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Transforming Growth Factor β in the Control of Epidermal Proliferation
Transforming Growth Factor {3 in the Control of Epidermal Proliferation BY JOHN A. BARNARD, MD,* CHARLES C. BASCOM, PHD,t RUSSETTE M. LYONS, PHD, t NANCY J. SIPES, PHD, t HAROLD L. MOSES, MOt
T
ransforming growth factors (TGFs) originally were named and operationally defined as polypeptides that induce morphologic transformation of fibroblastic cells in monolayer culture and stimulate colony formation in soft agar. I - 3 As a consequence of early work with TGFs, a new concept evolved to explain the uncontrolled proliferation characteristic of neoplastic cells. This concept was termed the autocrine hypothesis, which proposed that malignant cells secrete stimulatory polypeptide factors for which the cells themselves have functional surface receptors. 4 A large body of circumstantial and direct evidence has accumulated in support ofthe autocrine hypothesis as a mechanism for neoplastic cell growth. 5 More recently it has been suggested that TGFs like TGFa and TGF{1 also are involved in the growth and differentiation of normal cells,6 indicating that the autocrine hypothesis should be broadened to encompass a role for autocrine polypeptide growth factors in the modulation of normal growth. Much of this work has been performed in human and mouse keratinocytes. The present review summarizes the role of the growth inhibitory autocrine polypeptide TGF{1 in the regulation of normal keratinocyte growth and differentiation. Transforming Growth Factor fJ
TGF{1 is a 25 kD homodimer that is produced by a wide variety of normal and neoplastic cells. 7 Currently, two distinct molecular forms of TGF{1 have been identified. These molecules have been designated TGF{11 and TGF{12. Human and porcine TGF{11 are composed of two identical 12,500 MW subunits. Each monomer is synthesized as the COOH-terminal112 amino acids of a 391 amino acid precursor. s Phylogenetic comparisons with the human sequence show an exceptionally high degree of From the
* Departments of Cell Biology and Pediatrics and the
t Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee. Reprint requests: Dr. Harold L. Moses, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
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conservation.9 Eighty of the 112 mature TGF{12 amino acids are identical to TGF{11, 10 and all nine cysteines are conserved. 10 In the precursor region only 33% of the aligned residues are identical. The growth inhibitor from the African green monkey kidney cell line, BSC-l, recently has been cloned and the cDNA sequence indicates that it is identical to TGF{12. 11 Although the confusing terminology would imply otherwise, transforming growth factor alpha (TGFa) bears no structural similarity to any of the TGF{1 molecules. TGFa shares structural homology and a cell surface receptor with epidermal growth factor (EGF).12 This growth stimulatory autocrine polypeptide will not be discussed extensively in this review. The TGF{1s belong to a larger family of related polypeptides with structural and sequence homology, most of which possess growth regulatory functions. These include (among others) Mullerian inhibitory substance, a protein that induces regression of the Mullerian duct system in the developing male genitourinary system,13 inhibins (and their {1 chain dimers, activins),I4 and Vg-l, a protein produced in Xenopus that promotes mesodermal induction. 15 TGF{1 is secreted by cells in culture in an inactive, latent form that is irreversibly activated by nonphysiologic extremes ofpH. 16 Potential physiologic mechanisms of activation include the proteases plasmin and cathepsin D that appear to activate at least a portion of TGF{1 released in culture. 17 Identification of other possible cell or tissue-specific mechanisms for activation of latent TGF{1 will be of major importance in understanding regulation and specificity of TGF{1 action, as cell surface membrane receptors appear to be ubiquitously distributed. Three structurally distinct, glycosylated TGF{1 receptors have been identified in affinity cross-linking and photoaffinity labeling studies. 1s-2o The type I receptor is 65 kD and is found in most cell types examined. The type II receptor has a molecular weight of 95 kD (human and monkey cells) or 85 kD (other mammalian cells). The type III receptor contains a 280-330 kD ligand binding subunit that is associated with a larger (560-600 kD disulfide linked receptor 159
TGF.B In Epidermal
P~ollferation
TABLE 1 TGFfj Effects on Gene Expression Fibroblastic cells
Epithelial cells
Enhanced expression:
Enhanced expression: c-sis (unpublished) {3-actin 33 EGF receptor (unpublished)
C-SiS23 C-fOS 23
c_myc 23 KC (unpublished) JE 23 ~-actin46
"Y-actin46 type I procollagen 34 fibronectin 34 PAI_1 35.36 TGF~ (unpublished) Decreased expression: transin 36 U_PA35
U-pN5 PAI-1 35 TGF~ (unpublished) No effect:
C-fOS33
Decreased expression: c-myc 33 KC 33 Not expressed: JE 33
complex. 19 The type III receptor demonstrates high affinity for TGF,61 and TGF,62 whereas the type I and II receptors demonstrate a tenfold higher a~nity for ,61 than for TGF,62. Preliminary data suggest that each TGF,6 receptor type mediates a distinct biologic response. 20 The intracellular signaling mechanism activated by TGF,6 receptor interaction has not been elucidated. The TGF,6 receptor has no intrinsic tyrosine kinase activity21 and Fine et al 22 showed no alteration in Na+ /H+ exchanger activity in primary proximal renal tubular cells by the BSC-1 growth inhibitor (TGF,62). The biologic response to TGF,61 is cell-type specific and remarkably complex. In fibroblastic cells, TGF,6 appears to stimulate growth by an indirect mechani'sm. 23 Treatment of monolayer cultures of AKR-2B cells with TGF,6 caused a rapid induction of c-sis mRNA, followed by the appearance of platelet-derived growth factor (PDGF) radioreceptor competing material in the culture medium. Thus, TGF,6 may stimulate growth indirectly via the mitogenic polypeptide PDGF. By contrast, TGF,6 is a potent growth inhibitor in all normal epithelial cells studied. The growth of many neoplastic epithelial cells also is inhibited by TGF,6; however, Shipley et al 24 showed that clonal growth of the squamous cell carcinoma line, SCC-25, was not affected by the addition of TGF,6. 1251-TGF,6 binding studies revealed the absence of specific binding to SCC-25 cells, leading to speculation that absence of the negative control mechanism for growth contributed to the malignant phenotype. A large body of additional data also supports a pivotal role for TGF,6 in the regulation of neoplastic growth. For example, human A549 lung carcinoma cells ap-
160
pear unable to activate latent TGF,6,25 and the estrogen responsive human breast cancer cell line, MCF-7, demonstrates a decrease in TGF,6 protein production when treated with growth stimulatory concentrations of 17,6-estradio1. 26 The precise mechanism by which TGF,6 inhibits epithelial cell growth remains largely unknown. Inasmuch as the effect is reversible, the growth inhibitory properties ofTGF,6 do not appear to result from cytotoxicity. In a few selected cell types TGF,6 induces differentiation. 27-29 However, this is not a generalized phenomenon. In endothelial cells, TGF,6 inhibits epidermal growth factor (EGF)-induced expression of cmye, a nuclear protooncogene that is associated with cell proliferation. 30 In addition, the number of highaffinity EGF receptors was decreased in endothelial cells treated with TGF,6. EGF receptor density also was decreased 4 to 6 hours after treatment of NRK fibroblasts with TGF,6.31 Studies by Coffey6 and others32 have not demonstrated an effect of TGF,6 on EGF receptor-ligand interaction in cells of epithelial origin. Data from our laboratory indicate that expression of competence genes such as KC and c-mye is decreased rapidly (within 1 hour) in keratinocyte monolayers treated with TGF,6.33 This appears to be a selective phenomenon as expression of ,6-actin was increased and c-fos mRNA levels remained unchanged. The diverse and complex manner in which TGF,6 affects expression of genes relevant to growth, differentiation and extracellular matrix formation is summarized in Table 1. 23 ,34-38 Many of the effects appear to be unrelated to proliferation and are cell-type specific. The effect of TGF,6 on extracellular matrix formation 34 -36 coupled with the observations that TGF,6 is chemotactic for fibroblasts 39 and promotes angiogenesis 40 suggests a potential role in wound healing. Indeed, recent in vivo studies show that TGF,6 accelerates healing of incisional wounds in rats. 41 Undoubtedly, additional studies will further delineate the role of this molecule in the regulation of normal cell growth, embryogenesis, and in the process of cellular transformation. The Role of TGF~ in Keratlnocyte Growth
Much of the current understanding of the role of TGF,6 in the control of epithelial cell growth is derived from studies using normal keratinocytes. The addition of TGF,6 to cultures of proliferating human foreskin proketatinocytes caused a marked reduction in [3H]-thymidine labeling within 24 to 48 hours. 7 Clonal growth assays demonstrated that this growth inhibitory effect persisted for 10 days.24 Inhibition was first detectable at a TGF,6 concentration of 1 ng/ ml (40 pM), whereas in concentrations of more than 10 ng/ml, essentially no clonal growth was detectable. When growth-arrested normal prokeratinocytes in
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Barnard et al
monolayer were replated in clonal growth assays without TGF{3, no inhibition was noted, indicating the reversibility of growth arrest. Flow microfluorimetric studies showed that the majority of cells arrested in the G 1 phase of the cell cycle. 24 Weissman et al 42 showed that addition of epidermal growth factor (EGF) to primary cultures of BALB/c mouse epidermal cells resulted in establishment of a continuous, homogeneous cell line (BALB/MK) with an absolute requirement for EGF supplementation. These cells represent a useful model system for the study of epithelial cell growth control as they retain the capacity for differentiation in response to added extracellular calcium. 42•43 TGF{3 reversibly inhibited BALB/MK growth whereas total protein synthesis and RNA synthesis were unaffected. 6 TGF{31 and TGF{32 have been found to be equally effective in inhibiting proliferation in the BALB/MK cells (unpublished data). Quiescent cells that were restimulated with EGF showed a marked decrease in the percent of nuclei labeled with 3H-thymidine when 10 ng/ml TGF{3 was added concomitant with EGF restimulation. A potential mechanism for TGF{3 inhibition of EGF stimulation was investigated using 125I_EGF binding studies, but no alteration in EGF binding or internalization was found, suggesting that the TGF{3 effect occurred distal to EGF ligand-receptor binding or independent of EGF pathways. TGF{3 did not induce morphologic alterations typical of terminal differentiation of keratinocytes, and the differentiation markers pemphigus vulgaris antigen, filaggrin, or desmoplakin were not induced. Both morphologic and biochemical features of differentiation were detected in cells treated with 1.5 mM calcium for 24 hours. Thus, TGF{3 appeared to uncouple inhibition of proliferation and induction ofterminal differentiation. It could be theorized that autocrine TGF{3 production in the skin functions to maintain a nonproliferating pool of keratinocytes in the basal layers of the epidermis. This pool may serve as a reservoir for recruitment back into a proliferative compartment in response to a growth stimulatory signal. Coffey et al6 demonstrated TGF{3 gene expression by Northern blot analysis in BALB/MK cells and latent TGF{3 protein production by radioreceptor assay of serum-free BALB/MK conditioned medium. Unpublished experiments have demonstrated the presence of a high affinity (8.5 pM) class of TGF{3 receptors on BALB/MK cells, with a receptor density of 7000 binding sites/cell (unpublished data). Additional unpublished data show that TGF{31 treatment of rapidly growing BALB/MK cells results in an "autoinduction" of TGF{31 gene expression. A similar phenomenon has been described for TGFa in human keratinocytes. 44 Both EGF and TGFa induce TGFa gene expression and protein synthesis by human foreskin keratinocytes. These studies suggest
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ACllv.3lion of
~~e;~
!
.04--'
Figure 1. TGFp In the control of epidermal prollferallon. A growth Inhibitory TGFp autocrlne loop Is shown. TGFp decreases expression of the competence genes c-myc and Ke, a potenllal mechanism for TGFP-medlated growth Inhibition. TGFp also Induces Its own gene expression (autolnductlon). This may funcllon as a signal ampllHcalion mechanism. Acllvallon of TGFp Is required before receptor/ligand Interacllon.
that autoregulation of proliferation could be responsible for amplification of the growth factor response, a mechanism that may be relevant to pathophysiologic situations like wound healing. These studies show that all of the requisite elements for a TGF{3 autocrine growth control mechanism exist in normal keratinocytes. Similar observations have been reported for transforming growth factor a in human keratinocytes 44 and in BALB/MK cells. 6 Evidence for the relevance of these in vitro studies to in vivo epidermal homeostasis is accumulating. For example, TGFa mRNA was demonstrated in histological sections of neonatal foreskin by in situ hybridization, and TGFa protein was demonstrated by immunocytochemistry.44 Recently, TGF{3 mRNA was detected in the mouse epidermis by Northern blot analysis of RNA samples obtained from control and tumor promotor (12-tetra-decanoyl-phorbol-13-acetate, TP A) treated skin. 45 The control levels were quite low, but TPA treatment markedly induced TGF{3 expression within 6 hours of application. These observations were confirmed by in situ hybridization that showed marked induction ofTGF{3 mRNA localized predominantly in the immediate suprabasallayer of the epidermis. Summary
Transforming growth factor {3 is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF{3 in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF{3 and TGFa undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.
161
TGF,B In Epidermal Proliferation
References 1. Goustin AS, LeofEB, Shipley GD, Moses HL: Perspectives in cancer research: growth factors and cancer. Cancer Res 46: 1015-1029, 1986. 2. Delarco JE, Todaro GJ: Growth factors from murine sarcoma virus· transformed cells. Proc Natl Acad Sci USA 75:40014005,1978. 3. Todaro GJ, Fryling C, DeLarco JE: Transforming factors produced by certain human tumor cells: polypeptides that interact with epidermal growth fact receptors. Proc Natl Acad Sci USA 77:5258-5262, 1980. 4. Sporn MB, Todaro GJ: Autocrine secretion and malignant transformation of cells. N Engl J Med 303:878-880, 1980. 5. Keski·Oja J, Leof EB, Lyons RM, Coffey RJ, Moses HL: Transforming growth factors and control of neoplastic cell growth. J Cell Biochem 33:95-107, 1987. 6. Coffey RJ, Sipes NJ, Bascom CC, Graves-Deal R, Pennington CY, Weissman BE, Moses HL: Growth modulation of mouse keratinocytes by transforming growth factors. Cancer Res 48: 1596-1602, 1988. 7. Moses HL, Tucker RF, Leof EB, Coffey RJ, Halper J, Shipley GD: Type beta transforming growth factor is a growth stimulator and growth inhibitor, in Feramisco J, Ozanne B, Stiles C (eds): Growth Factors and Transformation, Cancer Cells 3. Cold Spring Harbor, New York, Cold Spring Harbor Press, 1985, pp 65-71. 8. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV: Human transform· ing growth factor fJ complementary DNA sequence and expression in normal and transformed cells. Nature 316:701-705, 1985. 9. Derynck R, Jarrett JA, Chen EY, Goeddel DV: The murine transforming growth factor-beta precursor. J Bioi Chem 261: 4377-4379, 1986. 10. Cheifetz S, Weatherbee JA, Tsang ML·S, Anderson JK, Mole JE, Lucas R, Massaque J: The transforming growth factorbeta system, a complex pattern of cross· reactive ligands and receptors. Cell 48:409-415, 1987. 11. Hanks SK, Armour R, Baldwin JH, Maldonado F, Spiess J, Holley RW: Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the eDNA. Proc Natl Acad Sci USA 85:79-82, 1988. 12. Derynck R: Transforming growth factor-alpha: Structure and biological activities. J Cell Biochem 32:293-304, 1986. 13. Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AZ, Gash DJ, Chow EP, Fisher A, Bertonis JB, Torres G, Wallner BP, Ramachandran KL, Ragin RC, Managanaro TF, MacLaughlin DT, Donahoe PK: Isolation of bovine and human genes for Miillerian inhibiting substance and expression of the human gene in animal cells. Cell 45:685-698, 1986. . 14. Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ying Y, Guillemin R, Niall H, Seeburg PH: Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta. Nature 318:659-663, 1985. 15. Weeks DC, Melton DA: A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGFfJ. Cell 51:861-867, 1987. 16. Lawrence DA, Pircher R, Krycene·Martinerie C, Jullien P: Normal embryo fibroblasts release transforming growth factors in a latent form. J Cell PhysioI121:184-188, 1984. 17. Lyons RM, Keski·Oja J, Moses HL: Proteolytic activation of latent transforming growth factor-beta from fibroblast conditioned medium. J Cell Bioi 106:1659-1665, 1988. 18. Cheifetz S, Like B, Massague J: Cellular distribution of type I and type II receptors for transforming growth factor fJ. J Bioi Chem 261:9972, 1986. 19. Massaque J: Subunit structure of a high affinity receptor for
162
type fJ transforming growth factor. Evidence for a disulfide linked glycosylated receptor complex. J Bioi Chem 260:70597066,1986. 20. Massaque J, Cheifetz S, Ignotz R: TGF-fJ receptors and actions. J Cell Bioi {SuppI12A):181, 1988. 21. Libby J, Mortinez R, Weber MJ: Tyrosine phosphorylation in cells treated with transforming growth factor-beta. J Cell Physiol129:159-166,1986. 22. Fine LG, Holley RW, Nasri H, Badie·Dezfooly B: BSC·l growth inhibitor transforms a mitogenic stimulus into a hyper· trophic stimulus for renal proximal tubular cells: Relationship to Na+ /H+ antiport activity. Proc Natl Acad Sci 82:6163-6166, 1985. 23. Leof EB, Proper JA, Goustin AS, Shipley GD, DiCorleto PE, Moses HL: Induction of c-sis mRNA factor by transforming growth factor fJ: a proposed model for indirect mitogenesis involving autocrine activity. Proc Natl Acad Sci USA 83:24532457, 1986. . 24. Shipley GD, Pittelkow MR, Willie JJ, Scott RE, Moses HL: Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum· free medium. Cancer Res 46:2068-2071, 1986. 25. Wakefield LM, Smith DM, Masui T, Harris CC, Sporn MB: Distribution and modulation of the cellular receptor for transforming growth factor beta. J Cell Bioi 105:965-975, 1987. 26. Knabbe C, Lippman ME, Wakefield LM, Flanders KC, Kasid A, Derynck R, Dickson RB: Evidence that transforming growth factor fJ is a hormonally regulated negative growth fac· tor in human breast cancer cells. Cell 48:417-428, 1987. 27. Masui T, Wakefield LM, Lechner JF, LaVeck MA, Sporn MB, Harris CC: Type beta transforming growth factor is the primary differentiation·inducing serum factor for normal human bronchial epithelial cells. Proc Natl Acad Sci USA 83:24382442,1986. 28. Kurokowa M, Lynch K, Podolsky DK: Effects of growth factors on an intestinal epithelial cell line: transforming growth factor fJ inhibits proliferation and stimulates differentiation. Biochem Biophys Res Commun 142:775-782, 1987. 29. Noda M, Rodan GA: Type fJ transforming growth factor (TGFfJ) regulation of alkaline phosphatase expression and other phenotype· related mRNAs in osteoblastic rat osteosarcoma cells. J Cell Physiol133:426-437, 1987. 30. Takehara K, LeRoy EC, Grotendorst GR: TGFfJ inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell 49:415-422, 1987. 31. Assoian RK, Frolik CA, Roberts AM, Miller DM, Sporn MB: Transforming growth factor fJ controls receptor levels for epidermal growth factor in NRK fibroblasts. Cell 36:35-41, 1984. 32. Like B, Massaque J: The antiproliferative effect of type fJ transforming growth factor occurs at a level distal from recep' tor for growth activating factors. J Bioi Chem 261:1342613429, 1986. 33. Coffey RJ, Bascom CC, Sipes NJ, Graves-Deal R, Weissman BE, Moses HL: Selective inhibition of growth related gene ex· pression in murine keratinocytes by transforming growth factor beta. Mol Cell Bioi, in press. 34. Ignotz RA, Massaque J: Transforming growth factor fJ stimulates expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Bioi Chem 261:43374345,1986. 35. Keski-Oja J, Blasi F, Leof EB, Moses HL: Regulation of the synthesis and activity of urokinase. plasminogen activation in A549 human lung carcinoma cells by transforming growth factor fJ. J Cell Bioi 106:451-459, 1988. 36. Laiho M, Saksela 0, Keski-Oja J: Transforming growth factor fJ alters plasminogen activator activity in human skin fibro· blasts. Exp Cell Res 164:399-407, 1986. 37. Feng P, Catt KJ, Knecht M: Transforming growth factor fJ reg-
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38.
39.
40.
41.
ulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation. J Bioi Chern 261:14167-14170, 1986. Matrisian LM, Leroy P, Ruhlmann C, Gesnel MC, Breathnach R: Isolation of the oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts. Mol Cell Bioi 6:1679-1986, 1986. Postlethwaite AE, Keski-Oja J, Moses HL, Kang AH: Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta. J Exp Med 165:251-256, 1987. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine VI, Liotta LA, Falanga V, Kehrl H, Fauci AS: Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proe Nat! Aead Sci USA 83:41674171,1986. Mustoe TA, Pierce GF, Thomason A, Gramates P, Sporn MB, Deuel TF: Accelerated healing of incisional wounds in rats in-
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42.
43.
44.
45.
46.
duced by transforming growth factor-/1. Science 237:13331336,1987. Weissman BE, Aaronson SA: BALB and Kirstin murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines. Cell 32:599606,1983. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH: Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245-254, 1980. Coffey RJ, Derynck R, Wilcox IN, Bringman TS, Goustin AS, Moses HL, Pittelkow MR: Production and auto-induction of transforming growth factor-alpha in human keratinocytes. Nature 328:817-820, 1987. Akhurst RJ, Fee F, Balmain A. Localized production ofTGF/1 mRNA in tumor promoter-stimulated mouse epidermis. Nature 331:363-365, 1988. Leof EB, Proper JA, Getz MJ, Moses HL: Transforming growth factor type /1 regulation of actin mRNA. J Cell Physiol 127:83-88, 1986.
163
T
ransforming growth factors (TGFs) originally were named and operationally defined as polypeptides that induce morphologic transformation of fibroblastic cells in monolayer culture and stimulate colony formation in soft agar. I - 3 As a consequence of early work with TGFs, a new concept evolved to explain the uncontrolled proliferation characteristic of neoplastic cells. This concept was termed the autocrine hypothesis, which proposed that malignant cells secrete stimulatory polypeptide factors for which the cells themselves have functional surface receptors. 4 A large body of circumstantial and direct evidence has accumulated in support ofthe autocrine hypothesis as a mechanism for neoplastic cell growth. 5 More recently it has been suggested that TGFs like TGFa and TGF{1 also are involved in the growth and differentiation of normal cells,6 indicating that the autocrine hypothesis should be broadened to encompass a role for autocrine polypeptide growth factors in the modulation of normal growth. Much of this work has been performed in human and mouse keratinocytes. The present review summarizes the role of the growth inhibitory autocrine polypeptide TGF{1 in the regulation of normal keratinocyte growth and differentiation. Transforming Growth Factor fJ
TGF{1 is a 25 kD homodimer that is produced by a wide variety of normal and neoplastic cells. 7 Currently, two distinct molecular forms of TGF{1 have been identified. These molecules have been designated TGF{11 and TGF{12. Human and porcine TGF{11 are composed of two identical 12,500 MW subunits. Each monomer is synthesized as the COOH-terminal112 amino acids of a 391 amino acid precursor. s Phylogenetic comparisons with the human sequence show an exceptionally high degree of From the
* Departments of Cell Biology and Pediatrics and the
t Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee. Reprint requests: Dr. Harold L. Moses, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
conservation.9 Eighty of the 112 mature TGF{12 amino acids are identical to TGF{11, 10 and all nine cysteines are conserved. 10 In the precursor region only 33% of the aligned residues are identical. The growth inhibitor from the African green monkey kidney cell line, BSC-l, recently has been cloned and the cDNA sequence indicates that it is identical to TGF{12. 11 Although the confusing terminology would imply otherwise, transforming growth factor alpha (TGFa) bears no structural similarity to any of the TGF{1 molecules. TGFa shares structural homology and a cell surface receptor with epidermal growth factor (EGF).12 This growth stimulatory autocrine polypeptide will not be discussed extensively in this review. The TGF{1s belong to a larger family of related polypeptides with structural and sequence homology, most of which possess growth regulatory functions. These include (among others) Mullerian inhibitory substance, a protein that induces regression of the Mullerian duct system in the developing male genitourinary system,13 inhibins (and their {1 chain dimers, activins),I4 and Vg-l, a protein produced in Xenopus that promotes mesodermal induction. 15 TGF{1 is secreted by cells in culture in an inactive, latent form that is irreversibly activated by nonphysiologic extremes ofpH. 16 Potential physiologic mechanisms of activation include the proteases plasmin and cathepsin D that appear to activate at least a portion of TGF{1 released in culture. 17 Identification of other possible cell or tissue-specific mechanisms for activation of latent TGF{1 will be of major importance in understanding regulation and specificity of TGF{1 action, as cell surface membrane receptors appear to be ubiquitously distributed. Three structurally distinct, glycosylated TGF{1 receptors have been identified in affinity cross-linking and photoaffinity labeling studies. 1s-2o The type I receptor is 65 kD and is found in most cell types examined. The type II receptor has a molecular weight of 95 kD (human and monkey cells) or 85 kD (other mammalian cells). The type III receptor contains a 280-330 kD ligand binding subunit that is associated with a larger (560-600 kD disulfide linked receptor 159
TGF.B In Epidermal
P~ollferation
TABLE 1 TGFfj Effects on Gene Expression Fibroblastic cells
Epithelial cells
Enhanced expression:
Enhanced expression: c-sis (unpublished) {3-actin 33 EGF receptor (unpublished)
C-SiS23 C-fOS 23
c_myc 23 KC (unpublished) JE 23 ~-actin46
"Y-actin46 type I procollagen 34 fibronectin 34 PAI_1 35.36 TGF~ (unpublished) Decreased expression: transin 36 U_PA35
U-pN5 PAI-1 35 TGF~ (unpublished) No effect:
C-fOS33
Decreased expression: c-myc 33 KC 33 Not expressed: JE 33
complex. 19 The type III receptor demonstrates high affinity for TGF,61 and TGF,62 whereas the type I and II receptors demonstrate a tenfold higher a~nity for ,61 than for TGF,62. Preliminary data suggest that each TGF,6 receptor type mediates a distinct biologic response. 20 The intracellular signaling mechanism activated by TGF,6 receptor interaction has not been elucidated. The TGF,6 receptor has no intrinsic tyrosine kinase activity21 and Fine et al 22 showed no alteration in Na+ /H+ exchanger activity in primary proximal renal tubular cells by the BSC-1 growth inhibitor (TGF,62). The biologic response to TGF,61 is cell-type specific and remarkably complex. In fibroblastic cells, TGF,6 appears to stimulate growth by an indirect mechani'sm. 23 Treatment of monolayer cultures of AKR-2B cells with TGF,6 caused a rapid induction of c-sis mRNA, followed by the appearance of platelet-derived growth factor (PDGF) radioreceptor competing material in the culture medium. Thus, TGF,6 may stimulate growth indirectly via the mitogenic polypeptide PDGF. By contrast, TGF,6 is a potent growth inhibitor in all normal epithelial cells studied. The growth of many neoplastic epithelial cells also is inhibited by TGF,6; however, Shipley et al 24 showed that clonal growth of the squamous cell carcinoma line, SCC-25, was not affected by the addition of TGF,6. 1251-TGF,6 binding studies revealed the absence of specific binding to SCC-25 cells, leading to speculation that absence of the negative control mechanism for growth contributed to the malignant phenotype. A large body of additional data also supports a pivotal role for TGF,6 in the regulation of neoplastic growth. For example, human A549 lung carcinoma cells ap-
160
pear unable to activate latent TGF,6,25 and the estrogen responsive human breast cancer cell line, MCF-7, demonstrates a decrease in TGF,6 protein production when treated with growth stimulatory concentrations of 17,6-estradio1. 26 The precise mechanism by which TGF,6 inhibits epithelial cell growth remains largely unknown. Inasmuch as the effect is reversible, the growth inhibitory properties ofTGF,6 do not appear to result from cytotoxicity. In a few selected cell types TGF,6 induces differentiation. 27-29 However, this is not a generalized phenomenon. In endothelial cells, TGF,6 inhibits epidermal growth factor (EGF)-induced expression of cmye, a nuclear protooncogene that is associated with cell proliferation. 30 In addition, the number of highaffinity EGF receptors was decreased in endothelial cells treated with TGF,6. EGF receptor density also was decreased 4 to 6 hours after treatment of NRK fibroblasts with TGF,6.31 Studies by Coffey6 and others32 have not demonstrated an effect of TGF,6 on EGF receptor-ligand interaction in cells of epithelial origin. Data from our laboratory indicate that expression of competence genes such as KC and c-mye is decreased rapidly (within 1 hour) in keratinocyte monolayers treated with TGF,6.33 This appears to be a selective phenomenon as expression of ,6-actin was increased and c-fos mRNA levels remained unchanged. The diverse and complex manner in which TGF,6 affects expression of genes relevant to growth, differentiation and extracellular matrix formation is summarized in Table 1. 23 ,34-38 Many of the effects appear to be unrelated to proliferation and are cell-type specific. The effect of TGF,6 on extracellular matrix formation 34 -36 coupled with the observations that TGF,6 is chemotactic for fibroblasts 39 and promotes angiogenesis 40 suggests a potential role in wound healing. Indeed, recent in vivo studies show that TGF,6 accelerates healing of incisional wounds in rats. 41 Undoubtedly, additional studies will further delineate the role of this molecule in the regulation of normal cell growth, embryogenesis, and in the process of cellular transformation. The Role of TGF~ in Keratlnocyte Growth
Much of the current understanding of the role of TGF,6 in the control of epithelial cell growth is derived from studies using normal keratinocytes. The addition of TGF,6 to cultures of proliferating human foreskin proketatinocytes caused a marked reduction in [3H]-thymidine labeling within 24 to 48 hours. 7 Clonal growth assays demonstrated that this growth inhibitory effect persisted for 10 days.24 Inhibition was first detectable at a TGF,6 concentration of 1 ng/ ml (40 pM), whereas in concentrations of more than 10 ng/ml, essentially no clonal growth was detectable. When growth-arrested normal prokeratinocytes in
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monolayer were replated in clonal growth assays without TGF{3, no inhibition was noted, indicating the reversibility of growth arrest. Flow microfluorimetric studies showed that the majority of cells arrested in the G 1 phase of the cell cycle. 24 Weissman et al 42 showed that addition of epidermal growth factor (EGF) to primary cultures of BALB/c mouse epidermal cells resulted in establishment of a continuous, homogeneous cell line (BALB/MK) with an absolute requirement for EGF supplementation. These cells represent a useful model system for the study of epithelial cell growth control as they retain the capacity for differentiation in response to added extracellular calcium. 42•43 TGF{3 reversibly inhibited BALB/MK growth whereas total protein synthesis and RNA synthesis were unaffected. 6 TGF{31 and TGF{32 have been found to be equally effective in inhibiting proliferation in the BALB/MK cells (unpublished data). Quiescent cells that were restimulated with EGF showed a marked decrease in the percent of nuclei labeled with 3H-thymidine when 10 ng/ml TGF{3 was added concomitant with EGF restimulation. A potential mechanism for TGF{3 inhibition of EGF stimulation was investigated using 125I_EGF binding studies, but no alteration in EGF binding or internalization was found, suggesting that the TGF{3 effect occurred distal to EGF ligand-receptor binding or independent of EGF pathways. TGF{3 did not induce morphologic alterations typical of terminal differentiation of keratinocytes, and the differentiation markers pemphigus vulgaris antigen, filaggrin, or desmoplakin were not induced. Both morphologic and biochemical features of differentiation were detected in cells treated with 1.5 mM calcium for 24 hours. Thus, TGF{3 appeared to uncouple inhibition of proliferation and induction ofterminal differentiation. It could be theorized that autocrine TGF{3 production in the skin functions to maintain a nonproliferating pool of keratinocytes in the basal layers of the epidermis. This pool may serve as a reservoir for recruitment back into a proliferative compartment in response to a growth stimulatory signal. Coffey et al6 demonstrated TGF{3 gene expression by Northern blot analysis in BALB/MK cells and latent TGF{3 protein production by radioreceptor assay of serum-free BALB/MK conditioned medium. Unpublished experiments have demonstrated the presence of a high affinity (8.5 pM) class of TGF{3 receptors on BALB/MK cells, with a receptor density of 7000 binding sites/cell (unpublished data). Additional unpublished data show that TGF{31 treatment of rapidly growing BALB/MK cells results in an "autoinduction" of TGF{31 gene expression. A similar phenomenon has been described for TGFa in human keratinocytes. 44 Both EGF and TGFa induce TGFa gene expression and protein synthesis by human foreskin keratinocytes. These studies suggest
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Figure 1. TGFp In the control of epidermal prollferallon. A growth Inhibitory TGFp autocrlne loop Is shown. TGFp decreases expression of the competence genes c-myc and Ke, a potenllal mechanism for TGFP-medlated growth Inhibition. TGFp also Induces Its own gene expression (autolnductlon). This may funcllon as a signal ampllHcalion mechanism. Acllvallon of TGFp Is required before receptor/ligand Interacllon.
that autoregulation of proliferation could be responsible for amplification of the growth factor response, a mechanism that may be relevant to pathophysiologic situations like wound healing. These studies show that all of the requisite elements for a TGF{3 autocrine growth control mechanism exist in normal keratinocytes. Similar observations have been reported for transforming growth factor a in human keratinocytes 44 and in BALB/MK cells. 6 Evidence for the relevance of these in vitro studies to in vivo epidermal homeostasis is accumulating. For example, TGFa mRNA was demonstrated in histological sections of neonatal foreskin by in situ hybridization, and TGFa protein was demonstrated by immunocytochemistry.44 Recently, TGF{3 mRNA was detected in the mouse epidermis by Northern blot analysis of RNA samples obtained from control and tumor promotor (12-tetra-decanoyl-phorbol-13-acetate, TP A) treated skin. 45 The control levels were quite low, but TPA treatment markedly induced TGF{3 expression within 6 hours of application. These observations were confirmed by in situ hybridization that showed marked induction ofTGF{3 mRNA localized predominantly in the immediate suprabasallayer of the epidermis. Summary
Transforming growth factor {3 is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF{3 in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF{3 and TGFa undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.
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TGF,B In Epidermal Proliferation
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