HIV-1 Transactivator Protein Tat Induces Proliferation and TGF Expression in Human Articular Chondrocytes

The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-/3 (TGFfl) and both inhibit lymphocyte proliferation. TGFfl is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGFfl and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF/L Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF~ and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF/~ showed that Tat increased synthesis and TGFfl activity and TGF/~I mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGFfl and by TGF~ antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGFfl. H U~AN immunodeficiency virus (HIV) ~ Tat is a virally encoded regulatory protein which stimulates HIV gene expression (8) through binding to a specific binding motif, tar, in the HIV promoter located in the long terminal repeat (LTR) (4). Tat is encoded by two exons and the mature protein contains 86 amino acids. Domains that are functionally important in HIV replication have been mapped to the first 72 amino acids encoded by exon 1 and include an acidic, a basic, and a cysteine-rich domain (9). The acidic NH2-terminal domain has been proposed to function as activation domain; the region between amino acids 22 and 37 contains 7 cysteine residues that are thought to mediate dimer formation and metal binding; and a basic domain between amino acids 49 and 57 is required for nuclear and nucleolar localization and binding to tar (5). The COOH-terminal 14 amino acids that are encoded by exon 2 do not appear to be essential in HIV replication but contain Address all correspondence to M. Lotz, UCSD School of Medicine, La Jolla, CA 92093-0663. 1. Abbreviations used in this paper: HIV, human immtmodeticiency virus; LTR, long terminal repeat. the RGD sequence which is a motif present in extracellular matrix proteins and involved with binding to cell surface adhesion receptors (2, 28). Tat requires interaction with cellular proteins to express its maximal effect on HIV replication and several intracellular proteins that participate in interactions with Tat have been identified (6, 14-18, 20, 23, 31, 32). Tat can activate the HIV promoter when it is synthesized in the same cell that contains the HIV LTR. In addition, a paracrine mechanism has been suggested that involves synthesis and secretion of Tat, binding to the cell surface, internalization and stimulation of the HIV LTR (12). A paracrine function of Tat may also be responsible for the development of Kaposi's sarcoma-like lesions in Tat transgenic mice which occurred in the absence of intralesional Tat-expressing cells (41). Furthermore, proliferation of Kaposi's sarcoma cells in vitro was increased by Tat (7). In contrast, growth-inhibitory effects have been reported in lymphocytes where Tat reduced antigen-induced T cell responses (38). Mechanisms responsible for these Tat effects are not completely characterized. Binding of Tat to cell surface antigens may be nonspecific, and adsorptive endocytosis has been suggested as a mechanism responsible for cellular uptake of Tat (10). However, Tat has also been shown to bind to specific cell surface receptors such as the otv/$5 integrin (39) and a © The Rockefeller University Press, 0021-9525/94/02/365/7 $2.00 The Journal of Cell Biology, Volume 124, Number 3, February 1994 365-371 365 on July 1, 2017 jcb.rress.org D ow nladed fom 90-kD protein which recognizes the region spanning amino acid residues 49-57 (42). Following active internalization, Tat can be detected in the cell nucleus. It may bind to promoters of cellular genes or possibly increase the DNA binding activity of cellular transcription factors and this may explain the activation or inhibition of cellular genes by Tat (13, 24, 25, 30, 33, 34, 44). HIV Tat and the cytokine TGF~ are qualitatively similar immunosuppressive factors and both inhibit lymphocyte proliferation (19, 38). One possible explanation for the antiproliferative effects in T ceils was that it induced the production of TGF/3 which is one of the most potent endogenous growth inhibitors for these cells (29). TGF/~ as a bifunctional regulator of cell function (26), is a potent stimulator of proliferation in human articular chondrocytes (11, 21). Three isoforms of TGF/3 are produced by chondrocytes and most other factors that can regulate chondrocyte proliferation have effects on the expression of TGF/3 (35, 36). These observations provided the basis for the hypothesis that an interaction of Tat and TGF/3 should be detectable as growth stimulation of Tat in chondrocytes. The present study shows that Tat and a peptide containing the basic domain are potent chondrocyte growth factors which act in part through the induction of TGF~. Materials and Methods Chondrocyte Isolation and Culture Primary human articular chondrocytes were isolated as described (37). Cartilage was obtained at autopsy and from the University of California, San Diego tissue bank from donors without known history of joint disease. For all experiments reported here, cartilage from the femoral condyles and tibial plateaus of the knee joints was used. Care was taken to obtain morphologically normal and full thickness cartilage and to avoid inclusion of subchondral bone. The cartilage surface was gently scraped with a scalpel to remove ceils from joint fluid potentially adhering to cartilage. The slices were washed with DMEM (Whittaker MAB Bioproducts, Walkersville, MD). They were cut into pieces (2-3 mm 3) and treated with trypsin (10% vol/vol) for 15 min in a 37°C waterbath. The tissues were transferred to DMEM containing 5 % FBS, penicillin-streptomycin-fungizone and 2 mg/ ml clostridial collagenase type IV (Sigma, St. Louis, MO) and digested for 3 h on a gyratory shaker until the tissue fragments were dissolved. The ceils were washed three times and cultured. For studies on TGF~ production, primary chondrocytes were cultured in T175 flasks for 24 h after isolation in DMEM 5% FBS. The cells were nonadherent at that time point; they were collected, washed two times in serum-free media, and plated in 96-well plates for studies on proliferation or TGF/~ production. Chondrocyte Proliferation Studies Cbondrocytes were plated in 96-well flat bottom tissue culture plates in DMEM containing 5 % FBS at 5,000 cells per well. Tat and growth factors were added and the cells were incubated for 96 h. Proliferation was determined by [3H]thymidine uptake during the final 12 h of culture. Cells were harvested on an automated cell harvester (Cambridge Tech, Watertown, MA) and incorporated radioactivity was quantified by liquid scintillation