and mesenchymal tissues are important during normal development (13). However, the specific factors responsible for such interactions have not been well established. Although EOF and TGFa are structurally related growth factors with epithelial as well as fibroblast specificity, neither shows a specific pattern of expression in stromal cells from epithelial tissues (14). The insulinlike growth factors are synthesized predominantly in cells of mesen-
1 From the Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland, the Department of Neurosurgery, Rhode Island Hospital, Providence, Rhode Island, t9-e Howard Hughes Medical Institute, Bethesda, Maryland, the Universita degli Studi Catania, Catania, Italy, and The Johns Hopkins Oncology Center, Baltimore, Maryland. 2 Correspondence and requests for reprints should be addressed to Stuart A. Aaronson, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Building 37, Room lE24, Bethesda, MD 20892. 3 Research Scholar of the National Institutes of Health.
58
AARONSON, RUBIN, FINCH, WONG, MARCHESE, FALCO, TAYLOR, AND KRAUS
TABLE 1 FOLD STIMULATION OF DNA SYNTHESIS AND FOLD INCREASE IN CELL NUMBER
Factor None Acidic FGF Basic FGF EGF
Fold Stimulation of DNA Synthesis'
Fold Increase in Cell Nurnber'l
Concentration (ngml-')
Alone
+1
+E
Alone
+1
+E
100 100 20
1.0 42 30 71
6.6 170 160 500
71 110 90
< 1 < 1
