MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 5222-5228

Vol. 11, No. 10

0270-7306/91/105222-07$02.00/0 Copyright © 1991, American Society for Microbiology

Overexpression of Transforming Growth Factor-1 in Transgenic Mice Carrying the Human T-Cell Lymphotropic Virus Type I tax Gene SEONG-JIN KIM,'* THOMAS S. WINOKUR,' HY-DE LEE,' DAVID DANIELPOUR,1 KYUNG YOUNG KIM,' ANDREW G. GEISER,' LIAN-SHENG CHEN,2 MICHAEL B. SPORN,' ANITA B. ROBERTS,' AND GILBERT JAY2

Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892,1 and Laboratory of Virology, Jerome H. Holland Laboratory, American Red Cross, Rockville, Maryland 208552 Received

5

April 1991/Accepted 3 July 1991

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-celi leukemia patients express high levels of the type 1 isoform of transforming growth factor-" (TGF-,B1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-f1 promoter. To understand further the regulation of TGF-Ill expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-l mRNA and protein. Moreover, TGF-pl significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-Il may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-, in vivo. as

Adult T-cell leukemia is an aggressive, usually fatal T-cell malignancy. Human T-cell lymphotropic virus type I (HTLV-I) has been identified as the responsible infectious agent (26, 34). Clinical features include skin infiltration by the leukemic cells, hypercalcemia, and immunosuppression manifested by a high incidence of opportunistic infections. HTLV-I encodes a 40-kDa protein (Tax) (8) which is a potent transactivator both of transcription directed by the viral long terminal repeat (LTR) and of transcription of specific cellular genes such as those for tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor (12, 23), interleukin-2 (14, 19, 31), interleukin-3 (22), interleukin-2 receptor alpha chain (4, 14, 31), and c-fos (10). Recently, we demonstrated that the Tax protein also activates the gene for transforming growth factor-, type 1 (TGF-41) through distinct elements in the 5' regulatory sequences of this gene; these sites have been identified as tetradecanoyl phorbol acetate-responsive elements containing a functional binding site for the transcription factor AP-1 (16). Fresh leukemic cells from adult T-cell leukemia patients as well as HTLV-I-infected T-cell lines express the TGF-,1l gene at elevated levels and secrete TGF-p1 into the medium (17, 25), which suggests that such trans-activation may be physiologically relevant in the pathogenesis of disease mediated by HTLV-I. To further explore the mechanisms of Tax activation of TGF-pl expression in vivo, we analyzed the patterns of TGF-131 expression in transgenic mice carrying the HTLV-I tax gene under the control of the viral LTR (13, 24). Here we report that tumors from these mice as well as other tissues, such as submaxillary gland and muscle, which also express high levels of tax mRNA selectively express high levels of TGF-1l mRNA and protein.

*

MATERIALS AND METHODS Transgenic mice. Transgenic mice carrying the HTLV-I LTR tax gene have been described previously (24). RNA isolation and blotting. Total RNAs from mouse tissues and tumors were extracted by acid guanidinium thiocyanate-phenol-chloroform extraction (2). Total RNAs (10 pLg each) were separated by electrophoresis through 1% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Prehybridization, hybridization, and washing of the membranes were as described previously (3). Rat TGF-P1 (27), mouse TGF-P2 (6), glyceraldehyde-3-phosphate dehydrogenase, and tax probes were prepared by random-primed labeling of excised cDNA inserts. S1 nuclease protection assay. The S1 probe was generated by end labeling a 1.7-kb XbaI-BglII mouse promoter fragment with [32P]ATP and isolating the fragment from an agarose gel. Probe hybridization and Si nuclease digestion buffers and conditions were as previously described (11). Immunohistochemical analysis. Tissues were fixed in buffered formalin, postfixed for 6 h in Bouin's fixative, and embedded in paraffin by standard techniques. Five-micrometer sections were incubated with isotype-specific peptide antibodies (9), subjected to an avidin-biotin procedure (Vectastin Elite), reacted with diaminobenzidine in hydrogen peroxide, and counterstained with Mayer's hematoxylin. Measurement of TGF-fr1 and TGF-132. Mice (4-week-old males) were sacrificed by suffocation with carbon dioxide. The excised tissues were homogenized with 8 volumes of acid-ethanol (88 p.g of phenylmethylsulfonyl fluoride per ml, 5 ,ug of pepstatin A per ml, 211 mM HCl, 88% ethanol), and the homogenates were extracted overnight at 4°C with rocking. Extracts were centrifuged at 10,000 x g for 30 min, concentrated to 1/10 their volume in a Speedvac, diluted threefold with H20, and lyophilized for 48 h. Samples were then reconstituted in solubilization buffer (4 mM HCl, 0.5 mg of bovine serum albumin per ml, and 150 mM NaCl; 5 ml/g of original tissue), extracted overnight at 4°C, and then soni-

Corresponding author. 5222

EXPRESSION OF TGF-11 BY HTLV-I tax

VOL. 11, 1991

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cated. After centrifugation, supernatants were analyzed for TGF-,11 and TGF-,B2 by sandwich enzyme-linked immunosorbent assay (ELISA) (5). Cell proliferation. For thymidine incorporation assays, PX-1, PX-2, or PX-8 cells (12) were plated at 5 x 104 per 0.5 ml in 24-well Costar dishes. After 1 h, TGF-1 was added, and 22 h later cells were pulsed with 0.25 ,uCi of [methyl3H]thymidine (40 to 60 Ci/mmol; NEN) for 2 h at 37°C. Cells were then fixed with 1 ml of methanol-acetic acid (3:1, vol/vol). After a minimum of 1 h at room temperature, the wells were washed twice with 2 ml of 80% methanol. Label was extracted by a 30-min treatment with 0.5 ml of 0.05% trypsin, followed by the addition of 0.5 ml of 1% sodium dodecyl sulfate. [methyl-3H]thymidine incorporation was determined by liquid scintillation counting.

FIG. 1. Northern blot analysis of TGF-,B mRNAs in tissues from transgenic mice. RNA was extracted from tissues and tumors by acid guanidinium thiocyanate-phenol-chloroform extraction (2). Total RNAs (10 ,ug each) were separated by electrophoresis through 1% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Prehybridization, hybridization, and washing of the membranes were as described previously (3). Rat TGF-p1, mouse TGF-f32, and tax probes were prepared by random-primed labeling of excised cDNA inserts. The organ or tissue source of the RNA is denoted above each lane. (A) TGF-p1, TGF-P2, and tax mRNA in tissues of either normal or tax-transgenic 4-week-old male mice; (B) TGF-p1 and tax mRNA in tissues of 5-week-old male tax-transgenic mice; and (C) TGF-p1 mRNA in submaxillary gland and skeletal muscle from 3-week-old normal (lanes C) or tax-transgenic (lanes TAX) female and male mice. The bottom blots (labeled "rRNA") of panels A and B show an ethidium bromide stain of the RNA sample used for Northern analysis. (Note that ear and tail RNAs from tax-transgenic mice are derived from tumor tissues.) RESULTS

Transgenic mice expressing HTLV-I tax show selective expression of the tax gene in nerves, muscles, bones, and salivary glands. With increasing age, they develop proliferative disorders arising from perineural fibroblasts and salivary ductal epithelial cells and frequently develop tumors classified as neurofibromas on the ears, tails, and legs (13). Overexpression of the TGF-f1 gene in tissues and tumors from HTLV-I tax-transgenic mice. Levels of TGF-p1, TGF132, and TGF-13 mRNAs in different tissues and tumor samples from adult tax-transgenic mice (approximately 3 to 5 weeks old) of two founder series were evaluated by Northern (RNA) blot analysis (Fig. 1). In 4-week-old control mice, TGF-pl mRNA was expressed predominantly in femur, spleen, lung, and placenta tissues (data not shown for spleen, lung, and placenta tissues), while the levels of expression in brain, skeletal muscle, submaxillary gland, and skin tissues were low to undetectable (32) (Fig. 1A). In contrast, levels of TGF-p1 transcripts were significantly elevated in skeletal muscle and submaxillary gland tissues

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MOL. CELL. BIOL.

KIM ET AL.

from the transgenic mice tested, correlating well with the levels of tax expression in these tissues (Fig. 1A). The levels of TGF-31 mRNA in skeletal muscle were significantly increased in 5-week-old mice compared with levels in 4-week-old transgenic mice (Fig. 1B); this response suggests that TGF-pl might be associated with the development of skeletal muscle degeneration. The levels of TGF-1l expression in skeletal muscle of 5-week-old control mice were also low to undetectable (data not shown). We also examined the expression of TGF-1l mRNA in younger transgenic and control mice (approximately 3 weeks old) in which neurofibromas had not yet developed. Even at this early stage, increased expression of TGF-pl transcripts was detected in the submaxillary glands and skeletal muscles from both female and male transgenic mice (Fig. 1C), suggesting that activation of TGF-fi1 gene expression by Tax is an early event.

To determine whether Tax activation was specific for we also examined the expression of TGF-42 and TGF-f33 mRNAs. Although it has been reported that expression of TGF-P2 and TGF-P3 mRNAs is high in the male submaxillary gland (6, 20, 21), no increase in their expression was observed in any of the tissues or in the peripheral tumors from the transgenic mice (Fig. 1A and unpublished results). Interestingly, contrary to the observed increase in the level of expression of TGF-,1 mRNA, expression of the

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TABLE 1. Levels of TGF-,B1 and TGF-,2 peptides in normal and tax-transgenic mice measured by specific and sensitive sandwich ELISAs (5) Concn (ng/g of tissue) of a: Tissue and source

TGF-,B1

TGF-132

Tail Normal mice (skin) tax-transgenic mice (neurofibroma)

2.7 ± 0.1 29.0 ± 5.0