Activation of Expression of Genes Coding for Extracellular Matrix Proteins in Tat-Producing Glioblastoma Cells J

Home , Tat (HIV)

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9617-9621, October 1992 Medical Sciences Activation of expression of genes coding for extracellular matrix proteins in Tat-producing glioblastoma cells J. PAUL TAYLOR*, CRYSTINA CuPP*, ARTURO DIAZt, MASHIUL CHOWDHURY*, KAMEL KHALILI*, SERGIO A. JIMENEZt, AND SHOHREH AMINI*t *Molecular Neurovirology Section, Jefferson Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107; and tRheumatology Research, Division of Rheumatology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107 Communicated by Darwin J. Prockop, July 10, 1992

ABSTRACT The Tat protein of human immunodeficiency ofthe infection and contributing to immunodeficiency and the virus type 1 has been increasingly implicated in directly con- formation of neoplasms. Considerable evidence has accumu- tributing to the disease AIDS by altering the expression of lated suggesting that Tat-mediated activation is responsible strategic cellular genes. In this study we demonstrate that the for some ofthe altered cellular gene expression seen in HIV-1 presence ofthe human immunodeficiency virus type 1 regulatory infection. Tat induces the expression oftumor necrosis factor protein Tat is associated with a significant induction in the ,( and transforming growth factor (3 (refs. 12 and 13 and C.C., expression of certain protein components of the extracellular unpublished data). Substantial quantities of Tat are released matrix in glial-derived cells. Northern blot analysis reveals that from HIV-1-infected cells, and this Tat can be taken up by in cells expressing Tat there is a marked elevation in the neighboring uninfected cells and thus exerts its functions in steady-state RNA levels for fibronectin and types I and mII these cells (14, 15). Tat specifically inhibits antigen-induced collagen. Metabolic labeling of the Tat-producing cells demon- proliferation ofuninfected peripheral blood lymphocytes (16). strates that this induction is also reflected at the level of protein Of great importance is the observation that Tat imparts a synthesis. Transient transfection experiments indicate that the growth stimulation to cell lines derived from Kaposi sarcoma presence of Tat results in increased transcription of fibronectin lesions in vitro (17). Further, transgenic mice expressing Tat and aI type I collagen promoters. Possible mechanisms for this develop dermal lesions similar to AIDS Kaposi sarcoma (18). phenomenon and their sigificance with regard to AIDS are These studies suggest a role for Tat in the pathogenesis of this discussed. particular common lesion in AIDS patients. Our interest lies in the regulation ofgene expression within the CNS. Noting that pathology within the CNS is a major The human immunodeficiency virus type 1 (HIV-1) is the component of HIV-1-associated disorders, we examined the etiologic agent of AIDS and has been directly implicated in influence of the Tat protein on cellular gene expression in a some of the clinical manifestations such as acquisition of an glial-derived cell line. We present detailed analysis of the immunodeficient state, neurological disorders, and the de- effect of Tat on the expression of components of the extra- velopment of malignancies (1-3). There is evidence for direct cellular matrix. infection by HIV-1 of a wide variety of cells including T and B lymphocytes, cells of the monocyte/macrophage lineage, MATERIALS AND METHODS neurons of the central nervous system (CNS), oligodendro- Cells, Plasmids, and Transfections. Cells (U87-MG from cytes, astrocytes, microglia, and endothelial cells (4-7). American Type Culture Collection) were maintained in Dul- Further, mounting evidence suggests that uninfected cells becco's modified Eagle's medium supplemented with 10% can be influenced at a distance by factors, both viral and (vol/vol) fetal bovine serum and plated in 60- or 100-mm cellular, released from infected cells (1, 4). dishes for 24 h prior to transfection. Plasmids pTat, pAct-Tat, In addition to the classic retroviral structural genes gag, pol, and pSVL-Tat contain the tat gene ofHIV-1 under the control and env, the HIV-1 genome encodes at least six regulatory of HIV-1 LTR, actin, and simian virus 40 late promoter genes (8). Among these, the regulator of expression of virion regions, respectively. Plasmid pCD15-CAT containing U3 protein (rev) and the transactivator (tat) genes are essential to and R regions of the HIV-1 LTR in front of the chloram- viral replication, and many studies have been directed at phenicol acetyltransferase (CAT) gene was kindly provided understanding the function of the encoded viral proteins and by Suresh Arya (National Cancer Institute/National Insti- how they contribute to AIDS. The Tat protein is a potent tutes of Health, Bethesda, MD). Plasmids pAC-171 (kindly activator of HIV-1 gene expression, by enhancing the rate of provided by Anne Olson, Lawrence Livermore Laborato- transcription from the viral long terminal repeat (LTR) (9). ries, Livermore, CA) and pO.6NF-CAT (kindly provided by Since production of Tat is augmented upon activation, it Suzanne Bourgeois, Salk Institute) contain 804- and 510- serves to establish a strong positive feedback loop that results base-pair fragments of human al type I collagen and human in a massive induction of HIV-1 gene expression and, conse- fibronectin upstream regulatory regions, respectively, cloned quently, further propagation of the infection (9). in front of the CAT gene. Plasmid pNeo is the neomycin- In addition to causing a direct cytopathic effect in infected resistance (neor) gene downstream from the bovine papillo- cells, HIV-1 infection results in the altered expression of mavirus promoter. Cells were transfected by the calcium strategic cellular genes (10, 11). Importantly, a number of phosphate coprecipitation method (19). Concentration of the immunoregulatory cytokines are released from infected cells transfected plasmid DNA was kept constant at 15 ,ug per in vitro and their serum levels are elevated in AIDS patients 60-mm dish and 30 ,ug per 100-mm dish with test plasmid plus (1, 4). The altered expression ofcellular factors is believed to be an integral part ofthe disease AIDS, facilitating the spread Abbreviations: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; CNS, central nervous system; CAT, chloram- The publication costs ofthis article were defrayed in part by page charge phenicol acetyltransferase; GAPDH, glyceraldehyde 3-phosphate payment. This article must therefore be hereby marked "advertisement" dehydrogenase; neor, neomycin resistance. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 9617 Downloaded by guest on September 27, 2021 9618 Medical Sciences: Taylor et al. Proc. Natl. Acad. Sci. USA 89 (1992) pUC19 plasmid DNA. Cell lines 3-8 and 3-10 express Tat under A control ofthe HIV-1 LTR. Cell lines 4-1, 4-4,4-9, 5-9, and 5-10 Cell lines express Tat under control of the simian virus 40 late promoter. 1-2 3-8 5-9 5-10 CAT Assay. All extracts were made 36-48 h after transfection and CAT enzyme assays were performed as described (20). RNA Preparation and Analysis. Total cellular RNA from two 100-mm plates was prepared by the hot acid/phenol procedure (21). RNA (20 /Lg) was electrophoresed in formaldehyde/agarose gels, transferred to nitrocellulose, and hybridized with cloned cDNA and DNA fragments (22, 23) B derived from a, type III and a, type I collagen, fibronectin, Cell lines \-ib a-;? lb.%Ha- r AN and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) labeled with [32P]dCTP by nick-translation. Protein Analysis. U87-MG and its derivatives were plated in 35-mm dishes to subconfluency. Cellular proteins were labeled with [14C]proline at 5 juCi/ml for 48 h (1 Ci = 37 GBq). Media were adjusted to final concentrations of the following components: 5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide, and 1 mM p-aminobenz- 1 2 3 4 5 6 amidine hydrochloride. The cell layers were detached and FIG. 1. Expression of HIV-1 Tat in transfected U87-MG glial sonicated in an ice-cold solution of 0.15 M NaCl/50 mM cells. (A) Visualization of Tat in neor Tat+ cells by indirect immu- Tris HCl, pH 7.4/protease inhibitors. The labeled proteins in nofluorescence. Cells were plated at low density in 35-mm dish and the medium and cell layers were analyzed by PAGE under after 16 h were prepared for indirect immunofluorescence with an reducing conditions. For collagen analyses, aliquots of pooled anti-Tat monoclonal antibody. (B) Biological activity ofTat produced media and cells were processed by limited digestion with in glial cells. Cells were plated in 60-mm dish and 16 h later were pepsin to remove the terminal propeptides of procollagen transfected with 0.5 jig of pCD15-CAT by the calcium phosphate (24) and then resolved on SDS/PAGE. method (19). The final amount of DNA was brought to 15 ,ug with molecules as described pUC DNA. Extracts were prepared 48 h after transfection and Immunofluorescence. Clonal U87-MG cells grown in 35-mm analyzed for CAT enzyme activity during a 60min reaction. dishes were rinsed, dried, and fixed with acetone. The cells were incubated with anti-Tat monoclonal antibody (1269-1.2 First, we examined the expression of the fibronectin gene by obtained from Patricia D'Souza, AIDS Program, National In- Northern blot hybridization (Fig. 2). Total RNA from control stitute ofAllergy and Infectious Diseases, National Institutes of 1-6 cells (Fig. 2A, lane 1) and five selected Tat-producing Health), stained with fluorescein isothiocyanate-conjugated clones [3-8 (lane 2), 3-10 (lane 3), 4-1 (lane 4), 4-4 (lane 5), and second antibody, and observed by fluorescence microscopy. 4-9 (lane 6)] were analyzed on formaldehyde/agarose gels. RESULTS Simultaneous hybridization with a fibronectin cDNA and a cDNA for GAPDH revealed a substantial increase in fibro- Generation of Tat-Producing Glial Cell Lines. To examine nectin expression in the Tat-producing cells. The extent of the role of the HIV-1 Tat protein in altering the pattern of the Tat+ cellular gene expression, we developed a panel of glial cells the induction ofmRNA for fibronectin varied among constitutively expressing Tat. Two expression systems were clones, whereas the levels of control GAPDH transcripts employed to create a spectrum of cell lines that produce remained constant in the Tat+ and Tat- clones (Fig. 2). different amounts of Tat. ptat expresses the 72-amino acid Clone 3-8 demonstrated very strong Tat activity, showed form of Tat under the control of the HIV-1 LTR and is the greatest induction in fibronectin expression, and was expected to produce high levels of Tat as a result of positive selected for further analysis. The results shown in Fig. 3 feedback. pSVL-tat expresses the 72-amino acid form of Tat indicate significant induction in the steady-state levels of under the control of the simian virus 40 late promoter and is RNA for both al type I and al type III collagen when expected to produce lower levels of Tat. Each of these compared to 1-2 (another G418-selected Tat- control cell constructs was cotransfected with a plasmid expressing the line). This experiment also demonstrates the increase in neor gene (pNeo, under control of the bovine papillomavirus fibronectin expression relative to an additional Tat- cell line. promoter) into the glial cell line U87-MG and G418-resistant Extracellular Matrix Protein Biosynthesis in Tat-Expressing colonies were selected. Expression of the tat gene was Cells. To directly examine collagen protein synthesis in the examined in single-cell clones by direct visualization of the Tat-producing cell lines, two neor Tat+ clones (3-8 and 4-4) protein by indirect immunofluorescence using an anti-Tat and two neor Tat- clones (1-2 and 1-6) were metabolically monoclonal antibody (Fig. 1A). Substantial accumulation of labeled for 48 h with [14C]proline. Total cellular proteins and Tat is observed and its nuclear localization is evident. Dif- conditioned media containing total secreted proteins were ferences in intensity of staining suggest that different analyzed by SDS/PAGE and fluorography. Comparison of amounts of Tat protein are produced in different clones. The [14C]proline-incorporated proteins from these cells reveals function ofthe stably expressed Tat protein was examined by enhancement of a major high molecular weight band comi- transiently transfecting the newly established cell lines with grating with fibronectin and two additional bands that migrate an HIV-1 LTR-CAT reporter plasmid PCD15-CAT. HIV-1 in positions consistent with al type I and a2 type I procol- LTR transcriptional activity is increased in Tat+ cells, and lagen chains (Fig. 4A). To confirm the identity ofthe proteins the level of induction roughly correlates with the intensity of enhanced in Tat+ cells, the [14C]proline-labeled proteins from immunofluorescence staining, indicating that these cells are cell line 3-8 were subjected to limited pepsin digestion and the producing biologically active Tat (Fig. 1B). products were examined by SDS/PAGE (Fig. 4B). This result RNA Expression of Genes Encoding Extracellular Matrix demonstrates the complete conversion of the procollagen Proteins. Several intriguing features ofthe Tat-producing cell molecules to collagen al and a2 chains after limited pepsin lines were observed, in particular, a slower rate of growth, treatment and the marked increase in the amounts of these and the ability to attach and grow in uncoated plastic dishes. chains in the Tat+ cell lines. To understand these phenomena, experiments were designed To directly investigate the level of fibronectin synthesis, to examine the expression phenotype of these cell lines. 14C-labeled proteins from the medium and cell layers from Downloaded by guest on September 27, 2021 Medical Sciences: Taylor et al. Proc. Natl. Acad. Sci. USA 89 (1992) %19 A U87-MG | Ieol neo ,Tat+ I

FIG. 2. Levels offibronectin gene

Fibronectin ** . expression in Tat-producing glioblas- i ... toma cells. (A) Total RNA (20 jzg) B from control neor (lane 1) and five Tat-producing (lanes 2-6) clones was fractionated by electrophoresis in a 1.0%1 agarose gel and transferred to a nitrocellulose membrane. The blot was hybridized to 32P-labeled cDNA GADPH- * fragments derived from fibronectin cDNA and GAPDH. (B) Densitomet- ric analysis ofbands corresponding to a fibronectin in control neor (bar 1) and five neor Tat+ cells (bars 2-6). The intensity of the bands corresponding to fibronectin RNA in various cell lines was normalized to GAPDH and rated arbitrarily on a scale of 0-150 adsorption units (ADU). Bars: 2, 3-8 cells; 3, 3-10 cells; 4, 4-1 cells; 5, 4-4 1 2 3 4 5 6 1 2 3 4 5 6 cells, 6, 4-9 cells. Tat- (1-2) and Tat' (3-8) cell lines were examined by SDS/ measured. The results demonstrate a dose-dependent increase PAGE. The results reveal that the level of fibronectin is in expression ofthis collagen promoter in response to Tat (Fig. increased in the Tat-producing cells (Fig. 4C). The identity of 5B). Similarly, the reporter plasmid p0.6FN-CAT containing fibronectin band was confirmed by immunoprecipitation us- 510 nucleotides of the 5' flanking sequences ofthe fibronectin ing human fibronectin antibody (results not shown). gene was examined for a transcriptional response to Tat. The Transcription of a, (I) Procoflagen and Fibronectin Genes. results demonstrate that fibronectin gene transcription is also Comparison of the Tat- and Tat+ cell lines clearly demon- increased in a dose-dependent manner in response to Tat (Fig. strates that the protein and mRNA levels of specific compo- SA). These results demonstrate that in the presence ofTat the nents ofthe extracellular matrix are increased in the cell lines transcriptional activity of these promoters is greatly induced; producing Tat. As each of these Tat+ cell lines represents an explaining, in part, the increased expression of these genes in isolated clone, one cannot rule out the possibility that matrix- Tat-producing cells. enriched clones were selected by chance. However, this seems unlikely since several control Tat- clones were used for DISCUSSION comparison (1-2 and 1-6 cells). Further, the observed increases In this report we have shown that the Tat protein of HIV-1 in collagen and fibronectin expression parallel the observed causes a significant increase in the expression of the extra- levels of Tat activity. However, to directly address this cellular matrix proteins fibronectin and types I and III question, we examined the transcriptional activity of the collagen when present in a glial-derived cell line. Further, the collagen and fibronectin promoters in the parental cell line results indicate that enhanced expression is mediated, at least U87-MG and evaluated the effect of Tat on transcription of in part, by transcriptional activation. It should be noted that these promoters. pAC-171 is a plasmid expressing the CAT the substantial enhancement seen in transcription of fibro- reporter gene under control of 804 nucleotides of 5' flanking nectin and collagen is reflected by an equal increase in sequences from the al type I collagen gene. This plasmid was steady-state RNA levels; however, the levels of the corre- transfected alone or cotransfected with a plasmid expressing sponding proteins are elevated to a lesser extent. This Tat into U87-MG cells, and the levels of CAT activity were suggests that posttranscriptional events, perhaps at the level

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.1 FIG. 3. Analysis of matrix- .fZ associated mRNAs in Tat-producing glial cells. RNAs from 1-2 (lanes 1) and 3-8 (lanes 2) cells were fractionated in formaldehyde/agarose gels, transferred to nitrocellulose, and hybridized to four cDNA probes [a1 type III, al type I, fibronectin, and control 0 (GAPDH)I. Lanes: 1, neor 1-2 1 2 1 2 2 1 2 1 2 cells; 2, neor Tat+ 3-8 clone. Downloaded by guest on September 27, 2021 %20 Medical Sciences: Taylor et al. Proc. NatL Acad Sci. USA 89 (1992) A B C c NIR, N'~'b1 N'9 'b' .,'~~~~C

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FIG. 4. Production ofcollagen and fibronectin. Subconfluent glial cells and Tat-producing glial cells were labeled with [14C]proline at 5 ,uCi/ml for 48 h and medium and cell layers were harvested. (A) Equal aliquots of media and cells were pooled and resolved by SDS/PAGE (7% gel) under reducing conditions. Lane 1 is a control reference obtained from normal human lung fibroblasts, illustrating the position of pro a, (1) and pro a2 (1) collagens and a major high molecular weight protein fibronectin that migrates above the collagens; lanes 2-5 are clones 1-2, 1-6, 3-8, and 4-4, respectively. (B) Samples of pooled media and cells containing equal cpm were digested with pepsin as described (24) and resolved by SDS/PAGE (7% gel) under reducing conditions. Lanes 1 and 2 are clones 1-2 and 3-8, respectively. Lane 3 is a control from normal human fibroblasts. (C) Clonal cell lines were labeled and extracted with 1 M urea and dialyzed. Aliquots of media (lanes 1 and 2) and cells (lanes 3 and 4) were separately sampled by SDS/PAGE (7% gel).

of translation or protein stability, may influence the net responsiveness of certain non-HIV-1 promoters (refs. 26 and output of the Tat-induced cellular genes. 27 and M.C., unpublished data). In addition, it has been It is unclear whether Tat functions directly at the promot- demonstrated that the cis-response element that binds the ers ofthe genes for flbronectin and the al chains oftype I and transcription factor NF-KB is capable of mediating Tat trans- type III collagen or whether the observed induction repre- activation in the appropriate cell type or after the appropriate sents a less-direct pathway. Within the HIV-1 LTR a se- stimulation (28, 29). The promoters ofthe genes forfibronectin quence termed the TAR (trans-activation responsive) ele- and the al chains of type I and type III collagen contain ment has been identified as a primary target for Tat (25). consensus binding sites for the transcription factor Spl (30, Recently, it has been shown that additional sequences within 31). Possibly, Tat induces transcription ofthese genes directly the viral promoter are involved in mediating transactivation through a Tat responsive element in their promoters. It seems by Tat. Specifically, the G+C-rich sequences capable of more likely, however, that enhanced expression of these binding transcription factor Spl mediate Tat transactivation matrix genes represents an indirect effect. Our results are in the HIV-1 promoter and may be responsible for the Tat consistent with a model in which Tat alters the expression of

FIG. 5. Transient transfection with m * Is collagen and fibronectin constructs by the calcium phosphate technique. pO.6- FN-CAT (A) and pAC-171 (B) (2.5 ,ug) a~~~~~~~~~~ were transfected alone (lane 1) or co- II-- transfected with 2.5, 5.0, and 10 jug (lanes I 2 3 4 2-4, respectively) of the Tat expression 1 2 3 4 vector pAct-Tat. The total amount of DNA transfected was made up to 15 ,ug B with pUC DNA. Forty-eight hours after D transfection, the cells were harvested and a crude protein extract was prepared by freezing and thawing. Equal amounts of protein (100 ,ug) were incubated for 1 h S.,., to assay for CAT activity. (C and D) Fold transactivation was quantified by mea- suring radioactivity by scintillation counting in spots cut from the TLC plate in A and B, respectively. Each experiment was replicated three times with independent plasmid preparations and 2 l 2 3 4 3 4 the results varied by <1%o. Downloaded by guest on September 27, 2021 Medical Sciences: Taylor et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9621 a limited number of cellular regulatory factors that in turn 7. Koenig, S., Gendelman, H. E., Orenstein, T. M., dal Canto, mediate the altered physiology of the cells. We (C.C., K.K., M. C., Pezeshkpour, G. H., Yungbluth, M., Janotta, F., Ak- and S.A., unpublished data) and others (13) have demon- samit, A., Martin, M. A. & Fauci, A. S. (1986) Science 233, strated the activation of transforming growth factor (3 expres- 1089-1093. sion by Tat. Because transforming growth factor is a potent 8. Haseltine, W. A. (1991) FASEB J. 5, 2349-2360. P3 9. Cullen, B. R. (1991) FASEB J. 5, 2361-2368. activator of matrix gene expression (32, 33), we speculate that 10. Merrill, J. E., Koyanagi, Y. & Chen, I. S. Y. (1989) J. Virol. 63, this factor mediates the activation of fibronectin and collagen 404 4408. expression. Interestingly, a transgenic mouse model of HIV- 11. Weeks, B. S., Klotman, M. E., Dhawan, S., Kibbey, M., associated nephropathy was recently reported in which an Rappaport, J., Kleinman, H. K., Yamada, K. M. & Klotman, important feature was accumulation of extracellular matrix P. E. (1991) J. Cell Biol. 114, 847-853. components (34). Although the authors could not attribute 12. Sastry, K. J., Reddy, R. H. R., Pandita, R., Totpal, K. & their observations to a particular viral factor, the tat gene was Aggarwal, B. B. (1990) J. Biol. Chem. 265, 20091-20093. among those introduced into the mice. 13. Lotz, M., Kekow, J., Cronin, M. T., McCutchan, J. A., Clark- The clinical course of AIDS is frequently complicated by Lewis, I., Carson, D. A. & Wachsman, W. (1990) FASEB J. 4, multiple CNS disorders (2, 35). There is a significant corre- 1861 (abstr.). between the level of HIV-1 expression in the brain and 14. Helland, D. E., Welles, J. L., Caputo, A. & Haseltine, W. A. lation (1991) J. Virol. 65, 4547-4549. clinical severity of the neurological complications (36-38). 15. Frankel, A. D. & Pabo, C. 0. (1988) Cell 55, 1189-1193. This strongly suggests that direct infection by HIV-1 or 16. Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. interaction with a viral product is responsible for the CNS (1989) Science 246, 1606-1608. pathology. 17. Ensoli, B., Barillari, G., Salahuddin, S. Z., Gallo, R. C. & Within the CNS, which is a large reservoir for HIV-1, Wong-Staal, F. (1990) Nature (London) 345, 84-86. microglial cells appear to carry the greatest viral load (2, 4, 18. Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A. & 36). It has been suggested that migration of microglial cells, Jay, G. (1988) Nature (London) 335, 606-611. which are derived from the monocyte/macrophage lineage, 19. Graham, F. L. & van der Eb, A. J. (1973) Virology 52,456-467. from peripheral blood to the brain is responsible for intro- 20. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. duction of HIV-1 to the CNS. Perhaps the presence of Tat in 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular certain tissues, such as the CNS, results in the production of Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold an extracellular matrix substrate that mediates the extravas- Spring Harbor, NY). cular dissemination of HIV-1. 22. Chu, M.-L., Myers, J. C., Bernard, M. P., Ding, J.-F. & The marked stimulation of expression of genes encoding Ramirez, F. (1982) Nucleic Acids Res. 10, 5925-5934. fibronectin and types I and III collagens in Tat-producing cells 23. Kornblihtt, A. R., Vibe-Pedersen, K. & Baralle, F. E. (1983) raises the possibility that a similar mechanism may be involved Proc. Natl. Acad. Sci. USA 80, 3218-3222. in the pathogenesis of certain idiopathic or autoimmune dis- 24. Bruckner, P. & Prockop, D. J. (1981) Anal. Biochem. 110, eases characterized by pathologic fibrogenesis such as sys- 360-368. 25. Rosen, C. A., Sodroski, J. & Haseltine, W. A. (1985) Cell 41, temic sclerosis or idiopathic pulmonary fibrosis. This remains 813-823. an attractive possibility, although no apparent transactivator 26. Berkhout, B., Gatignol, A., Rabson, A. B. & Jeang, K.-T. similar to Tat has been identified in those fibrotic diseases. (1990) Cell 62, 757-767. 27. Kamine, J., Subramanian, T. & Chinnadurai, G. (1991) Proc. We express our appreciation to Dr. Jay Rappaport for providing Natl. Acad. Sci. USA 88, 8510-8514. reagents and for his valuable suggestions, Dr. R. C. Gallo and 28. Harrich, D., Garcia, J., Mitsuyasu, R. & Gaynor, R. (1990) members of his lab for sharing materials, and Drs. Anne Olson and EMBO J. 9, 4417-4423. Suzanne Bourgeois for supplying collagen and fibronectin con- 29. Taylor, J. P. Pomerantz, R., Bagasra, O., Chowdhury, M., structs, respectively. We are grateful to Drs. Patricia D'Souza and Rappaport, J., Khalili, K. & Amini, S. (1992) EMBO J. 11, Gregory Milman of the Pathogenesis Branch, Division of AIDS, 3395-3403. National Institute of Allergy and Infectious Diseases, National 30. Dean, D. C., Bowlus, C. L. & Bourgeois, S. (1987) Proc. Natl. Institutes of Health, for providing anti-Tat antibody. We thank Kerry Acad. Sci. USA 84, 1876-1880. Bosley for his technical assistance, members of the Molecular 31. Bornstein, P., McKay, J., Morishima, J. K., Devarayalu, S. & Neurovirology Section for helpful discussion, and Cynthia Schriver Gelinas, R. E. (1987) Proc. Natl. Acad. Sci. USA 84,8869-8873. for preparing this manuscript. This work was supported in part by 32. Dean, D. C., Newby, R. F. & Bourgeois, S. (1988) J. Cell Biol. Grants AM-19616 and HL-41214 awarded by the National Institutes 106, 2159-2170. of Health to S.A.J. and Grants CA47996, A128272 (from National 33. Ignotz, R. A., Endo, T. & Massague, J. (1987) J. Biol. Chem. Institutes of Health), and RG2054-A-1, RG232-A-25 from National 262, 6443-6446. Multiple Sclerosis to K.K. and S.A. 34. Kopp, J. B., Adler, S. H., Klotman, M. E., Bruggeman, L. A., Dickie, P., Marinos, N. J., Eckhaus, M., Bryant, J. L., Not- 1. Rosenberg, Z. F. & Fauci, A. S. (1991) FASEB J. 5, 2382-2390. kins, A. L. & Klotman, P. E. (1992) Proc. Natl. Acad. Sci. 2. Price, R. W., Brew, B. J. & Rosenblum, M. (1990) in Immu- USA 89, 1577-1581. nologic Mechanisms in Neurologic and Psychiatric Disease, 35. Price, R. W., Brew, B. J., Sidtis, J. J., Rosenblum, M., ed. Waksman, B. H. (Raven, New York), pp. 269-289. Scheck, A. C. & Cleary, P. (1988) Science 239, 586-592. 3. Centers for Disease Control (1990) HIV/AIDS Surveillance 36. Rosenblum, M. L., Levy, R. M. & Bredesen, D. E. (1988) (Centers for Disease Control, Atlanta). AIDS and the Nervous System (Raven, New York). 4. Merrill, J. E. & Chen, I. S. Y. (1991) FASEB J. 5, 2391-2397. 37. Navia, B. A. & Price, R. W. (1987) Arch. Neurol. 44, 65-69. 5. Pumarola-Sune, T., Navia, B. A., Cordon-Cardo, C., Cho, 38. Weiser, B., Peress, N., La Neve, D., Eilbott, D. J., Seidman, E.-S. & Price, R. W. (1987) Ann. Neurol. 21, 490-496. R. & Burger, H. (1990) Proc. Natl. Acad. Sci. USA 87, 6. Wiley, C. A. & Nelson, J. A. (1988) Am. J. Pathol. 133,73-81. 3997-4001. Downloaded by guest on September 27, 2021