Regulation of Activity Levels of Glycolipid Sulfotransferases

Accumulation of sulfolipids associated with markedly elevated levels of glycolipid sulfotransferase activities was previously demonstrated in human renal cell carcinoma cells. To explore the regulation mechanisms of sulfoglycolipid synthesis in renal cancer, effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated by using a human renal cell carcinoma cell line, SMKT-R3. Among the growth factors tested, transforming growth factor a (TGF-a) and epidermal growth factor 0EGF) were found to increase the sulfotransferase activity markedly (about 300%), but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. The augmented effects of TGF-ot was abolished by cycloheximide. Since TGF-a is known to bind to the same receptor as EGF, SMKT-R3 cells were investigated for the EGF receptor by affinity cross-linking with IzSI-EGF. A radiolabeled protein with a molecular mass of 175 kDa corresponding to the ligand-receptor complex was immunoprecipitated with a monoclonal anti-EGF receptor antibody. When production of the growth factors was examined immunochemicaily, the cells were found to secrete TGF-a at a low level and retain it in a membrane-bound form, whereas EGF was not detected. These observations suggest that the sulfotransferase activities are regulated through the autocrine, paracrine, and/or juxtacrine modes of intercellular stimulation by TGF-a in human renal cancer cells. I N T R O D U C T I O N Structural modifications of carbohydrates on cell surface glycolipids appear to be implicated in the genetically programmed process of tissue differentiation and neoplastic transformation (1). Synthesis of abnormal glycolipids is due at least in part to aberrant expression of the responsible glycosyl transferases. Sulfoglycolipids were found to increase in human cancer tissues originating from several organs (2-9). In particular, the activity levels of glycolipid sulfotransferases, which catalyze the transfer of sulfate from PAPS 2 to galactose on GalCer and LacCer (10, 11), were markedly elevated in human renal cell carcinoma tissue, resulting in the accumulation of sulfoglycolipids (8). Furthermore, the levels of sulfotransferase activity were significantly increased in sera from patients with renal cell carcinoma compared to that of normal serum (12). In our recent study, a series of cell lines established from human renal cell carcinoma was found to possess high activity levels of sulfotransferases (13), confirming our previous observations on tumor tissues (8). This has enabled us to investigate the regulatory mechanism of the sulfotransferase activities at the cellular level. Various growth factors and growth factor receptors have been implicated in carcinogenesis of tumor cells (14, 15). TGF-c~ is a secreted Received 7/12/93; accepted 9/30/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Kita-ku N15 W7, Sapporo 060, Japan. 2 The abbreviations used are: PAPS, 3'-phosphoadenosine-5'-phosphosulfate; GalCer, galactosylceramide; LacCer, lactosylceramide; EGF, epidermal growth factor; TGF-ct, transforming growth factor or; PDGF, platelet-derived growth factor; IL-6, interleukin 6; IGF, insulin-like growth factor; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DSS, disuccinimidyl suberate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay. polypeptide that interacts with the same receptor as EGF and induces a mitogenic response in a wide variety of cells (16, 17). The EGF receptor gene, which is a cellular protooncogene c-erbB, encodes a 170-kDa transmembrane glycoprotein with intracellular tyrosine kinase activity and an extracellular binding site for EGF and TGF-ot (18, 19). Increased expression of TGF-a and the EGF receptor have been demonstrated in a variety of human cancer cell lines and tumor specimens, including renal cell carcinoma (20--29). In addition, it was demonstrated that proliferation of human renal cell carcinoma cells was regulated by endogenously produced TGF-o~ through the EGF receptor (30). These findings suggest that TGF-ot contributes to the growth and characteristics of renal cancer cells by an autocrine mechanism. Therefore, we attempted to evaluate whether TGF-a participates in the regulation of glycolipid sulfotransferase activities in human renal cancer cells. M A T E R I A L S A N D M E T H O D S Materials. [35S]PAPS (1.5 Ci/mmol), and Na125I (17 Ci/mg) were purchased from New England Nuclear. Unlabeled PAPS and p-nitrocatechol sulfate were from Sigma, DSS was from Pierce, mouse EGF was from Collaborative Research, a monoclonal anti-human EGF receptor mouse IgG was from Amersham, human recombinant TGF-o~ was from GIBCO BRL, human recombinant IL-6 was from Boehringer Mannheim, human recombinant PDGF-BB was from Oncogene Science, human recombinant insulin was from Shionogi Pharmaceuticals, human recombinant IGF-I was from Genzyme, and a monoclonal anti-human TGF-ct mouse IgG and human recombinant IGF-II were from Wakunaga Pharmaceuticals. Other reagents were of analytical grade. Cell Culture. SMKT-R3 cells were established from human renal cell carcinoma as described previously (31), and were cultured in Dulbecco's modified minimal essential medium supplemented with 10% fetal bovine serum. When effects of growth factors were examined, cells grown to subconfluence were washed 3 times with serum-free Dulbecco's modified minimal essential medium containing 0.1% bovine serum albumin, and cultured in the same serum-free medium with growth factors for an appropriate period. Cell viability was estimated by the trypan blue exclusion test, and was always greater than 95%. Enzyme Assay of Renal Cell Carcinoma Cells. Activities of glycolipid sulfotransferases and arylsulfatase A of SMKT-R3 cells were assayed as described previously (13). Briefly, cell monolayers were washed, harvested, and suspended in TBS containing 0.1% Lubrol PX, followed by sonication on ice. Glycolipid sulfotransferase activities of the cell homogenate were assayed by using GalCer and LacCer individually as substrates according to the previously reported method (32). Arylsulfatase A activity of the cell homogenate was assayed by using p-nitrocatechol sulfate as a substrate by the method of Baum et al. (33). To enable comparisons between the separate experiments, the measured enzyme activity was expressed as a percentage of the mean of the control value within each experiment. After the incubation of SMKT-R3 cells for 12 h under serum-free conditions with neither growth factors nor reagents, the specific activities (mean SD) of glycolipid sulfotransferase toward GalCer and LacCer, and arylsulfatase A were 8020 _+ 780 pmol/h/mg protein, 2960 _ 250 pmol/h/mg protein, and 220 _+ 18 nmol/h/mg protein, respectively. Immunohistochemical Staining. SMKT-R3 cells were cultured on Multitest slides (Flow Laboratories) for 12 h. The slides were immersed in PBS and fixed in 100% ethanol for 10 min, rinsed in PBS, and air dried. The cells were then reacted with a monoclonal anti-human TGF-a mouse IgG that does not react with EGF (34) for 1 h at 37~ After washing with PBS, the cells were incubated with a fluorescein isothiocyanate-conjugated F(ab')2 fragment of