MINIREVIEW
Interactions of Retinoids and Transforming Growth Factor-/? in Regulation of Cell Differentiation and Proliferation
Michael B. Sporn and Anita B. Roberts Laboratory of Chemoprevention National Cancer Institute Bethesda, Maryland 20892
Introduction Retinoids control cell differentiation and proliferation by regulating gene expression. The important questions are. which genes and how? During the 7 yr since we last reviewed retinoid action (1) there have been great advances. This article reviews new information about mechanistic relationships between retinoids and transforming growth factor-/3 (TGF-/3), including their respective receptors. We consider these molecules, together with oncogenes and suppressor genes, to be components of a central, unifying regulatory system, flexibly connected, and used in many ways to integrate information relating to the state of differentiation and proliferation of the cell. Here we consider the following topics: 1) a summary of the role of retinoids in cellular differentiation and proliferation, especially as related to their role in carcinogenesis; 2) a brief review of the current knowledge of the role of newly discovered retinoic acid receptors in modulating the above processes; 3) a brief summary of the structure of the genes and peptides comprising the immediate TGF-/3 family and their role in modulating cellular differentiation and proliferation; and 4) the interface between retinoids and TGF-/3, and the role of the products of oncogenes and suppressor genes in this interface. Role of Retinoids in Differentiation, Proliferation, and Carcinogenesis Virchow eloquently noted that pathology illustrates normal physiology, more than 100 years ago. The study of retinoids provides a classic example, since their discovery and the definition of their normal function was in the context of elucidating their role in preventing or reversing the pathological state of vitamin A deficiency. More0888-8809/91 /0003-0007$02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
over, in their landmark paper on the histopathology of retinoid deficiency in the rat, Wolbach and Howe (2) noted important relationships between the effects of retinoids on both cellular differentiation and proliferation and indicated that retinoids would be significant molecules for the study of carcinogenesis They clearly recognized that during retinoid deficiency there was a failure of stem cells to mature into appropriate differentiated cells; this was accompanied by enhanced cellular proliferation, with formation of lesions resembling those in malignant or premalignant tissues. Subsequently, it was shown that retinoids are required to maintain normal differentiation and proliferation of almost all cells in the mammalian organism, during both embryogenesis and adult life (3, 4). Epithelial tissues that depend on retinoids for appropriate cell differentiation and growth account for more than threequarters of the total primary cancer in both men and women; the organ and tissue sites include bronchi and trachea, stomach, intestine, uterus, bladder, testis, breast, prostate, pancreas, and skin (3, 4). In order to quantify the requirements of an epithelial tissue for various retinoids, we developed a serum-free tracheal organ culture system to study relationships between structure and activity of several hundred natural and synthetic retinoids (5, 6). In the absence of retinoids, tracheal epithelial organ cultures lose their normal columnar ciliated and mucus cells and develop proliferative lesions characterized by aberrant differentiation of squamous cells with heavy keratinization (squamous metaplasia), resembling the epithelium found in the bronchial tree of heavy smokers. Addition of retinoids to the tracheal organ cultures after development of such lesions causes reversal of keratinization, reappearance of normal ciliated and mucus cells, and suppression of the excessive proliferation of basal cells. In this system it is clear that the processes of proliferation and differentiation are intimately linked to each other, both in the absence and presence of retinoids. Of all the naturally occurring retinoids, including retinol,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 4
retinal, retinoic acid, and many of their metabolites, none was found to be more active than alkrans-retinoic acid, which reversed abnormal keratinization in picomolar concentrations; in particular, retinoic acid was 10- to 20-fold more active than retinol or retinal, suggesting that it was indeed the proximately active metabolite (6). Because retinoids exert this hormone-like control of cell differentiation and cell proliferation, and particularly because retinoids also reverse premalignant epithelial lesions in prostatic organ cultures (7) and suppress malignant transformation of mesenchymal cells induced by a chemical carcinogen (8), it was logical to investigate whether they might also be useful agents for chemoprevention of carcinogenesis in various organs in vivo. This basic approach thus involves an enhancement of the intrinsic physiological mechanisms that protect the organism against the development of mutant clones of new cells that could potentially invade and destroy it (9, 10). Without such intrinsic defense mechanisms, the incidence of invasive cancer would be much higher, since man is constantly exposed to both endogenous and exogenous mutagenic agents. Since the process of carcinogenesis is fundamentally characterized by loss or arrest of cellular differentiation and growth control, and since retinoids both suppress growth and induce or enhance cellular differentiation, chemoprevention of cancer with retinoids represents a physiological, rather than cytotoxic approach to arrest or reverse the process of carcinogenesis (10). By now there are many examples of the ability of synthetic retinoids, mostly analogs of retinoic acid, to block the development of many forms of epithelial cancer in experimental animals (see Ref. 11 for a review); target sites include mammary gland, bladder, lung, pancreas, skin, and others. The validity of this approach is not limited to animal studies, since important clinical results have been obtained in prevention of head and neck cancer, as well as skin cancer, with 13-c/s-retinoic acid (12, 13); this same retinoid is also effective in arresting or reversing premalignant lesions of the oral mucosa, known as leukoplakia (14). At present there are many other ongoing clinical trials of retinoic acid analogs for prevention of carcinogenesis, the most notable of which involves the use of 4-hydroxyphenyl retinamide for prevention of breast cancer in more than a thousand Italian women at high risk (15). This retinoid has previously been shown to be highly effective and relatively nontoxic in animal studies, in which it has been shown to have effects on both proliferation and differentiation of mammary epithelium (16), resulting in significant inhibition of mammary carcinogenesis. It remains to be determined whether this retinoid will have a similar effect in women at risk. Role of Retinoic Acid Receptors (RARs) At a Ciba Foundation Symposium held in 1984 (17), we proposed that the term, "retinoid," be defined, not in terms of classical chemical structure of a ligand, but in
Vol 5 No. 1
terms of the ability of a ligand (or set of ligands) to elicit specific biological responses by binding to and activating a specific receptor or set of receptors. This definition was important for two reasons: 1) it allowed new molecules, such as synthetic retinoidal benzoic acid derivatives (17) that were highly active in both in vitro and in vivo assays to be classified as retinoids; and 2) it focused attention on a receptor system as the locus for future research to define the molecular mechanism of action of retinoids. Interestingly, the ultimate molecular characterization of functional retinoic acid receptors, which was achieved in 1987 (18, 19), did not come from retinoid studies themselves, but rather from other investigations, concerned with the cloning and molecular genetics of members of the steroid receptor superfamily (20, 21), particularly with attempts to identify the true ligands for several orphan receptors (22). At the present time, there are at least five distinct molecular species of RARs, and they all appear to be nuclear transcription factors, serving to regulate transcription of target genes by binding to specific regulatory sequences known as retinoic acid response elements (RAREs). Three of the receptors, known as RAR-«, RAR-/3, and RAR-y, have striking homology to each other, particularly in their ligand-binding domains and in their highly conserved DNA-binding regions (23). Most recently, two new retinoic acid receptors, called RXR-a and RXR-/3, have been cloned and characterized (24), again in searches for ligands for orphan receptors. It is of interest that retinoidal benzoic acid derivatives such as TTNPB (17) can activate RAR-a; in contrast, this synthetic retinoid only had minimal ability to activate RXR-« in transfection studies in mammalian cells (24). All of these RAR subtypes are expressed in distinct patterns during embryonic development and in the adult animal, indicating that they regulate different functions (23, 24). Thus, high levels of mRNA for RAR-a are found in adult human lung, while essentially no RAR-7 mRNA is found in this tissue; conversely levels of RAR7 mRNA are high in adult skin, while RAR-a mRNA is absent (23). Studies of the mechanism of selective gene regulation by these activated receptors have just begun. It has already been demonstrated that the mouse laminin B1 gene contains a 46-base pair RARE in its upstream regulatory region; RARs-a, -/3, and -7 all serve as transcriptional activators for this gene, while the related activated thyroid hormone receptor does not (25). Most recently, further evidence implicating RARs in control of cell differentiation has come from studies of chromosomal translocation in human leukemia. It has been shown that the translocation between the long arms of chromosomes 15 and 17, which is diagnostic for acute promyelocytic leukemia, occurs within the RAR-a gene (26-28). As noted (27), it will be most interesting to clarify the apparent paradox between the therapeutically useful differentiating effect of retinoic acid in acute promyelocytic leukemia and the proposed leukemogenic role of RAR-a in this disease.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
Minireview
TGF-0 At the beginning of this review, we noted that it would be essential to define the relationships between retinoids and TGF-8, and this topic is now one of major current interest. Indeed, our laboratory began its investigations of peptide growth factors, which culminated in the isolation and characterization of TGF-B as a new molecule (29-31), because we realized that it was not possible to understand the mechanism of action of retinoic acid in control of cell differentiation, proliferation, and carcinogenesis without considering the potential role of peptide growth factors in regulating these processes (1). There are several current reviews covering the structure and function of TGF-8 in detail (32-36); here we will provide only the briefest summary. There are three known isoforms of TGF-8 in mammalian organisms, referred to as TGF-/?s 1, 2, and 3. The original isolation of TGF-/3 from human platelets, human placenta, and bovine kidney (29-31) resulted in the identification of a single form of the peptide, a 25,000 mol wt homodimer, now called TGF-/31. Since then, a second and third form of the peptide, known as TGF-/32 and TGF-/33, have been isolated and/or cloned from multiple mammalian tissues or cDNA libraries (see Refs. 32-36 for details). The three isoforms are interchangeable in most biological assays, although they are encoded by distinct genes located on separate chromosomes in both man and mouse. All three isoforms are homodimeric molecules, with 112 amino acids in each monomer chain, each of which contains 9 cysteine residues in identical locations, although the location of the disulfide bridges has not yet been established. The regulatory response elements in the promoters of the genes of each of the three isoforms differ markedly (37, 38) and provide a basis for understanding their differential expression observed in several studies of embryonic development (39, 40). TGF-B is the prototypical multifunctional growth factor (32, 34, 41). The nature of its action on a particular target cell is critically dependent on many parameters, including the cell type, its state of differentiation, the growth conditions, and on the presence or absence of other peptide growth factors. There are numerous examples of TGF-8 acting as either a proliferative or an antiproliferative agent. Similarly, it can stimulate or arrest cell differentiation, all depending on the context of its action. The diversity of the effects of TGF-8, together with the almost universal ability of cells to respond to this peptide (essentially all cells have functional receptors for TGF-6) place this peptide in a unique position with regard to regulation of both normal and pathologic physiology. Detailed descriptions of the effects of TGF8 on differentiation and proliferation of cells found in connective tissue, muscle, bone, cartilage, the immune system, ovary, testis, adrenal, large and small blood vessels, liver, and other tissues, as well as its role in embryogenesis, can be found in Refs. 32-36.
The Interface between Retinoids, TGF-8, Oncogenes, and Suppressor Genes In light of the multifunctional actions of both retinoids and TGF-8, it is hardly surprising to suggest that there must be an important interface between these two sets of molecules. Indeed in the Introduction we suggested that retinoids, TGF-8, as well as oncogenes and suppressor genes should be regarded as a central, unifying regulatory system used by the cell for integration of information relating to its state of differentiation and proliferation. This is an area of current intense interest, and we can only give a few indications of the excitement of research efforts in this area. The transcriptional and posttranscriptional regulation of the several isoforms of TGF-8 is now a central focus of investigation, and several members of the steroid receptor superfamily exert significant effects in this regard (32, 33, 42). For example, it has recently been shown that retinoic acid is a potent inducer of TGF-02 in mouse keratinocytes, both in vitro and in vivo (43). Northern blot analysis showed that retinoic acid caused a major increase in the steady-state level of each of four different mRNA transcripts of the TGF-/32 gene, while barely affecting the level of mRNA for TGF-/31. Nuclear run-on transcription experiments indicated that this increase in TGF-/32 transcripts occurred at a posttranscriptional, rather than transcriptional, level. Parallel to these efforts on mRNA, typing of the isoforms of TGF-8 secreted by keratinocytes treated with retinoic acid showed a greater than 100-fold increase in the level of TGF-/32 peptide, but virtually no change in TGFj81. Similar results were also obtained in vivo after topical application of retinoic acid to mouse skin (43). It is now essential to develop an integrated metabolic map to explain the various interactions between other regulatory molecules and TGF-8. Such a map would be analogous to the pathways of carbohydrate, amino acid, and fatty acid metabolism that provided the foundation for classical biochemistry a generation ago. It is already known that TGF-8 genes can up-regulate or down-regulate each other (44-46), and that such regulation is mediated, in part, by interactions between TGF-8 and the jun-fos oncogene complex (47). With respect to retinoic acid, the important discovery has been made that Jun-Fos (AP-1) and the RAR-a recognize a common response element in the human osteocalcin gene (48); the actions of RAR-a and Jun-Fos appear to be antagonistic at this AP-1 site. Recent evidence suggests that there may be additional mechanisms of gene control by retinoid receptors, apart from direct DNA binding. Thus, it has been shown in several laboratories (49-52) that there is reciprocal repression of transcriptional activation by the glucocorticoid receptor and members of the Jun-Fos (AP-1) complex, mediated by protein-protein interactions rather than by direct DNA binding. Based on these results, it can now be suggested that expression of certain genes that are reciprocally regulated by retinoic acid and phorbol esters might also be controlled by protein-protein inter-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 6
actions between RAR-a and the Jun-Fos (AP-1) complex. Taken together, these new studies demonstrate that there will be multiple levels of cross-coupling of transcriptional control by members of the steroid receptor and leucine-zipper classes of transcriptional regulators. These mechanisms directly couple these two major signal transduction systems and provide a basis for integrated control of gene expression by growth factors, steroids and retinoids, and oncogenes. In a similar manner, the interface between retinoids, TGF-/3, and suppressor genes needs further investigation. Although it has been reported that there is an association between treatment with retinoic acid and the state of phosphorylation of RB (retinoblastoma) protein during induction of differentiation of leukemia cells (53), there is no evidence yet for a direct interface between retinoic acid and Rb gene expression, or posttranslational modification of the RB protein. Similarly, the interface between TGF-/3 and suppressor genes, such as Rb, needs further definition. Important first observations have been made in this area (54, 55), but evidence for a direct mechanistic connection is not yet available. Finally, since it has now been shown that the RB protein can repress c-fos expression and AP-1 transcriptional activity in both serum-induced and cycling 3T3 cells, there is a new interface between suppressor genes and oncogenes (56). We would conclude that the names that have been given to all of these nuclear regulatory elements are only of historical interest, and that the task now at hand is to determine how they all interact in a common, integrated pathway to regulate cellular differentiation and proliferation. Hopefully, this new knowledge will also provide new insights for the prevention of carcinogenesis as well as for the treatment of many other diseases characterized by excessive cellular proliferation.
Acknowledgments We thank Dianna Jessee for expert assistance with the manuscript. This minireview owes its inception to a Symposium on "Cell to Cell Interaction" held in Basel, Switzerland in September 1990. Another version of this review will be published by S. Karger AG, Basel, as part of the proceedings of that Symposium. We gratefully acknowledge the support of Johnson & Johnson, Genentech, and Schering AG for our research in this area.
Received September 23, 1990. Revision received October 23,1990. Accepted October 23,1990.
REFERENCES
1. Sporn MB, Roberts AB 1983 Role of retinoids in differentiation and carcinogenesis. Cancer Res 43:3034-3040 2. Wolbach SB, Howe PR 1925 Tissue changes following deprivation of fat soluble A vitamin. J Exp Med 42:753777 3. Sporn MB, Dunlop NM, Newton DL, Smith JM 1976 Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 35:1332-1338
Vol 5 No. 1
4. Moore T 1967 Effects of vitamin A deficiency in animals: pharmacology and toxicology of vitamin A. In: Volume I, Sebrell WH, Harris RS (eds) The Vitamins, ed 2. Academic Press, New York, pp 245-266; 280-294 5. Sporn MB, Dunlop NM, Newton DL, Henderson WR 1976 Relationships between structure and activity of retinoids. Nature 263:110-113 6. Newton DL, Henderson WR, Sporn MB 1980 Structure activity relationships of retinoids in hamster tracheal organ culture. Cancer Res 40:3413-3425 7. Lasnitzki I 1955 The influence of A hyper-vitaminosis on the effect of 20-methylcholanthrene on mouse prostate glands grown in vitro. Br J Cancer 9:434-441 8. Merriman RL, Bertram JS 1979 Reversible inhibition by retinoids of 3-methylcholanthrene-induced neoplastic transformation in C3H/10T1/2 CL8 cells. Cancer Res 39:1661-1666 9. Cairns J 1975 Mutation selection and the natural history of cancer. Nature 255:197-200 10. Sporn MB, Newton DL 1979 Chemoprevention of cancer with retinoids. Fed Proc 38:2528-2534 11. Moon, RC, Itri LM 1984 Retinoids and cancer. In: Sporn MB, Roberts AB, Goodman DS (eds). The Retinoids. Academic Press, New York, vol 2. 327-371 12. Hong WK, Lippman SM, Itri LM, Karp DD, Lee JS, Byers RM, Schantz SP, Kramer AM, Lotan R, Peters LJ, Dimery IW, Brown BW, Goepfert H 1990 Prevention of second primary tumors in squamous cell carcinoma of the head and neck with 13-c/s-retinoic acid. N Engl J Med 323:795801 13. Kraemer KH, DiGiovanna JJ, Moshell AN, Tarone RE, Peck GL 1988 Prevention of skin cancer in xeroderma pigmentosum with the use of oral isotretinoin. N Engl J Med 318:1633-1637 14. Hong WK, Endicott J, Itri LM, Doos W, Batsakis JG, Bell R, Byers R, Atkinson EN, Vaughan C, Toth B, Kramer A, Dimeryl W, Strong S 1986 13-c/s-Retinoic acid in the treatment of oral leukoplakia. N Engl J Med 315:15011502 15. Costa A, Malone W, Perloff M, Buranelli F, Campa T, Dossena G, Magni A, Pizzichetta M, Andreoli C, Vecchio MD, Formelli F, Barbieri A 1989 Tolerability of the synthetic retinoid fenretinide (HPR). Eur J Cancer Clin Oncol 25:805-808 16. Moon RC, Thompson HJ, Becci PJ, Grubbs CH, Gander RJ, Newton DL, Smith JM, Phillips SL, Henderson WR, Mullen LT, Brown CC, Sporn MB 1979 N-(4-hydroxyphenyl)retinamide, A new retinoid for prevention of breast cancer in the rat. Cancer Res 39:1339-1346 17. Sporn MB, Roberts AB 1985 What is a retinoid? In: Retinoids, differentiation and disease. Ciba Foundation Symposium 113, Pitman, London, pp 1-5 18. Petkovich M, Brand NJ, Krust A, Chambon P 1987 A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444-450 19. Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629 20. Green S, Chambon P 1986 A superfamily of potentially oncogenic hormone receptors. Nature 324:615-617 21. Evans RM 1988 The steroid and thyroid hormone receptor super-family. Science 240:889-895 22. O'Malley B 1990 The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 4:363-369 23. Krust A, Kastner PH, Petkovich M, Zelent A, Chambon P 1989 A third human retinoic acid receptor, hRAR-7. Proc Natl Acad Sci USA 86:5310-5314 24. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 354:224-229 25. Vasios GW, Gold JD, Petkovich M, Chambon P, Gudas LJ 1989 A retinoic acid-responsive element is present in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
Minireview
the 5' flanking region of the laminin B1 gene. Proc Natl Acad Sci USA 86:9099-9103 26. Borrow J, Goddard AD, Sheer D, Solomon E 1990 Molecular analysis Of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science 249:15771580 27. de The H, Chomienne C, Lanotte M, Degos L, Dejean A 1990 The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor a gene to a novel transcribed locus. Nature 347:558-561 28. Alcalay M, Longo L, Zangrilli D, Pandolfi PP, Mencarelli A, Biondi A, Rambaldi A, Lo Coco F, Diverio D, Donti E, Grignani F, Pelicci PG 1990 The chromosome 17 breakpoint in the APL t(15;17) lies within the RARa locus. Proceedings of AACR Conference on Chromosomal and Growth Factor Abnormalities in Leukemia, Chatham, MA 29. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB 1983 Transforming growth factor-/? in human platelets: identification of a major storage site, purification, and characterization. J Biol Chem 258:7155-7160 30. Frolik CA, Dart LL, Meyers CA, Smith DM, Sporn MB 1983 Purification and initial characterization of a type /? transforming growth factor from human placenta. Proc Natl Acad Sci USA 80:3676-3680 31. Roberts AB, Anzano MA, Meyers CA, Wideman J, Blacher R, Pan YCE, Stein S, Lehrman SR, Smith JM, Lamb LC, Sporn MB 1983 Purification and properties of a type /3 transforming growth factor from bovine kidney. Biochemistry 22:5692-5698 32. Roberts AB, Sporn MB 1990 The transforming growth factors-/?. In: Sporn MB, Roberts AB (eds) Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, vol 95/I 419-472 33. Sporn MB, Roberts AB 1990 TGF-/3: problems and prospects. Cell Regul 1:875-882 34. Masague J 1990 The transforming growth factor-/? family. Annu Rev Cell Biol 6:597-641 35. Piez KA, Sporn MB (eds) 1990 Transforming Growth Factor /3s: Chemistry, Biology, and Therapeutics. Annals NY Acad Sci 593: 379 pp 36. Bock G, Marsh J 1991 Clinical Applications of TGF-/3. Ciba Foundation Symposium 157. John Wiley, Chichester and New York, in press 37. Kim S-J, Glick A, Sporn MB, Roberts AB 1989 Characterization of the promoter region of the human transforming growth factor-(81 gene. J Biol Chem 264:402-408 38. Lafyatis R, Lechleider R, Kim S-J, Jakowlew S, Roberts AB, Sporn MB 1990 Structural and functional characterization of the transforming growth factor-/33 promoter: a cAMP responsive element regulates basal and induced transcription. J Biol Chem 265:19128-19136 39. Fitzpatrick DR, Denhez F, Kondaiah P, Akhurst RJ 1990 Differential expression of TGF beta isoforms in murine palatogenesis. Development 109:585-595 40. Pelton RW, Hogan BLM, Miller DA, Moses HL 1990 Differential expression of genes encoding TGFs /31, /32, and j33 during murine palate formation. Dev Biol 141:456460 41. Sporn MB, Roberts AB 1990 The multifunctional nature of peptide growth factors. In: Sporn MB, Roberts AB (eds) Handbook of Experimental Pharmacology. Peptide
Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, vol 95/1: 3-15 42. Wakefield LM, Sporn MB 1990 Suppression of carcinogenesis: a role for TGF-/3 and related molecules in prevention of cancer. In: Klein G (ed) Tumor Suppressor Genes. Marcel Dekker, New York, pp 217-243 43. Glick AB, Flanders KC, Danielpour D, Yuspa SH, Sporn MB 1989 Retinoic acid induces transforming growth fact o r - ^ in cultured keratinocytes and mouse epidermis. Cell Regul 1:87-97 44. Van Obberghen-Schilling E, Roche NS, Flanders KC, Sporn MB, Roberts AB 1988 Transforming growth factor/81 positively regulates its own expression in normal and transformed cells. J Biol Chem 263:7741-7746 45. Bascom CC, Wolfshohl JR, Coffey RJ, Madisen L, Webb NR, Purchio AR, Derynck R, Moses HL 1989 Complex regulation of transforming growth factor /31, /32, /33 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors /31 and 02. Mol Cell Biol 9:5508-5515 46. Kim S-J, Jeang K-T, Glick A, Sporn MB, Roberts AB 1989 Promoter sequences of the human TGF-/3 gene responsive to TGF-/31 autoinduction. J Biol Chem 264:70417045 47. Kim S-J, Angel P, Lafyatis R, Hattori K, Kim K-Y, Sporn MB, Karin M, and Roberts AB 1990 Autoinduction of transforming growth factor (31 is mediated by the AP-1 complex. Mol Cell Biol 10:1492-1497 48. Schule R, Umesono K, Mangelsdorf DJ, Bolado J, Pike JW, Evans RM 1990 Jun-fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene. Cell 61:497-504 49. Diamond Ml, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249:1266-1272 50. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189-1204 51. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205-1215 52. Schule R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:1217-1226 53. Mihara K, Cao X-R, Yen A, Chandler S, Driscoll B, Murphree AL, T'Ang A, Fung Y-K 1989 Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246:1300-1303 54. Pietenpol JA, Stein RW, Moran E, Yaciuk P, Schlegel R, Lyons RM, Pittelkow MR, Munger K, Howley PM, Moses HL 1990 TGF-/31 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell 61:777-785 55. Laiho M, DeCaprio JA, Ludlow JW, Livingston DM, Massague J 1990 Growth inhibition by TGF-/3 linked to suppression of retinoblastoma protein phosphorylation. Cell 62:175-185 56. Robbins PD, Horowitz JM, Mulligan RC 1990 Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature 346:668-671
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
Interactions of Retinoids and Transforming Growth Factor-/? in Regulation of Cell Differentiation and Proliferation
Michael B. Sporn and Anita B. Roberts Laboratory of Chemoprevention National Cancer Institute Bethesda, Maryland 20892
Introduction Retinoids control cell differentiation and proliferation by regulating gene expression. The important questions are. which genes and how? During the 7 yr since we last reviewed retinoid action (1) there have been great advances. This article reviews new information about mechanistic relationships between retinoids and transforming growth factor-/3 (TGF-/3), including their respective receptors. We consider these molecules, together with oncogenes and suppressor genes, to be components of a central, unifying regulatory system, flexibly connected, and used in many ways to integrate information relating to the state of differentiation and proliferation of the cell. Here we consider the following topics: 1) a summary of the role of retinoids in cellular differentiation and proliferation, especially as related to their role in carcinogenesis; 2) a brief review of the current knowledge of the role of newly discovered retinoic acid receptors in modulating the above processes; 3) a brief summary of the structure of the genes and peptides comprising the immediate TGF-/3 family and their role in modulating cellular differentiation and proliferation; and 4) the interface between retinoids and TGF-/3, and the role of the products of oncogenes and suppressor genes in this interface. Role of Retinoids in Differentiation, Proliferation, and Carcinogenesis Virchow eloquently noted that pathology illustrates normal physiology, more than 100 years ago. The study of retinoids provides a classic example, since their discovery and the definition of their normal function was in the context of elucidating their role in preventing or reversing the pathological state of vitamin A deficiency. More0888-8809/91 /0003-0007$02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
over, in their landmark paper on the histopathology of retinoid deficiency in the rat, Wolbach and Howe (2) noted important relationships between the effects of retinoids on both cellular differentiation and proliferation and indicated that retinoids would be significant molecules for the study of carcinogenesis They clearly recognized that during retinoid deficiency there was a failure of stem cells to mature into appropriate differentiated cells; this was accompanied by enhanced cellular proliferation, with formation of lesions resembling those in malignant or premalignant tissues. Subsequently, it was shown that retinoids are required to maintain normal differentiation and proliferation of almost all cells in the mammalian organism, during both embryogenesis and adult life (3, 4). Epithelial tissues that depend on retinoids for appropriate cell differentiation and growth account for more than threequarters of the total primary cancer in both men and women; the organ and tissue sites include bronchi and trachea, stomach, intestine, uterus, bladder, testis, breast, prostate, pancreas, and skin (3, 4). In order to quantify the requirements of an epithelial tissue for various retinoids, we developed a serum-free tracheal organ culture system to study relationships between structure and activity of several hundred natural and synthetic retinoids (5, 6). In the absence of retinoids, tracheal epithelial organ cultures lose their normal columnar ciliated and mucus cells and develop proliferative lesions characterized by aberrant differentiation of squamous cells with heavy keratinization (squamous metaplasia), resembling the epithelium found in the bronchial tree of heavy smokers. Addition of retinoids to the tracheal organ cultures after development of such lesions causes reversal of keratinization, reappearance of normal ciliated and mucus cells, and suppression of the excessive proliferation of basal cells. In this system it is clear that the processes of proliferation and differentiation are intimately linked to each other, both in the absence and presence of retinoids. Of all the naturally occurring retinoids, including retinol,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 4
retinal, retinoic acid, and many of their metabolites, none was found to be more active than alkrans-retinoic acid, which reversed abnormal keratinization in picomolar concentrations; in particular, retinoic acid was 10- to 20-fold more active than retinol or retinal, suggesting that it was indeed the proximately active metabolite (6). Because retinoids exert this hormone-like control of cell differentiation and cell proliferation, and particularly because retinoids also reverse premalignant epithelial lesions in prostatic organ cultures (7) and suppress malignant transformation of mesenchymal cells induced by a chemical carcinogen (8), it was logical to investigate whether they might also be useful agents for chemoprevention of carcinogenesis in various organs in vivo. This basic approach thus involves an enhancement of the intrinsic physiological mechanisms that protect the organism against the development of mutant clones of new cells that could potentially invade and destroy it (9, 10). Without such intrinsic defense mechanisms, the incidence of invasive cancer would be much higher, since man is constantly exposed to both endogenous and exogenous mutagenic agents. Since the process of carcinogenesis is fundamentally characterized by loss or arrest of cellular differentiation and growth control, and since retinoids both suppress growth and induce or enhance cellular differentiation, chemoprevention of cancer with retinoids represents a physiological, rather than cytotoxic approach to arrest or reverse the process of carcinogenesis (10). By now there are many examples of the ability of synthetic retinoids, mostly analogs of retinoic acid, to block the development of many forms of epithelial cancer in experimental animals (see Ref. 11 for a review); target sites include mammary gland, bladder, lung, pancreas, skin, and others. The validity of this approach is not limited to animal studies, since important clinical results have been obtained in prevention of head and neck cancer, as well as skin cancer, with 13-c/s-retinoic acid (12, 13); this same retinoid is also effective in arresting or reversing premalignant lesions of the oral mucosa, known as leukoplakia (14). At present there are many other ongoing clinical trials of retinoic acid analogs for prevention of carcinogenesis, the most notable of which involves the use of 4-hydroxyphenyl retinamide for prevention of breast cancer in more than a thousand Italian women at high risk (15). This retinoid has previously been shown to be highly effective and relatively nontoxic in animal studies, in which it has been shown to have effects on both proliferation and differentiation of mammary epithelium (16), resulting in significant inhibition of mammary carcinogenesis. It remains to be determined whether this retinoid will have a similar effect in women at risk. Role of Retinoic Acid Receptors (RARs) At a Ciba Foundation Symposium held in 1984 (17), we proposed that the term, "retinoid," be defined, not in terms of classical chemical structure of a ligand, but in
Vol 5 No. 1
terms of the ability of a ligand (or set of ligands) to elicit specific biological responses by binding to and activating a specific receptor or set of receptors. This definition was important for two reasons: 1) it allowed new molecules, such as synthetic retinoidal benzoic acid derivatives (17) that were highly active in both in vitro and in vivo assays to be classified as retinoids; and 2) it focused attention on a receptor system as the locus for future research to define the molecular mechanism of action of retinoids. Interestingly, the ultimate molecular characterization of functional retinoic acid receptors, which was achieved in 1987 (18, 19), did not come from retinoid studies themselves, but rather from other investigations, concerned with the cloning and molecular genetics of members of the steroid receptor superfamily (20, 21), particularly with attempts to identify the true ligands for several orphan receptors (22). At the present time, there are at least five distinct molecular species of RARs, and they all appear to be nuclear transcription factors, serving to regulate transcription of target genes by binding to specific regulatory sequences known as retinoic acid response elements (RAREs). Three of the receptors, known as RAR-«, RAR-/3, and RAR-y, have striking homology to each other, particularly in their ligand-binding domains and in their highly conserved DNA-binding regions (23). Most recently, two new retinoic acid receptors, called RXR-a and RXR-/3, have been cloned and characterized (24), again in searches for ligands for orphan receptors. It is of interest that retinoidal benzoic acid derivatives such as TTNPB (17) can activate RAR-a; in contrast, this synthetic retinoid only had minimal ability to activate RXR-« in transfection studies in mammalian cells (24). All of these RAR subtypes are expressed in distinct patterns during embryonic development and in the adult animal, indicating that they regulate different functions (23, 24). Thus, high levels of mRNA for RAR-a are found in adult human lung, while essentially no RAR-7 mRNA is found in this tissue; conversely levels of RAR7 mRNA are high in adult skin, while RAR-a mRNA is absent (23). Studies of the mechanism of selective gene regulation by these activated receptors have just begun. It has already been demonstrated that the mouse laminin B1 gene contains a 46-base pair RARE in its upstream regulatory region; RARs-a, -/3, and -7 all serve as transcriptional activators for this gene, while the related activated thyroid hormone receptor does not (25). Most recently, further evidence implicating RARs in control of cell differentiation has come from studies of chromosomal translocation in human leukemia. It has been shown that the translocation between the long arms of chromosomes 15 and 17, which is diagnostic for acute promyelocytic leukemia, occurs within the RAR-a gene (26-28). As noted (27), it will be most interesting to clarify the apparent paradox between the therapeutically useful differentiating effect of retinoic acid in acute promyelocytic leukemia and the proposed leukemogenic role of RAR-a in this disease.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
Minireview
TGF-0 At the beginning of this review, we noted that it would be essential to define the relationships between retinoids and TGF-8, and this topic is now one of major current interest. Indeed, our laboratory began its investigations of peptide growth factors, which culminated in the isolation and characterization of TGF-B as a new molecule (29-31), because we realized that it was not possible to understand the mechanism of action of retinoic acid in control of cell differentiation, proliferation, and carcinogenesis without considering the potential role of peptide growth factors in regulating these processes (1). There are several current reviews covering the structure and function of TGF-8 in detail (32-36); here we will provide only the briefest summary. There are three known isoforms of TGF-8 in mammalian organisms, referred to as TGF-/?s 1, 2, and 3. The original isolation of TGF-/3 from human platelets, human placenta, and bovine kidney (29-31) resulted in the identification of a single form of the peptide, a 25,000 mol wt homodimer, now called TGF-/31. Since then, a second and third form of the peptide, known as TGF-/32 and TGF-/33, have been isolated and/or cloned from multiple mammalian tissues or cDNA libraries (see Refs. 32-36 for details). The three isoforms are interchangeable in most biological assays, although they are encoded by distinct genes located on separate chromosomes in both man and mouse. All three isoforms are homodimeric molecules, with 112 amino acids in each monomer chain, each of which contains 9 cysteine residues in identical locations, although the location of the disulfide bridges has not yet been established. The regulatory response elements in the promoters of the genes of each of the three isoforms differ markedly (37, 38) and provide a basis for understanding their differential expression observed in several studies of embryonic development (39, 40). TGF-B is the prototypical multifunctional growth factor (32, 34, 41). The nature of its action on a particular target cell is critically dependent on many parameters, including the cell type, its state of differentiation, the growth conditions, and on the presence or absence of other peptide growth factors. There are numerous examples of TGF-8 acting as either a proliferative or an antiproliferative agent. Similarly, it can stimulate or arrest cell differentiation, all depending on the context of its action. The diversity of the effects of TGF-8, together with the almost universal ability of cells to respond to this peptide (essentially all cells have functional receptors for TGF-6) place this peptide in a unique position with regard to regulation of both normal and pathologic physiology. Detailed descriptions of the effects of TGF8 on differentiation and proliferation of cells found in connective tissue, muscle, bone, cartilage, the immune system, ovary, testis, adrenal, large and small blood vessels, liver, and other tissues, as well as its role in embryogenesis, can be found in Refs. 32-36.
The Interface between Retinoids, TGF-8, Oncogenes, and Suppressor Genes In light of the multifunctional actions of both retinoids and TGF-8, it is hardly surprising to suggest that there must be an important interface between these two sets of molecules. Indeed in the Introduction we suggested that retinoids, TGF-8, as well as oncogenes and suppressor genes should be regarded as a central, unifying regulatory system used by the cell for integration of information relating to its state of differentiation and proliferation. This is an area of current intense interest, and we can only give a few indications of the excitement of research efforts in this area. The transcriptional and posttranscriptional regulation of the several isoforms of TGF-8 is now a central focus of investigation, and several members of the steroid receptor superfamily exert significant effects in this regard (32, 33, 42). For example, it has recently been shown that retinoic acid is a potent inducer of TGF-02 in mouse keratinocytes, both in vitro and in vivo (43). Northern blot analysis showed that retinoic acid caused a major increase in the steady-state level of each of four different mRNA transcripts of the TGF-/32 gene, while barely affecting the level of mRNA for TGF-/31. Nuclear run-on transcription experiments indicated that this increase in TGF-/32 transcripts occurred at a posttranscriptional, rather than transcriptional, level. Parallel to these efforts on mRNA, typing of the isoforms of TGF-8 secreted by keratinocytes treated with retinoic acid showed a greater than 100-fold increase in the level of TGF-/32 peptide, but virtually no change in TGFj81. Similar results were also obtained in vivo after topical application of retinoic acid to mouse skin (43). It is now essential to develop an integrated metabolic map to explain the various interactions between other regulatory molecules and TGF-8. Such a map would be analogous to the pathways of carbohydrate, amino acid, and fatty acid metabolism that provided the foundation for classical biochemistry a generation ago. It is already known that TGF-8 genes can up-regulate or down-regulate each other (44-46), and that such regulation is mediated, in part, by interactions between TGF-8 and the jun-fos oncogene complex (47). With respect to retinoic acid, the important discovery has been made that Jun-Fos (AP-1) and the RAR-a recognize a common response element in the human osteocalcin gene (48); the actions of RAR-a and Jun-Fos appear to be antagonistic at this AP-1 site. Recent evidence suggests that there may be additional mechanisms of gene control by retinoid receptors, apart from direct DNA binding. Thus, it has been shown in several laboratories (49-52) that there is reciprocal repression of transcriptional activation by the glucocorticoid receptor and members of the Jun-Fos (AP-1) complex, mediated by protein-protein interactions rather than by direct DNA binding. Based on these results, it can now be suggested that expression of certain genes that are reciprocally regulated by retinoic acid and phorbol esters might also be controlled by protein-protein inter-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 6
actions between RAR-a and the Jun-Fos (AP-1) complex. Taken together, these new studies demonstrate that there will be multiple levels of cross-coupling of transcriptional control by members of the steroid receptor and leucine-zipper classes of transcriptional regulators. These mechanisms directly couple these two major signal transduction systems and provide a basis for integrated control of gene expression by growth factors, steroids and retinoids, and oncogenes. In a similar manner, the interface between retinoids, TGF-/3, and suppressor genes needs further investigation. Although it has been reported that there is an association between treatment with retinoic acid and the state of phosphorylation of RB (retinoblastoma) protein during induction of differentiation of leukemia cells (53), there is no evidence yet for a direct interface between retinoic acid and Rb gene expression, or posttranslational modification of the RB protein. Similarly, the interface between TGF-/3 and suppressor genes, such as Rb, needs further definition. Important first observations have been made in this area (54, 55), but evidence for a direct mechanistic connection is not yet available. Finally, since it has now been shown that the RB protein can repress c-fos expression and AP-1 transcriptional activity in both serum-induced and cycling 3T3 cells, there is a new interface between suppressor genes and oncogenes (56). We would conclude that the names that have been given to all of these nuclear regulatory elements are only of historical interest, and that the task now at hand is to determine how they all interact in a common, integrated pathway to regulate cellular differentiation and proliferation. Hopefully, this new knowledge will also provide new insights for the prevention of carcinogenesis as well as for the treatment of many other diseases characterized by excessive cellular proliferation.
Acknowledgments We thank Dianna Jessee for expert assistance with the manuscript. This minireview owes its inception to a Symposium on "Cell to Cell Interaction" held in Basel, Switzerland in September 1990. Another version of this review will be published by S. Karger AG, Basel, as part of the proceedings of that Symposium. We gratefully acknowledge the support of Johnson & Johnson, Genentech, and Schering AG for our research in this area.
Received September 23, 1990. Revision received October 23,1990. Accepted October 23,1990.
REFERENCES
1. Sporn MB, Roberts AB 1983 Role of retinoids in differentiation and carcinogenesis. Cancer Res 43:3034-3040 2. Wolbach SB, Howe PR 1925 Tissue changes following deprivation of fat soluble A vitamin. J Exp Med 42:753777 3. Sporn MB, Dunlop NM, Newton DL, Smith JM 1976 Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 35:1332-1338
Vol 5 No. 1
4. Moore T 1967 Effects of vitamin A deficiency in animals: pharmacology and toxicology of vitamin A. In: Volume I, Sebrell WH, Harris RS (eds) The Vitamins, ed 2. Academic Press, New York, pp 245-266; 280-294 5. Sporn MB, Dunlop NM, Newton DL, Henderson WR 1976 Relationships between structure and activity of retinoids. Nature 263:110-113 6. Newton DL, Henderson WR, Sporn MB 1980 Structure activity relationships of retinoids in hamster tracheal organ culture. Cancer Res 40:3413-3425 7. Lasnitzki I 1955 The influence of A hyper-vitaminosis on the effect of 20-methylcholanthrene on mouse prostate glands grown in vitro. Br J Cancer 9:434-441 8. Merriman RL, Bertram JS 1979 Reversible inhibition by retinoids of 3-methylcholanthrene-induced neoplastic transformation in C3H/10T1/2 CL8 cells. Cancer Res 39:1661-1666 9. Cairns J 1975 Mutation selection and the natural history of cancer. Nature 255:197-200 10. Sporn MB, Newton DL 1979 Chemoprevention of cancer with retinoids. Fed Proc 38:2528-2534 11. Moon, RC, Itri LM 1984 Retinoids and cancer. In: Sporn MB, Roberts AB, Goodman DS (eds). The Retinoids. Academic Press, New York, vol 2. 327-371 12. Hong WK, Lippman SM, Itri LM, Karp DD, Lee JS, Byers RM, Schantz SP, Kramer AM, Lotan R, Peters LJ, Dimery IW, Brown BW, Goepfert H 1990 Prevention of second primary tumors in squamous cell carcinoma of the head and neck with 13-c/s-retinoic acid. N Engl J Med 323:795801 13. Kraemer KH, DiGiovanna JJ, Moshell AN, Tarone RE, Peck GL 1988 Prevention of skin cancer in xeroderma pigmentosum with the use of oral isotretinoin. N Engl J Med 318:1633-1637 14. Hong WK, Endicott J, Itri LM, Doos W, Batsakis JG, Bell R, Byers R, Atkinson EN, Vaughan C, Toth B, Kramer A, Dimeryl W, Strong S 1986 13-c/s-Retinoic acid in the treatment of oral leukoplakia. N Engl J Med 315:15011502 15. Costa A, Malone W, Perloff M, Buranelli F, Campa T, Dossena G, Magni A, Pizzichetta M, Andreoli C, Vecchio MD, Formelli F, Barbieri A 1989 Tolerability of the synthetic retinoid fenretinide (HPR). Eur J Cancer Clin Oncol 25:805-808 16. Moon RC, Thompson HJ, Becci PJ, Grubbs CH, Gander RJ, Newton DL, Smith JM, Phillips SL, Henderson WR, Mullen LT, Brown CC, Sporn MB 1979 N-(4-hydroxyphenyl)retinamide, A new retinoid for prevention of breast cancer in the rat. Cancer Res 39:1339-1346 17. Sporn MB, Roberts AB 1985 What is a retinoid? In: Retinoids, differentiation and disease. Ciba Foundation Symposium 113, Pitman, London, pp 1-5 18. Petkovich M, Brand NJ, Krust A, Chambon P 1987 A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444-450 19. Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629 20. Green S, Chambon P 1986 A superfamily of potentially oncogenic hormone receptors. Nature 324:615-617 21. Evans RM 1988 The steroid and thyroid hormone receptor super-family. Science 240:889-895 22. O'Malley B 1990 The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 4:363-369 23. Krust A, Kastner PH, Petkovich M, Zelent A, Chambon P 1989 A third human retinoic acid receptor, hRAR-7. Proc Natl Acad Sci USA 86:5310-5314 24. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 354:224-229 25. Vasios GW, Gold JD, Petkovich M, Chambon P, Gudas LJ 1989 A retinoic acid-responsive element is present in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
Minireview
the 5' flanking region of the laminin B1 gene. Proc Natl Acad Sci USA 86:9099-9103 26. Borrow J, Goddard AD, Sheer D, Solomon E 1990 Molecular analysis Of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science 249:15771580 27. de The H, Chomienne C, Lanotte M, Degos L, Dejean A 1990 The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor a gene to a novel transcribed locus. Nature 347:558-561 28. Alcalay M, Longo L, Zangrilli D, Pandolfi PP, Mencarelli A, Biondi A, Rambaldi A, Lo Coco F, Diverio D, Donti E, Grignani F, Pelicci PG 1990 The chromosome 17 breakpoint in the APL t(15;17) lies within the RARa locus. Proceedings of AACR Conference on Chromosomal and Growth Factor Abnormalities in Leukemia, Chatham, MA 29. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB 1983 Transforming growth factor-/? in human platelets: identification of a major storage site, purification, and characterization. J Biol Chem 258:7155-7160 30. Frolik CA, Dart LL, Meyers CA, Smith DM, Sporn MB 1983 Purification and initial characterization of a type /? transforming growth factor from human placenta. Proc Natl Acad Sci USA 80:3676-3680 31. Roberts AB, Anzano MA, Meyers CA, Wideman J, Blacher R, Pan YCE, Stein S, Lehrman SR, Smith JM, Lamb LC, Sporn MB 1983 Purification and properties of a type /3 transforming growth factor from bovine kidney. Biochemistry 22:5692-5698 32. Roberts AB, Sporn MB 1990 The transforming growth factors-/?. In: Sporn MB, Roberts AB (eds) Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, vol 95/I 419-472 33. Sporn MB, Roberts AB 1990 TGF-/3: problems and prospects. Cell Regul 1:875-882 34. Masague J 1990 The transforming growth factor-/? family. Annu Rev Cell Biol 6:597-641 35. Piez KA, Sporn MB (eds) 1990 Transforming Growth Factor /3s: Chemistry, Biology, and Therapeutics. Annals NY Acad Sci 593: 379 pp 36. Bock G, Marsh J 1991 Clinical Applications of TGF-/3. Ciba Foundation Symposium 157. John Wiley, Chichester and New York, in press 37. Kim S-J, Glick A, Sporn MB, Roberts AB 1989 Characterization of the promoter region of the human transforming growth factor-(81 gene. J Biol Chem 264:402-408 38. Lafyatis R, Lechleider R, Kim S-J, Jakowlew S, Roberts AB, Sporn MB 1990 Structural and functional characterization of the transforming growth factor-/33 promoter: a cAMP responsive element regulates basal and induced transcription. J Biol Chem 265:19128-19136 39. Fitzpatrick DR, Denhez F, Kondaiah P, Akhurst RJ 1990 Differential expression of TGF beta isoforms in murine palatogenesis. Development 109:585-595 40. Pelton RW, Hogan BLM, Miller DA, Moses HL 1990 Differential expression of genes encoding TGFs /31, /32, and j33 during murine palate formation. Dev Biol 141:456460 41. Sporn MB, Roberts AB 1990 The multifunctional nature of peptide growth factors. In: Sporn MB, Roberts AB (eds) Handbook of Experimental Pharmacology. Peptide
Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, vol 95/1: 3-15 42. Wakefield LM, Sporn MB 1990 Suppression of carcinogenesis: a role for TGF-/3 and related molecules in prevention of cancer. In: Klein G (ed) Tumor Suppressor Genes. Marcel Dekker, New York, pp 217-243 43. Glick AB, Flanders KC, Danielpour D, Yuspa SH, Sporn MB 1989 Retinoic acid induces transforming growth fact o r - ^ in cultured keratinocytes and mouse epidermis. Cell Regul 1:87-97 44. Van Obberghen-Schilling E, Roche NS, Flanders KC, Sporn MB, Roberts AB 1988 Transforming growth factor/81 positively regulates its own expression in normal and transformed cells. J Biol Chem 263:7741-7746 45. Bascom CC, Wolfshohl JR, Coffey RJ, Madisen L, Webb NR, Purchio AR, Derynck R, Moses HL 1989 Complex regulation of transforming growth factor /31, /32, /33 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors /31 and 02. Mol Cell Biol 9:5508-5515 46. Kim S-J, Jeang K-T, Glick A, Sporn MB, Roberts AB 1989 Promoter sequences of the human TGF-/3 gene responsive to TGF-/31 autoinduction. J Biol Chem 264:70417045 47. Kim S-J, Angel P, Lafyatis R, Hattori K, Kim K-Y, Sporn MB, Karin M, and Roberts AB 1990 Autoinduction of transforming growth factor (31 is mediated by the AP-1 complex. Mol Cell Biol 10:1492-1497 48. Schule R, Umesono K, Mangelsdorf DJ, Bolado J, Pike JW, Evans RM 1990 Jun-fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene. Cell 61:497-504 49. Diamond Ml, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249:1266-1272 50. Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189-1204 51. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205-1215 52. Schule R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:1217-1226 53. Mihara K, Cao X-R, Yen A, Chandler S, Driscoll B, Murphree AL, T'Ang A, Fung Y-K 1989 Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246:1300-1303 54. Pietenpol JA, Stein RW, Moran E, Yaciuk P, Schlegel R, Lyons RM, Pittelkow MR, Munger K, Howley PM, Moses HL 1990 TGF-/31 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell 61:777-785 55. Laiho M, DeCaprio JA, Ludlow JW, Livingston DM, Massague J 1990 Growth inhibition by TGF-/3 linked to suppression of retinoblastoma protein phosphorylation. Cell 62:175-185 56. Robbins PD, Horowitz JM, Mulligan RC 1990 Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature 346:668-671
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 07:05 For personal use only. No other uses without permission. . All rights reserved.
