Fetal Growth Factors as Determinants of Intrauterine Hepatic Growth PHILIP A. GRUPPUSO, THOMAS R. CURRAN, JANET E. MEAD, NELSON FAUSTO, AND WILLIAM OH
To understand mechanisms at the cellular level that may lead to the selective organomegaly seen in fetuses of diabetic mothers, we examined the role of insulin and autocrine-paracrine growth factors in the regulation of hepatic growth in the fetal rat. Analyses of fetal liver from the last one-third of gestation demonstrated the presence of specific mRNAs for the transforming growth factors (TGFs) TGF-ex and TGF-p. TGF-o, a homologue of epidermal growth factor (EGF), acts through EGF receptors. Levels of mRNA for TGF-90% of the cells in the resultant pellet, were plated in 35-mm wells coated with rat tendon collagen (Vitrogen 100, Collagen, Palo Alto, CA) at a density of 2 x 105 cells/well. After a 2-h attachment period in minimum essential medium (MEM; Gibco, Grand Island, NY) with 5% fetal bovine serum, cells were maintained in MEM without serum and supplemented with the following (final concn): 2 mM glutamine, 0.2 mM aspartate, 1 mM proline, 0.2 mM serine, 2 mM glutamate, 0.03 mM ornithine, 1 mM pyruvate, 50 |xg/ml gentamycin, and 1 fxg/ml hydrocortisone. Studies were done over the first 48 h in culture.
52
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
P.A. GRUPPUSO AND ASSOCIATES 17 d fetal [Hormone], nM: 0
21 d fetal »~80
0
adult «-80
EGFr-
FIG. 1. Ligand-dependent phosphorylation of epidermal growth factor receptor (EGFr) and insulin-receptor p-subunit (INSr) from fetal rat liver membranes (17 and 21 days gestation) and adult rat liver membranes. Receptors, partially purified by wheat-germ lectin-affinity chromatography, were phosphorylated with [32P]MgATP in presence of various concentrations of EGF and insulin (0, 2.5, 5,10, 20, 40, and 80 nM). After separation by polyacrylamide gel electrophoresis under reducing conditions, 32P-labeled proteins were detected by autoradiography. Seventeen-day fetal autoradiogram was exposed for twice as long (4 vs. 2 days) as 21-day fetal and adult analyses. EGF-receptor and insulin-receptor p-subunit were identified by their dose dependency and characteristic molecular weights (170,000 and 95,000 Mr, respectively).
we demonstrated that binding studies with this polypeptide represent a measure of IGF-I receptor expression, not binding to the IGF-II receptor or IGF binding proteins (25). Thus, our binding data indicate a gradual increase in IGF-I receptor binding capacity as term approaches (Table 1). Other investigators have demonstrated the presence of the IGF-II receptors in liver throughout postembryonic development in the rat (26). Similar to the case for the IGFs, interpretation of TGF-p binding studies must take into account the existence of multiple TGF-p receptors (for review, see ref. 27). With affinity labeling, we demonstrated that the predominant TGF-p receptor in fetal (6) and adult (18) rat liver is the 85,000-Mr type II receptor. TGF-p binding and affinitylabeled type II receptors are present in higher levels in fetal rat liver (17-21 days gestation) than in adult rat liver. Note that TGF-p binding exhibits ~ 100-fold higher affinity than binding of insulin, TGF-a, or IGF-I to fetal liver membranes. A 10- to 30-fold lower abundance of TGF-p receptors coincides with this higher affinity. These binding kinetics are in keeping with the ED50 for TGF-p inhibition of fetal hepatocyte growth (1-10 pM). EGF and TGF-a receptors, insulin receptors, and IGF-I receptors are protein tyrosine kinases that undergo hormone-dependent self-phosphorylation (28). Through phosphorylation with [32P]ATP followed by gel electrophoresis and autoradiography, we demonstrated the functional presence (tyrosine kinase activity) of these receptors. The EGF receptor is detected as an EGF- or TGF-a-dependent 170,000-/Wr phosphoprotein. Our studies confirmed a marked increase in functional EGF receptors after day 19 of gestation (Fig. 1). The phosphorylated 95,000-Mr insulin receptor p-subunit is identified by its characteristic molecular weight and dose dependence on insulin. Insulin-receptor kinase activity increases approximately twofold from fetal day 17 to day 21 (Fig. 1). Both insulin-receptor and EGFreceptor kinase activity, measured by autophosphorylation, are present at comparable levels in full-term fetal rat liver
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
mm mm*
-••###
and adult rat liver. In other studies (not shown), we found that IGF-l-receptor kinase activity nearly doubled from day 17 to day 21 but, in contrast to insulin-receptor activity, declined by 60-70% in the first 2 wk of life (25). Given their structural similarities and ligand cross-reactivity, care must be taken in assigning kinase activity to insulin receptors versus IGF-I receptors. We have taken the approach of using dose response to specific ligands (e.g., insulin vs. IGF-I) to study specific receptor kinase activities. CO
£ ID
100-
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S D
o
yA
2 E
10-
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10 3
100
H-thymidine incorporation: No additions cpm x 10~ 3
FIG. 2. Effect of fetal bovine serum (FBS) on DNA synthesis in primary cultures of fetal and adult rat hepatocytes. Data represent counts per minute (cpm) of [3H]thymidine incorporated into DNA per 3.5-cm well in absence (abscissa) or presence (ordinate) of 5% FBS. Dashed line, line of identity between results with and without serum. Cells isolated from fetuses on 17 (O, • ) , 19 (D, • ) , or 20-21 days gestation (A, • ) were incubated with [3H]thymidine for 24 (closed symbols) or 48 h (open symbols).
53
FETAL GROWTH FACTORS
175
12
"35 "5 J= $ 150 c o > O
w «*•
< ? 125 100 1 -
I 0.1
1.0
10
100
[Insulin], nM
I
10
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[TGF-beta], pM FIG. 3. Inhibition of DNA synthesis in cultured fetal hepatocytes by transforming growth factor-3 (TGF-beta). Hepatocytes from fetuses (20 days gestation) were cultured in presence of 5% fetal bovine serum (FBS) plus various concentrations of TGF-p v TGF-p and [3H]thymidine were present concomitantly for 24 h in culture, after which cells were harvested for determination of [3H]thymidine incorporation into DNA. Similar results were obtained in absence of FBS. cpm, Counts per minute.
The detection of mRNA for a growth factor and the presence of a functional growth factor receptor still do not establish a biological function for that growth factor. We have used the system of fetal rat hepatocytes in primary culture to study the in vitro actions of insulin and the autocrine-paracrine growth factors. We initially anticipated that we would be able to study the mitogenic effects of TGF-a and the IGFs by adding them to fetal hepatocytes cultured in defined media. However, our studies showed that fetal hepatocytes, unlike adult rat hepatocytes, actively synthesize DNA in culture in the absence of added mitogens (Fig. 2). This was in sharp contrast to the behavior of adult hepatocytes in culture, which, with our cell culture conditions, require a combination of insulin and EGF or TGF-a to promote DNA synthesis (5). Interestingly, the capacity of fetal hepatocytes for DNA synthesis seemed to diminish with advancing gestation. We are undertaking studies aimed at identifying autocrine-paracrine factors that may be produced in these cultures, accounting for growth in the absence of added mitogens or serum. Although primary cultures of fetal hepatocytes did not require addition of mitogens to proliferate, TGF-p acted as a potent inhibitor of DNA synthesis in this system (Fig. 3). Furthermore, DNA synthesis could be increased above "mitogen-independent" levels by physiological concentrations of insulin (Fig. 4). Thus, TGF-p and insulin represent negative and positive modulators, respectively, of fetal hepatocyte growth in vitro. Our studies have implications for the augmented fetal growth seen with diabetes in pregnancy. The hypothetical action of fetal hyperinsulinemia in producing augmented fetal hepatic growth may involve potentiation of fetal hepatic
54
FIG. 4. Potentiation of fetal hepatocyte DNA synthesis by insulin. Fetal hepatocytes (17-20 days gestation) were cultured with defined medium (no serum) in presence of various concentrations of insulin. DNA synthesis was measured as incorporation of [3H]thymidine over 24 h in culture. Results are expressed as percentage of control (no insulin). Each data point is mean of triplicate determinations. Results represent studies on 4 separate primary fetal hepatocyte cultures. Results were similar in cultures of cells obtained from fetuses of various gestational ages.
growth similar to that found in vitro. However, the in vitro effect could represent either a direct mitogenic action of insulin or effects mediated by potentiation of the actions of locally produced growth factors, most notably the IGFs. The latter (indirect) pathway could involve effects on growth factor synthesis, growth factor bioavailability (through modulation of growth factor processing or binding proteins), receptor expression, receptor tyrosine kinase specific activity, or modulation of signal transmission pathways distal to cell surface receptors. Alternatively, hyperinsulinism and/or other effects of maternal diabetes could lead to attenuation of the inhibitory effects of TGF-p on fetal hepatocyte proliferation, thus resulting in augmented growth. ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants HD-24455, CA-23226, and CA-35249. We thank Marilyn Panzica and Patricia Carter for assistance in these studies.
REFERENCES 1. Pedersen J: The Pregnant Diabetic and Her Newborn. Baltimore, MD, Williams & Wilkins, 1977 2. Susa JB, Schwartz R: Effects of hyperinsulinemia in the primate fetus. Diabetes 34 (Suppl. 2):36-41, 1985 3. Mclntyre N: Hepatotrophic Factors. New York, Elsevier, 1977 (Ciba Found. Symp. no. 55) 4. Fausto N, Mead JE: Regulation of liver growth: protooncogenes and transforming growth factors. Lab Invest 60:4-13, 1989 5. Mead JE, Fausto N: Transforming growth factor alpha may be a physiologic regulator of liver regeneration by means of an autocrine mechanism. Proc NatlAcad Sci USA 86:1558-62, 1989 6. Gruppuso PA: Expression of hepatic transforming growth factor receptors during late gestation in the fetal rat. Endocrinology 125:3037-43, 1989 7. Gruppuso PA, Boylan, Posner Bl, Faure R, Brautigan DL: Hepatic protein phosphotyrosine phosphatase: dephosphorylation of insulin and epidermal growth factor receptors in normal and alloxan diabetic rats. J Clin Invest 85:1754-60, 1990 8. Fausto N, Mead JE, Gruppuso PA, Braun L: TGF-beta in liver development, regeneration and carcinogenesis. Ann NY Acad Sci 593:231-42, 1990
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
P.A. GRUPPUSO AND ASSOCIATES 9. Massague J: Transforming growth factors: isolation, characterization and interaction with cellular receptors. Prog Med Virol 32:142-58, 1985 10. Braun L, Mead JE, Panzica M, Mikumo R, Bell Gl, Fausto N: Transforming growth factor beta mRN A increases during liver regeneration: a possible paracrine mechanism of growth regulation. Proc Natl Acad Sci USA 85:1539-43, 1988 11. Sporn MB, Roberts AB, Wakefield LM, Assoian RK: Transforming growth factor-beta: biological function and chemical structure. Science 233: 532-34, 1986 12. Gluckman PD, Butler JH: Insulin-like growth factors in the fetus. In The Physiologic Development of the Fetus and Newborn. Jones CT, Nathanielsz PW, Eds. London, Academic, 1985, p. 21-25 13. D'Ercole AJ: Somatomedins/insulin-like growth factors: relationship to insulin and diabetes. In Report 93rd Ross Conf. Pediatric Res. Gabbe SG, Oh W, Eds. Columbus, OH, Ross Laboratories, 1987, p. 50-60 14. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D'Ercole AJ, Van Wyk JJ: Somatomedin-C/insulin-like growth factor-l and insulinlike growth factor-ll mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-44, 1986 15. Han VKM, D'Ercole AJ, Lund PK: Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-97, 1987 16. Gonzalez A-M, Buscaglia M, Ong M, Baird A: Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues. J Cell Biol 110:753-65, 1990 17. Neufeld ND, Scott M, Kaplan SA: Ontogeny of the mammalian insulin receptor: studies of human and rat fetal liver plasma membranes. Dev 6/0/78:151-60, 1980 18. Gruppuso PA, Mead JE, Fausto N: Transforming growth factor receptors in liver regeneration following partial hepatectomy in the rat. Cancer Res 50:1464-69, 1990
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
19. Massague J: Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells. J Biol Chem 258:13614-20, 1983 20. Rechler MM, Nissley SP: The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 47:425-42, 1985 21. Hintz RL: Plasma forms of somatomedin and the binding protein phenomenon. Clin Endocrinol Metab 13:31-42, 1984 22. Rubin JB, Shia MA, Pilch PF: Stimulation of tyrosine-specific phosphorylation in vitro by insulin-like growth factor I. Nature (Lond) 305:438-40, 1983 23. Ullrich A, Gray A, Tarn AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y: Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-12, 1986 24. Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WJ: Insulin-like growth factor II receptor as a multifunctional binding protein. Nature (Lond) 329:301-307, 1987 25. Gruppuso PA, Walker TD, Carter PA: Ontogeny of hepatic type I insulin-like growth factor receptors in the rat. Pediatr Res 29:226-30, 1991 26. Senior PV, Byrne S, Brammar WJ, Beck F: Expression of the IGF-II/ mannose-6-phosphate receptor mRN A and protein in the developing rat. Development 109:67-73, 1990 27. Massague J, Cheifetz S, Boyd FT, Andres JL: TGF-beta receptors and TGF-beta binding proteoglycans: recent progress in identifying their functional properties. Ann NY Acad Sci 593:59-72, 1990 28. Sibley DR, Benovic JL, Carbon MG, Lefkowitz RJ: Regulation of transmembrane signalling by receptor phosphorylation. Cell48:913-22,1987
55
To understand mechanisms at the cellular level that may lead to the selective organomegaly seen in fetuses of diabetic mothers, we examined the role of insulin and autocrine-paracrine growth factors in the regulation of hepatic growth in the fetal rat. Analyses of fetal liver from the last one-third of gestation demonstrated the presence of specific mRNAs for the transforming growth factors (TGFs) TGF-ex and TGF-p. TGF-o, a homologue of epidermal growth factor (EGF), acts through EGF receptors. Levels of mRNA for TGF-90% of the cells in the resultant pellet, were plated in 35-mm wells coated with rat tendon collagen (Vitrogen 100, Collagen, Palo Alto, CA) at a density of 2 x 105 cells/well. After a 2-h attachment period in minimum essential medium (MEM; Gibco, Grand Island, NY) with 5% fetal bovine serum, cells were maintained in MEM without serum and supplemented with the following (final concn): 2 mM glutamine, 0.2 mM aspartate, 1 mM proline, 0.2 mM serine, 2 mM glutamate, 0.03 mM ornithine, 1 mM pyruvate, 50 |xg/ml gentamycin, and 1 fxg/ml hydrocortisone. Studies were done over the first 48 h in culture.
52
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
P.A. GRUPPUSO AND ASSOCIATES 17 d fetal [Hormone], nM: 0
21 d fetal »~80
0
adult «-80
EGFr-
FIG. 1. Ligand-dependent phosphorylation of epidermal growth factor receptor (EGFr) and insulin-receptor p-subunit (INSr) from fetal rat liver membranes (17 and 21 days gestation) and adult rat liver membranes. Receptors, partially purified by wheat-germ lectin-affinity chromatography, were phosphorylated with [32P]MgATP in presence of various concentrations of EGF and insulin (0, 2.5, 5,10, 20, 40, and 80 nM). After separation by polyacrylamide gel electrophoresis under reducing conditions, 32P-labeled proteins were detected by autoradiography. Seventeen-day fetal autoradiogram was exposed for twice as long (4 vs. 2 days) as 21-day fetal and adult analyses. EGF-receptor and insulin-receptor p-subunit were identified by their dose dependency and characteristic molecular weights (170,000 and 95,000 Mr, respectively).
we demonstrated that binding studies with this polypeptide represent a measure of IGF-I receptor expression, not binding to the IGF-II receptor or IGF binding proteins (25). Thus, our binding data indicate a gradual increase in IGF-I receptor binding capacity as term approaches (Table 1). Other investigators have demonstrated the presence of the IGF-II receptors in liver throughout postembryonic development in the rat (26). Similar to the case for the IGFs, interpretation of TGF-p binding studies must take into account the existence of multiple TGF-p receptors (for review, see ref. 27). With affinity labeling, we demonstrated that the predominant TGF-p receptor in fetal (6) and adult (18) rat liver is the 85,000-Mr type II receptor. TGF-p binding and affinitylabeled type II receptors are present in higher levels in fetal rat liver (17-21 days gestation) than in adult rat liver. Note that TGF-p binding exhibits ~ 100-fold higher affinity than binding of insulin, TGF-a, or IGF-I to fetal liver membranes. A 10- to 30-fold lower abundance of TGF-p receptors coincides with this higher affinity. These binding kinetics are in keeping with the ED50 for TGF-p inhibition of fetal hepatocyte growth (1-10 pM). EGF and TGF-a receptors, insulin receptors, and IGF-I receptors are protein tyrosine kinases that undergo hormone-dependent self-phosphorylation (28). Through phosphorylation with [32P]ATP followed by gel electrophoresis and autoradiography, we demonstrated the functional presence (tyrosine kinase activity) of these receptors. The EGF receptor is detected as an EGF- or TGF-a-dependent 170,000-/Wr phosphoprotein. Our studies confirmed a marked increase in functional EGF receptors after day 19 of gestation (Fig. 1). The phosphorylated 95,000-Mr insulin receptor p-subunit is identified by its characteristic molecular weight and dose dependence on insulin. Insulin-receptor kinase activity increases approximately twofold from fetal day 17 to day 21 (Fig. 1). Both insulin-receptor and EGFreceptor kinase activity, measured by autophosphorylation, are present at comparable levels in full-term fetal rat liver
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
mm mm*
-••###
and adult rat liver. In other studies (not shown), we found that IGF-l-receptor kinase activity nearly doubled from day 17 to day 21 but, in contrast to insulin-receptor activity, declined by 60-70% in the first 2 wk of life (25). Given their structural similarities and ligand cross-reactivity, care must be taken in assigning kinase activity to insulin receptors versus IGF-I receptors. We have taken the approach of using dose response to specific ligands (e.g., insulin vs. IGF-I) to study specific receptor kinase activities. CO
£ ID
100-
Z
S D
o
yA
2 E
10-
eo
I
10 3
100
H-thymidine incorporation: No additions cpm x 10~ 3
FIG. 2. Effect of fetal bovine serum (FBS) on DNA synthesis in primary cultures of fetal and adult rat hepatocytes. Data represent counts per minute (cpm) of [3H]thymidine incorporated into DNA per 3.5-cm well in absence (abscissa) or presence (ordinate) of 5% FBS. Dashed line, line of identity between results with and without serum. Cells isolated from fetuses on 17 (O, • ) , 19 (D, • ) , or 20-21 days gestation (A, • ) were incubated with [3H]thymidine for 24 (closed symbols) or 48 h (open symbols).
53
FETAL GROWTH FACTORS
175
12
"35 "5 J= $ 150 c o > O
w «*•
< ? 125 100 1 -
I 0.1
1.0
10
100
[Insulin], nM
I
10
I 25
[TGF-beta], pM FIG. 3. Inhibition of DNA synthesis in cultured fetal hepatocytes by transforming growth factor-3 (TGF-beta). Hepatocytes from fetuses (20 days gestation) were cultured in presence of 5% fetal bovine serum (FBS) plus various concentrations of TGF-p v TGF-p and [3H]thymidine were present concomitantly for 24 h in culture, after which cells were harvested for determination of [3H]thymidine incorporation into DNA. Similar results were obtained in absence of FBS. cpm, Counts per minute.
The detection of mRNA for a growth factor and the presence of a functional growth factor receptor still do not establish a biological function for that growth factor. We have used the system of fetal rat hepatocytes in primary culture to study the in vitro actions of insulin and the autocrine-paracrine growth factors. We initially anticipated that we would be able to study the mitogenic effects of TGF-a and the IGFs by adding them to fetal hepatocytes cultured in defined media. However, our studies showed that fetal hepatocytes, unlike adult rat hepatocytes, actively synthesize DNA in culture in the absence of added mitogens (Fig. 2). This was in sharp contrast to the behavior of adult hepatocytes in culture, which, with our cell culture conditions, require a combination of insulin and EGF or TGF-a to promote DNA synthesis (5). Interestingly, the capacity of fetal hepatocytes for DNA synthesis seemed to diminish with advancing gestation. We are undertaking studies aimed at identifying autocrine-paracrine factors that may be produced in these cultures, accounting for growth in the absence of added mitogens or serum. Although primary cultures of fetal hepatocytes did not require addition of mitogens to proliferate, TGF-p acted as a potent inhibitor of DNA synthesis in this system (Fig. 3). Furthermore, DNA synthesis could be increased above "mitogen-independent" levels by physiological concentrations of insulin (Fig. 4). Thus, TGF-p and insulin represent negative and positive modulators, respectively, of fetal hepatocyte growth in vitro. Our studies have implications for the augmented fetal growth seen with diabetes in pregnancy. The hypothetical action of fetal hyperinsulinemia in producing augmented fetal hepatic growth may involve potentiation of fetal hepatic
54
FIG. 4. Potentiation of fetal hepatocyte DNA synthesis by insulin. Fetal hepatocytes (17-20 days gestation) were cultured with defined medium (no serum) in presence of various concentrations of insulin. DNA synthesis was measured as incorporation of [3H]thymidine over 24 h in culture. Results are expressed as percentage of control (no insulin). Each data point is mean of triplicate determinations. Results represent studies on 4 separate primary fetal hepatocyte cultures. Results were similar in cultures of cells obtained from fetuses of various gestational ages.
growth similar to that found in vitro. However, the in vitro effect could represent either a direct mitogenic action of insulin or effects mediated by potentiation of the actions of locally produced growth factors, most notably the IGFs. The latter (indirect) pathway could involve effects on growth factor synthesis, growth factor bioavailability (through modulation of growth factor processing or binding proteins), receptor expression, receptor tyrosine kinase specific activity, or modulation of signal transmission pathways distal to cell surface receptors. Alternatively, hyperinsulinism and/or other effects of maternal diabetes could lead to attenuation of the inhibitory effects of TGF-p on fetal hepatocyte proliferation, thus resulting in augmented growth. ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants HD-24455, CA-23226, and CA-35249. We thank Marilyn Panzica and Patricia Carter for assistance in these studies.
REFERENCES 1. Pedersen J: The Pregnant Diabetic and Her Newborn. Baltimore, MD, Williams & Wilkins, 1977 2. Susa JB, Schwartz R: Effects of hyperinsulinemia in the primate fetus. Diabetes 34 (Suppl. 2):36-41, 1985 3. Mclntyre N: Hepatotrophic Factors. New York, Elsevier, 1977 (Ciba Found. Symp. no. 55) 4. Fausto N, Mead JE: Regulation of liver growth: protooncogenes and transforming growth factors. Lab Invest 60:4-13, 1989 5. Mead JE, Fausto N: Transforming growth factor alpha may be a physiologic regulator of liver regeneration by means of an autocrine mechanism. Proc NatlAcad Sci USA 86:1558-62, 1989 6. Gruppuso PA: Expression of hepatic transforming growth factor receptors during late gestation in the fetal rat. Endocrinology 125:3037-43, 1989 7. Gruppuso PA, Boylan, Posner Bl, Faure R, Brautigan DL: Hepatic protein phosphotyrosine phosphatase: dephosphorylation of insulin and epidermal growth factor receptors in normal and alloxan diabetic rats. J Clin Invest 85:1754-60, 1990 8. Fausto N, Mead JE, Gruppuso PA, Braun L: TGF-beta in liver development, regeneration and carcinogenesis. Ann NY Acad Sci 593:231-42, 1990
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
P.A. GRUPPUSO AND ASSOCIATES 9. Massague J: Transforming growth factors: isolation, characterization and interaction with cellular receptors. Prog Med Virol 32:142-58, 1985 10. Braun L, Mead JE, Panzica M, Mikumo R, Bell Gl, Fausto N: Transforming growth factor beta mRN A increases during liver regeneration: a possible paracrine mechanism of growth regulation. Proc Natl Acad Sci USA 85:1539-43, 1988 11. Sporn MB, Roberts AB, Wakefield LM, Assoian RK: Transforming growth factor-beta: biological function and chemical structure. Science 233: 532-34, 1986 12. Gluckman PD, Butler JH: Insulin-like growth factors in the fetus. In The Physiologic Development of the Fetus and Newborn. Jones CT, Nathanielsz PW, Eds. London, Academic, 1985, p. 21-25 13. D'Ercole AJ: Somatomedins/insulin-like growth factors: relationship to insulin and diabetes. In Report 93rd Ross Conf. Pediatric Res. Gabbe SG, Oh W, Eds. Columbus, OH, Ross Laboratories, 1987, p. 50-60 14. Lund PK, Moats-Staats BM, Hynes MA, Simmons JG, Jansen M, D'Ercole AJ, Van Wyk JJ: Somatomedin-C/insulin-like growth factor-l and insulinlike growth factor-ll mRNAs in rat fetal and adult tissues. J Biol Chem 261:14539-44, 1986 15. Han VKM, D'Ercole AJ, Lund PK: Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science 236:193-97, 1987 16. Gonzalez A-M, Buscaglia M, Ong M, Baird A: Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues. J Cell Biol 110:753-65, 1990 17. Neufeld ND, Scott M, Kaplan SA: Ontogeny of the mammalian insulin receptor: studies of human and rat fetal liver plasma membranes. Dev 6/0/78:151-60, 1980 18. Gruppuso PA, Mead JE, Fausto N: Transforming growth factor receptors in liver regeneration following partial hepatectomy in the rat. Cancer Res 50:1464-69, 1990
DIABETES, VOL. 40, SUPPL. 2, DECEMBER 1991
19. Massague J: Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells. J Biol Chem 258:13614-20, 1983 20. Rechler MM, Nissley SP: The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 47:425-42, 1985 21. Hintz RL: Plasma forms of somatomedin and the binding protein phenomenon. Clin Endocrinol Metab 13:31-42, 1984 22. Rubin JB, Shia MA, Pilch PF: Stimulation of tyrosine-specific phosphorylation in vitro by insulin-like growth factor I. Nature (Lond) 305:438-40, 1983 23. Ullrich A, Gray A, Tarn AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Le Bon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y: Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-12, 1986 24. Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WJ: Insulin-like growth factor II receptor as a multifunctional binding protein. Nature (Lond) 329:301-307, 1987 25. Gruppuso PA, Walker TD, Carter PA: Ontogeny of hepatic type I insulin-like growth factor receptors in the rat. Pediatr Res 29:226-30, 1991 26. Senior PV, Byrne S, Brammar WJ, Beck F: Expression of the IGF-II/ mannose-6-phosphate receptor mRN A and protein in the developing rat. Development 109:67-73, 1990 27. Massague J, Cheifetz S, Boyd FT, Andres JL: TGF-beta receptors and TGF-beta binding proteoglycans: recent progress in identifying their functional properties. Ann NY Acad Sci 593:59-72, 1990 28. Sibley DR, Benovic JL, Carbon MG, Lefkowitz RJ: Regulation of transmembrane signalling by receptor phosphorylation. Cell48:913-22,1987
55
