Proteoglycan synthesis is increased in cells with impaired clathrin-dependent endocytosis

Alicia Llorente 3 Kristian Prydz 2 Mieke Sprangers 2 Grethe Skretting 3 Svein Olav Kolset 0 Kirsten Sandvig ) 1 3 0 Institute for Nutrition Research, University of Oslo , Box 1046 Blindern, 0316 Oslo , Norway 1 Author for correspondance ( 2 Department of Biochemistry, University of Oslo , Box 1041 Blindern, 0316 Oslo , Norway 3 Department of Biochemistry, The Norwegian Radium Hospital , Montebello, 0310 Oslo , Norway SUMMARY Overexpression of a GTPase deficient dynamin mutant in HeLa dynK44A cells causes a block in clathrin-dependent endocytosis. When endocytosis is inhibited, these cells incorporate higher levels of [35S]sulfate into both cellular and secreted macromolecules and larger amounts of proteoglycans such as syndecan and perlecan are immunoprecipitated from [35S]sulfate-labelled lysates. Gel filtration and ion-exchange chromatography revealed that the increased [35S]sulfate incorporation into proteoglycans was not due to significant differences in size or density of negative charge of glycosaminoglycan chains attached to proteoglycan core proteins. On the other hand, measurements of the syndecan-1 mRNA level and of [3H]leucine-labelled perlecan after immunoprecipitation Endocytosis of membrane, ligands and fluid occurs both by clathrin-dependent and - independent mechanisms (for review see Schmid, 1992; Smythe and Warren, 1991; Lamaze and Schmid, 1995; Sandvig and van Deurs, 1994; van Deurs et al., 1989). During the last years clathrin-dependent endocytosis has been extensively characterized. Also several approaches have been used to block this process, thus facilitating the study of clathrin-independent endocytosis, the endocytic pathway used by different molecules, and the role of clathrin-dependent endocytosis for the physiology of the cells. Initially, K+depletion (Moya et al., 1985) and cytosol acidification (Sandvig et al., 1987) were used to block clathrin-dependent endocytosis. More recently the 100-kDa GTPase dynamin was shown to play an important role in clathrin-dependent endocytosis (for review see Damke, 1996; De Camilli et al., 1995; Urrutia et al., 1997), and transiently and stably transfected cell lines overexpressing GTPase defective dynamin mutants are now commonly used to block this endocytic mechanism (Damke et al., 1994; Herskovits et al., 1993; van der Bliek et al., 1993). The role of endocytosis in signal transduction has recently been investigated in stably transfected HeLa cells where the overexpression of one of these mutants, dynK44A, is regulated by tetracycline. The results from these studies suggest that endocytosis is important supported the idea that the increased [35S]sulfate incorporation into proteoglycans was due to a selective increase in the synthesis of proteoglycan core proteins. Interestingly, the activity of protein kinase C was increased in cells expressing mutant dynamin and inhibition of protein kinase C with BIM reduced the differences in [35S]sulfate incorporation between cells with normal and impaired clathrin-dependent endocytosis. Thus, the activation of protein kinase C observed upon inhibition of clathrin-dependent endocytosis may be responsible for the increased synthesis of proteoglycans. not only to attenuate signalling, but also for certain aspects of the signalling process itself (Ceresa and Schmid, 2000). We have previously observed that HeLa dynK44A cells incubated with radioactive sulfate to label a modified ricin containing a sulfation site (Llorente et al., 1998) incorporate a larger amount of radioactive sulfate into high molecular mass molecules, presumably proteoglycans (PG)s, when clathrindependent endocytosis is impaired. However, the incorporation of sulfate into several newly synthetized proteins is unchanged. PGs are formed by addition of one or more glycosaminoglycan (GAG) chains to core proteins. The GAG chains are built of repeating dissaccharide units and are classified according to the nature of these units and by the degree and position of sulfation. PGs seem to be synthesized by all vertebrate cell types and have been found at cell surfaces, in vesicles, and in the extracellular matrix (for review see Hardingham and Fosang, 1992; Kjelln and Lindahl, 1991). These molecules have been ascribed a large variety of functions that are often mediated by electrostatic interactions of the GAG chains with other molecules such us growth factors, extracellular matrix molecules, or enzymes. In this paper we have investigated the nature of the high molecular mass molecules giving rise to increased incorporation of radioactive sulfate upon inhibition of clathrindependent endocytosis. We show that the synthesis of PG core proteins is increased when clathrin-dependent endocytosis is inhibited, whereas the total protein synthesis is not affected. Protein kinase C (PKC) regulates PG synthesis in a number of cell lines (Tao et al., 1997; Fagnen et al., 1999; Thibot et al., 1999), and this seems also to be the case in HeLa dynK44A cells. Expression of mutant dynamin and reduction of endocytosis in HeLa dynK44A cells leads to activation of PKC. Evidence is presented that PKC activation is responsible for the increased synthesis of PGs. MATERIALS AND METHODS Tetracycline, bovine serum albumin (BSA), puromycin, guanidine, Tris-HCl, bisindolylmaleimide (BIM), phorbol 12-myristate 13acetate (TPA) and Triton X-100 were from Sigma Chemical Co., St Louis, MO, USA. Chondroitinase ABC was purchased from Seikagaku Corp., Tokyo, Japan. Ba(NO2)2 was from Merck, Darmstadt, Germany. Na235SO4, [32P]dCTP, Na125I Sephadex G-50 Fine, Superose 6, and Protein A-Sepharose CL-4B were obtained from Amersham Pharmacia Biotech, Uppsala, Sweden. Geneticin was obtained from Saveen Biotech, Malm, Sweden. [3H]Leucine was from NEN Life Science Products, Boston, MA, USA. Econo-pac high Q Cartridges were from Bio-Rad Laboratories, Hercules, CA. Acidic fibroblast growth factor (FGF) was produced in bacteria, purified on a heparin-Sepharose column (Wiedlocha et al., 1996), and iodinated by the iodogen method (Fraker and Speck, 1978). Transferrin was iodinated by the same method. The HeLa cell lines stably transformed with the cDNAs for dynWT or dynK44A were kindly provided by Dr S. L. Schmid, The Scripps Research Institute, La Jolla, CA, USA (Damke et al., 1994). The cells were grown in Falcon (Franklin Lakes, NJ, USA) or Nunc (Naperville, IL, USA) flasks and maintained in DMEM (Flow Laboratories, Irvine, Scotland) supplemented with 10% FCS, 100 units/ml penicillin, 100 m g/ml streptomycin, 2 mM glutamine, 400 m g/ml geneticin, 200 ng/ml puromycin and 1 m g/ml tetracycline. BHK21 cells which in an inducible manner produce antisense mRNA clathrin heavy chain (CHC) (BHK21-tTa/anti-CHC) (G. Skretting, unpublished) were grown in DMEM supplemented with 7.5% FCS, 100 units/ml penicillin, 100 m g/ml streptomycin, 2 mM L-glutamine, 200 m g/ml geneticin, 200 ng/ml puromycin and 2 m g/ml tetracycline. For experiments, these cells were grown (...truncated)