Transforming Growth Factor β3: Pharmacological Properties and Physiological Functions

Immune Growth

Regulation by Transforming Factor Beta 1

Gregory P. Boivin D.V.M., M.S. Department

of

Pathology,

University of Cincinnati,

Introduction

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ransforming growth factor beta- 1 (TGFBl) is a polypeptide homodimer with a molecular weight of 25 kDa. The TGFB’s are members of a larger superfamily of peptides that include the activins and inhibins, the bone morphogenesis proteins, and the decapentaplegic and veg-related proteins. Three TGFB isoforms, named TGFB 1, TGFl32, and TGFl33, have been identified in mammals. There is pronounced expression of all three isoforms during development, with both overlapping and distinct spatial and temporal patterns of expression. The distinct roles played by each of the TGFg’s are evident in the different phenotypes seen in mice deficient for these proteins. l-5 This review will examine the regulation of the immune system by TGFB 1, primarily concentrating

Cincinnati,

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on advances made using murine models with a brief summary of in vitro based data. Role of TGFM in vitro Numerous authors have examined the role of TGFl31 in vitro. However, caution must be used in interpretation of these studies, because in some experiments the TGFB isoform is not identified, nor is it clear whether procedures to differentiate the three isofotms were used. Based on these studies, previous reviews have concluded that TGFBl affects numerous physiological processes and elicits diverse cellular responses, having potentially both inhibitory and stimulatory effects on the same cell depending on TGFBl concentration,

Transforming Pharmacological Physiological

cell type, state of differentiation, and other environmental signals.6V7Thus, it has been suggested that TGFBl acts as a biological switch, a signalling molecule coupling the cell to its environment and providing for plasticity of response.* One of the primary regulatory roles TGFBl plays is as a potent inhibitor of immune and inflammatory responses9 A brief synopsis of some of the regulatory roles of TGFBl in the immune system follows: TGFBl inhibits cytotoxic T cell formation,” is an autocrine regulatory agent in the suppression of T and B lymphocyte proliferation, and suppresses immunoglobContinued

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Growth Factor p3: Properties and Functions

Philip N. Howles, Ph.D. University of Cincinnati Cincinnati, Ohio

College of Medicine, Department of Pathology and Laboratory

T

he transforming growth factor-beta (TGF-l3) superfamily encompasses a large set of structurally and functionally related polypeptides, including the TGF-B’s, the bone morphogenetic proteins (BMP’s), the activins and inhibins, Mtillerian inhibitory substance (MIS), and the decapentaplegic peptides (dpp) from Drosophila. These proteins share striking CIMNDC

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homology in their amino acid as well as their nucleic acid sequences. Functionally, they share the common general properties of being mitogenic for mesenchymal cells and inhibitory for epithelial cells. They also promote the deposition of extracellular matrix (ECM) proteins. The general Continued

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and specific properties of these growth factors and their receptors have been the focus of a decade of intense research effort by many investigators. The results have been well summarized in several reviews and the reader is referred to these articles for background information.‘-3 In this brief review I will focus specifically on TGF-P3. I will draw on recent in vivo and in vitro studies to illustrate its various pharmacological properties and discuss the lessons learned from analysis of “knockout” mice. TGF-P’s 1,2, and 3 are the three mammalian members of the TGF-0 family. Their numerical designation is historical and reflects the order in which they were discovered. TGF-P3 was first isolated from cDNA libraries prepared from various reproductive tissues and tumor-derived cell lines after low stringency hybridization with TGF-Bl probes.4-6 Like the first two members of the family, TGF-B3 is a 112 amino acid peptide which represents the carboxy terminus cleaved from a 4 12 amino acid precursor protein at the tetrabasic processing site RKKR (.arg.lys.lys.arg.). The mature peptide shares 72% identity with TGF-Bl and 76% identity with TGF-B2.

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everal cell culture experiments have delineated pharmacological properties of TGF-(3 that have implications for possible clinical applications.

Interspecies homologies of the mature peptides are greater than 95% in most cases. The precursor portion of TGF-P3 shares 40% to 47% identity with the corresponding region from the other two family

members, and this similarity is clustered in regions of near identity between the three proteins. In each case, the secondary and tertiary structures of the mature peptides are nearly identical, and the precursor protein stays complexed with the cleaved peptide and renders it inactive until it is released by acid pH or further proteolytic processing.le3 The potential significance of these similarities and differences will be discussed later. Since its discovery, TGF-03 has been actively studied by several investigators. These studies have used three different approaches. The first utilized various cell culture systems to compare its activity as a cytokine with TGF-P’s 1 and 2. The second approach involved characterization of the expression profiles of these genes using in situ hybridization, immunocytochemistry, and Northern blot analysis to determine where and when the mRNA’s and/or the proteins are found in various tissues. The third approach has used gene targeting to create strains of mice devoid of a specific TGF-P activity and to characterize the different pathologies that result. The data generated from these different studies have been at times complementary and at other times contradictory, reflecting the strengths and limitations of each of these individual approaches and pointing to areas for further research. Several cell culture experiments have delineated pharmacological properties of TGF-03 that have implications for possible clinical applications. Although all three TGF-B’s have similar biological properties, their relative potencies vary depending on the function being tested and the assay being used. Thus, while TGF-Pl is 100 times more potent than TGF-P2 in inhibiting hematopoietic cells,’ TGF-P3 is 5 to 10 times more potent than TGF-P2 and TGF-B 1 in suppressing DNA synthesis in keratinocytes and mink lung epithelial cells.* TGF-P3 and TGF-l32 are both 10 to 20 times more potent than TGF-Pl in stimulating proliferation of fibroblasts8 TGF-B3, as well as TGF-P’s 1 and 2 and FGF-2 (fibrobast growth factor-2) promotes dedifferentiation and moderate cell proliferation in cultured neonatal Schwann cells.’ In an assay of in vitro motoneuron survival, TGF-P3 was found

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to act synergistically with FGF-2 to prolong the survival of embryonic motoneurons.” TGF-P3 is also mitogenic for rat retinal cells” and, along with TGF-P2, can serve as a survival factor for dopaminergic neurons. ‘* Additionally, TGF-P3 can prevent myoblast fusion and differentiation in vitro and also inhibits its own synthesis and secretion.13 Thus, TGF-P3 may prove useful in studies aimed at promoting nerve and muscle regeneration after injury. The potent inhibitory properties of TGF-[33 have been used to reduce scar formation in a rat cutaneous injury model.14 Topical application of TGF-P3 reduced scar formation and improved the architecture of the neodermis by inhibiting excess deposition of ECM proteins and by inhibiting the recruitment of inflammatory cells. Interestingly, neutralizing antibodies against TGF-P’s 1 and 2 had a similar effect but only when added together. When TGF-PI or 2 are put on the wounds, no decrease in scarring is seen and there is increased ECM deposition and cell recruitment in the early stages of healing. This study highlights the competing and compensatory activities of the three TGF-P’s and also holds great promise in the area of cosmetic surgery. A related effect of TGF-P3 holds promise in the area of cancer chemotherapy. Cytotoxic effects on normal, rapidly dividing cells is often the dose-limiting factor in chemotherapy. In a hamster model of oral mucositis, the effect of 5-fluorouracil on oral mucosal cells was dramatically reduced after topical pretreatment with TGF-P3.15 The epithelial cells were arrested in the G 1 phase of the cell cycle and were protected from the cytotoxic effects of the drug. Mucositis, weight loss and mortality were all reduced about 50%. Studies of particular interest to immunologists showed that TGF-P3 could protect human bone marrow stem cells from the cytotoxic effects of alkylating agents in a manner similar to TGF-P1.16 In a comparative study, TGF-fi3 was found to be about ten-fold more potent than TGF-Pl in downregulating interleukin-3 (IL-3) receptors and inhibiting IL-3 induced colony formation by light density bone marrow (LDBM) cells.” In this same

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study, TGF-/33 was found to be two-fold less potent than TGF-P 1 at promoting GM-CSF receptor expression and colony formation by lineage-negative LDBM cells. Thus, TGF-l33 and TGF-Pl may play similar roles in promoting the survival of hematopoietic stem cells but may play opposing roles in regulating and balancing the response of mature hematopoietic cells to various cytokines. While the above studies suggest possible biological functions, they have been performed with preactivated TGF-P3 and/ or have used isolated cell systems that may not accurately reflect the in vivo situation. To define the physiological roles of TGF-B3 and the other TGF-P’s, several early studies were focused on characterizing the expression profiles of each of these genes during mouse embryogenesis. A combination of in situ hybridization and immunohistochemistry demonstrated overlapping expression patterns of the TGF-P’s,-while each was either unique or predominant in some subset of tissues or cell types. ‘*,19Thus, high TGF-B3 levels appear in the perichondrial tissue of nonossifying cartilage in various locations. It is expressed at high levels in skin epidermis and is the predominant isoform seen in developing muscle tissue, mesothelia and the liver capsule. During lung development, TGF-p3 is highly expressed in tracheal mesenchyme, bronchiolar epithelium, and prepleural mesoderm cells in a pattern that changes as lung development proceeds. Messenger RNA patterns for TGF-P3 in the embryonic nervous system suggest that it plays a role in the proliferation, migration and differentiation of both neural and glial cells.” In the secondary palate, TGF-P3 is expressed at higher levels than either of the other two TGF-P’s and cell culture experiments have shown that TGF-P3 mRNA levels can be increased by the other two TGF-B’s or decreased by epidermal growth factor (EGF).*l A role for TGF-P3 in formation of the valves and septa of the heart was suggested by its presence in the developing myocardium and by the results of an in vitro study showing inhibited migration of cardiac mesenchymal cells in the presence of antisense oligonucleotides to TGF-P3.*’ In many cases, different members of the

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TGF-P family are present in adjacent but different cell types. In some cases the RNA and protein for a given TGF-l3 are present in adjacent cells but not the same cells, suggesting both paracrine and autocrine regulation by these factors.18*‘9 Taken together, these findings led investigators to predict that TGF-j33, TGF-Pl, and TGF-/32 play critical roles in several developmental processes. While there has been less rigorous characterization of expression in adult tissues, several interesting findings have been reported. Consistent with the fact that TGF-P3 was cloned from testicular and ovarian cDNAlibraries, both the mRNA and the proteins for all three TGF-0’s have been detected in rat and human uterine tissue at all stages of estrus.23TGF-P3 and -pl are most highly expressed during diestrus II and proestrus with less expression during diestrus I and estrus. TGF-P3 expression in the adult lung of mice and humans has also been reported.% High levels of the message were reported in bronchiolar epithelium, mesenchymal cells and alveolar macrophages, along with the message for TGF-P 1. By contrast, only TGF-l31 mRNA was found in endothelial cells of the lung. Each of these findings is consistent with the proposed role of these growth factors in epitheliaY mesenchymal interactions and tissue remodeling. Two recent reports of significant clinical interest describe decreased TGF-P3 mRNA levels in several cases of basal cell carcinoma*’ and increased TGF-p3 mRNAlevels in some cases of malignant melanoma.*(j Interestingly, in the latter cases there was decreased expression in the normal epidermis overlying the tumor tissue. Together, these studies suggest that disregulation of the TGF-P’s may be important in tumor progression and metastasis. Perhaps the most exciting report for the immunologist showed that TGF-P3 is expressed in the thymic epithelium along with TGF-P’s 1 and 2.*’ Both TGF-P3 and TGF-Pl were found to be upregulated when primary cultures of thymic epithelial cells were stimulated with epithelial growth factor (EGF), but only TGF-P3 was secreted into the medium by these cultured epithelial cells. This study sug0 Elsevier

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gests that TGF-fi3 may play a role in T-cell maturation or thymic function. The other approach used to determine the physiological role of TGF-l33 has been to use gene targeting to create strains of mice lacking all TGF-j33 activity. In these experiments, a DNA construct is made which contains several kilobases of the

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n spite of its broad spectrum of embryonic expression, the only apparent phenotype seen in the TGF-p3 knockout animals are cleft palate and delayed pulmonary development.

target gene with a portion of the coding region either disrupted or replaced by the DNA coding for the marker gene neomycin acetyl transferase (which confers resistance to the drug geneticin). This DNA is then introduced into embryonic stem (ES) cells where it recombines with the host genome. In some proportion of the cells (0.1 to 10 %) the DNA will undergo homologous recombination with its cognate locus in the genome and thereby replaces the endogenous gene with the mutated copy. After a screening process to detect the mutated gene, the ES cells are injected into host blastocysts (4.5 day) where they contribute to many of the tissues of the resulting embryo. The mice thus derived are screened to determine which individuals carry germ cells derived from the injected ES cells, and these animals serve as the founders of the strain of “knockout mice.” In this manner, all three of the TGF-/3 genes have been knocked out with very interesting and somewhat unexpected results. In spite of its broad spectrum of embryonic expression, the only apparent phenotypes seen in the TGF-P3 knockout animals are cleft palate and delayed pulmonary development.28~29These defects are severe, however, and the affected mice

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die within hours of birth. The lungs show hypoplastic alveoli, decreased septal formation, and mesenchymal thickening as well as extreme intrapulmonary and pleural hemorrhage. Clefting of the secondary palate is seen in all of the TGF-B3(-/-) mice. Interestingly, the palate grows normally and the palatal shelves elevate and come into apposition. The clefting results from a failure of the shelves to fuse successfully. Both the lung and palate phenotypes are in keeping with the known activities of TGF-P’s in epithelial/mesenchymal interactions and transformations. In spite of the studies suggesting its involvement in embryogenesis, the major defect in the TGF-l31 knockout mice has proven to be an autoimmune syndrome that leads to wasting and death before the animals reach adulthood.30,3’ While the TGF-P3 and the TGF-01 knockout animals suffer defects far less severe than was predicted, the TGF-P2 knockout animals display a wide array of developmental abnormalities including conotruncal defects, bone and cartilage malformations, and several other defects involving a variety of internal organs.32 In this case, a likely explanation for the wide spectrum of developmental abnormalities is that the absence of TGF-B2 activity results in defective or inappropriate cell migration (especially of neural crest derived lineages) during critical stages of embryogenesis and organogenesis. These phenotypes are in keeping with the known property of TGF-P2 to regulate ECM deposition and chemotaxis as well as epithelial/mesenchymal interactions. One of the striking lessons learned from the TGF-P knockout mouse models is that these three peptides, which have similar expression patterns, similar biological activities and even share the same receptors, appear to have very dissimilar physiological functions. These differences may be explained in part by the slight differences in quaternary structure that have been revealed by comparison of the crystal structures of TGF-P3 and TGF-B2.33 These minor differences, which result from divergence of the primary amino acid sequences, may have profound effects on the relative affinity of the growth factors for the three ceI1 surface

receptors known to be common to the TGF-~‘s.~~Additionally, recent work by several groups has described a family of intracellular proteins that are differentially effected/activated in response to TGF-P stimulation of cells and these proteins are differentially expressed in various tissues and cell types. 35Detailed descriptions of the TGF-P receptors and their signaling pathways are beyond the scope of this article and the reader is referred to previous reviews34 and the current literature.35 Another explanation for the limited phenotypes of the TGF-PI and 43 knockouts is compensatory upregulation of one or more of the other family members. However, this possibility was ruled out using RT-PCR analysis of knockout tissues.28,30

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iven the intriguing findings regarding thewidespread effects of TGF-p3 on neural, hematopoietic, reproductive, and regenerating tissues, as well as its altered expression in certain tumors, tissue specific knockouts of this gene are clearly needed to determine the physiological roles of this molecule in adult organisms.

Finally, how complete are the physiological lessons we have learned from the TGF-p knockout animals? In spite of all the in vitro data discussed above, does TGF-P3 function solely to assure proper lung and palate development? The unfortunate limitation of knockout mice is that pathologies resulting in developmental or neonatal lethality preclude many physiological studies that might be desired. Another aspect of gene knockouts that must be considered is a careful choice of the genetic background of the parent strain of mice. Due to technical reasons, knock-

out mice are derived on a hybrid genetic background. Although backcrossing the mutation onto one or more inbred strains takes up to two years to complete, problems of “incomplete penetrance” and variable phenotypes often disappear. For example, approximately 50% of TGF-Pl (-/-) conceptuses fail during embryogenesis.30,31When this knockout was bred onto the C57BL/6 strain, no knockout pups were born. 32On other backgrounds, one might expect 100% of the (4) pups to be born. These genetic studies have the potential to yield important information about subtle gene interactions during specific physiological situations. In the case of the developmental lethal phenotype, it becomes necessary to refine the knockout strategy to use the CRE/lox system. 36In these experiments, the construct is designed to insert recombinase recognition sequences into the target locus so that the target gene is completely functional until acted upon by this enzyme. The recombinase is introduced as a transgene under the expression of a tissueor developmentally-specific promoter so that only in the desired tissues or cell lineages will the recombinase be produced and then inactivate the target gene. In this way, specific physiological questions can be asked in the context of an otherwise completely normal and healthy animal. Secondary and systemic effects are eliminated or greatly reduced. Given the intriguing findings regarding the widespread effects of TGF-P3 on neural, hematopoietic, reproductive, and regenerating tissues, as well as its altered expression in certain tumors, tissue specific knockouts of this gene are clearly needed to determine the physiological roles of this molecule in adult organisms. A comparison of the phenotypes seen with the TGF-/33 knockout versus the TGF-fil knockout would provide important insight into the relative roles and interactions of these two cytokines in hematopoiesis and immune function. Thus, rather than providing final answers to questions concerning the physiological role(s) of TGF-P3, the knockout mouse model for this gene has underscored the need for further investigations using both whole animal genetic

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models and in vitro experimentation. A focus on TGF-03 function in studies of immune function, chemotherapy, nerve regeneration and wound repair is likely to yield significant biomedical advances in the coming years. References 1. Roberts AB, Spom MB: The transforming growth factor&. In: Spom MB, Roberts AB (eds.): Peptide growth factors and their receptors handbook of experimental pathology. SpringerVerlag, New York, pp. 419-472, 1990. 2. Kingsley DM: The TGF-8 superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133-146, 1994. 3. Cox DA: Transforming growth factor-M. Cell Biol lntematl9:357-371, 1995. 4. Derynck R, Lindquist PB, Lee A, et al.: A new type of transforming growth factor-8 TGF-l33. EMBO J 7:3737-3743, 1988. 5. Jakowlew SB, Dillard PJ, Kondaiah P, Spom MB, Roberts AB: Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-8 messenger ribonucleic acid from chick embryo chondrocytes. Molec Endocrin 71747-755,

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ten Dijke P, Hansen P, lwata KK, Pieler C, Foulkes JG: Identification of another member of the transforming growth factor type I3 gene family. Proc Nat1 Acad Sci USA 85:4715-4719, 1988. 7. Ohta M, Greenberg JS, Anklesaria P, Bassols A, Massagut J: Two forms of transforming growth factor-8 distinguished by multipotential hematopoietic progenitor cells. Nature 329:539541.1987. 8. Graycar JL, Miller DA, Arrick BA, et al.: Human transforming growth factor-03: recombinant expression, purification, and biological activities in comparison with transforming growth factors131 and -82. Molec Endocrin 89:1977-1986, 1989. 9. Morgan L, Jessen KR, Mirsky R: Negative regulation of the P, gene in Schwann cells: suppression of P, mRNA and protein induction in cultured Schwann cells by FGF2 and TGF 81, TGF 82 and TGF 83. Development 120: 13991409, 1994. 10. Gouin A, Bloch-Gallego E, Tanaka H, Rosenthal A, Henderson CE: Transforming growth factor83, glial cell line-derived neurotrophic factor, and tibroblast growth factor-2 act in different manners to promote motoneuron survival in vitro. J Neurosci Res 43:454-464, 1996. 11. Anchan RM, Reh TA: Transforming growth factor-B3 is mitogenic for rat retinal progenitor 6.

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cells in vitro. J Neurobiol28:133-145, 1995. 12. Poulsen KT, Armanini MP, Klein RD, et al.: TGF-I32 and TGF-I33 are potent survival factors for midbrain dopaminergic neurons. Neuron 13:1245-1252, 1994. 13. Lafyatis R, Lechleider R, Roberts AB, Spom MB: Secretion and transcriptional regulation of transforming growth factor-B3 during myogenesis. Molec Cell Biol 11:3795-3803, 199 1. 14. Shah M, Foreman DM, Ferguson Mw: Neutralisation of TGF-81 and TGF-62 or exogenous addition of TGF-83 to cutaneous rat wounds reduces scarring. J Cell Sci 108:9851002.1995. 15. Sonis ST, Lindquist L, Van Vugt A, et al.: Prevention of chemotherapy-induced ulcerative mucositis by transforming growth factor 83. Cancer Research 54:1135-1138, 1994. 16. Lemoli RM, Strife A, Clarkson BD, Haley JD, Gulati SC: TGF-83 protects normal hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide in vitro. Exptl Hematol 20:1252-1256, 1992. 17. Jacobsen SE, Ruscetti FW, Roberts AB, Keller JR: TGF-8 is a bidirectional modulator of cytokine receptor expression on murine bone marrow cells. Differential effects of TGF-I31 and TGF-83. J. lmmunol 151:4534-4544, 1993. 18. Pelton RW, Saxena B, Jones M, Moses HL, Gold Ll: lmmunohistochemical localization of TGFD 1, TGFl32, and TGFl33 in the mouse embryo: Expression patterns suggest multiple roles during embryonic development. J Cell Biol 115:10911105,199l. 19. Schmid P, Cox D, Bilbe G, Maier R, McMaster G: Differential expression of TGF 81, 82 and 83 genes during mouse embryogenesis. Development 111:117-130, 1991. 20. Flanders KC, Ltidecke G, Engels S, et al.: Localization and actions of transforming growth factor-ps in the embryonic nervous system. Development 113:183-191, 1991. 2 1. Gehris AL, Pisano MM, Nugent P, Greene RM: Regulation of TGFl33 gene expression in embryonic palatal tissue. In Vitro Cellular & Dev Biol Animal 30A:671-679,1994. 22. Potts JD, Dagle JM, Walder JA, et al: Epithelialmesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor 83. Proc Natl Acad Sci USA 88:1516-1520, 1991. 23. Chegini N, Zhao Y, Williams RS, Flanders KC: Human uterine tissue throughout the menstrual cycle expresses transforming growth factor-8 I (TGFBl), TGF82, TGFl33, and TGFl3 type II receptor messenger ribonucleic acid and protein

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and contains [ 125l]TGFRl-binding sites. Endocrinology 135:439-449, 1994. Coker RK, Laurent GJ, Shahzeidi S, et al.: Diverse cellular TGF-I31 and TGF-83 gene expression in normal human and murine lung. Eur Resp J 9:2501-2507, 1996. S&mid P, ltin P, Rufli T: In situ analysis of transforming growth factors-8 (TGF-81, TGF-82, TGF-B3) and TGF-8 type II receptor expression in basal cell carcinomas. British Journal of Dermatology 134:1044-1051, 1996. Schmid P, ltin P, Rufli T: In situ analysis of transforming growth factor-l% (TGF-8 1, TGF-82, TGF-83). and TGF-R type II receptor expression in malignant melanoma. Carcinogenesis 16:1499-1503, 1995. Schluns KS, Grutkoski PS, Cook JE, Engelmann GL, Le PT: Human thymic epithelial cells produce TGF-83 and express TGF-8 receptors. International Immunology 7: 1681-1690, 1995. Proetzel G, Pawlowski SA, Wiles MV, et al.: Transforming growth factor-83 is required for secondary palate fusion. Nature Genetics 11:409-414, 1995. Kaartinen V, Voncken JW, Shuler C, et al.: Abnormal lung development and cleft palate in mice lacking TGF-83 indicates defects of epithelial-mesenchymal interaction. Nature Genet 11:415-421, 1995. Shull MM, Ormsby I, Kier AB, et al.: Targeted disruption of the mouse transforming growth factor-81 gene results in multifocal inflammatory disease. Nature 359:693-699, 1992. Kulkarni AB, Huh CG, Becker D, et al.: Transforming growth factor-81 null mutation in mice causes excessive inflammatory response and early death. Proc Nal Acad Sci USA 901770-774,

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LP, Ormsby I, Gittenberger-de Groot AC, et al: TGFl32 knockout mice have multiple developmental defects that are non-overlapping with other TGFl3 knockout phenotypes. Development 124: l-12, 1997. Mitt1 PR, Priestle JP, Cox DA, et al.: The crystal structure of TGF-03 and comparison to TGF-82: implications for receptor binding. Protein Science 5:1261-1271, 1996. Massague J, Attisano L, Wrana J: The complex interactions of TGF-8 receptors. Trends Cell Biol 4:172-178, 1994. Nakao A, Roijer E, lmamura T, et al.: Identification of Smad2, a human Mad-related protein in the transforming growth factor I3 signaling pathway. J Biol Chem 272:2896-2900, 1997. Rajewsky K, Gu H, Kuhn R, et al.: Conditional gene targeting. J Clin Invest 98:600-603, 1996.

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