Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.

Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.

Growth, morphology, function, and morphogenetic properties of rat renal glomerular epithelial cells in vitro: effects of retinyl acetate.

We report here that retinyl acetate (RA) regulates growth, morphology, function and cell organization in rat renal glomerular epithelial cells (SGE1). SGE1 cells are able to grow in a serum-free medium (DHFs medium) which is supplemented with insulin, transferrin, selenium, bovine serum albumin (BSA), linoleic acid and epidermal growth factor (EGF). When 0.1 and 1 micrograms/ml RA were added to the medium, the growth rates in the sparse culture were noticeably increased, compared to those in DHFs alone, whereas more than 10 micrograms/ml RA was cytotoxic to the cells. In the confluent culture, addition of 0.1, 1.0 and 10 micrograms/ml RA prolonged the cell survival. Since 10 micrograms/ml RA is not cytotoxic to the confluent culture, the cytotoxic action of RA seems to be dependent on cell density as well as RA dose. Ultrastructural observation revealed that RA treatment caused an increase of microvilli and alteration of cell shape, from flattened to columnar. Biochemical and immunological studies revealed that RA treatment increased the activity of r-glutamyl transpeptidase (GGT) and an amount of the membrane component with molecular mass (Mr) of 108,000 which is identical to one of nephritogenic antigens, Fx1A. By using fluorescence phalloidin stain, it was found that RA treatment increased content and organization of F-actin fibers. Furthermore, in collagen-embedding culture, RA induced 3-dimensional (3D) growth of SGE1 cells leading to the formation of organoids, cystic spheres with central lumen, in a serum-free condition; the addition of DHFs to collagen gel alone was ineffective for the 3D growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro.

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl(3)) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 µg/ml, whereas a 10-fold excess of iron concentration was required with FeCl(3), Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.

Fatty acid composition of ganglioside GM3 of renal glomerular epithelial SGE1 cells during spontaneous dome formation in vitro.

Ganglioside GM3, one of acidic components of membrane glycosphingolipids (GSL), has been known to change its content quantitatively during growth and differentiation of various cells in vitro. Detailed analysis of lipid portion of GM3 of rat renal glomerular SGE1 cells revealed that fatty acids with long carbon chains, especially that of C24:0 and C24:1 increased, while that of short C18:0 and C20:0 decreased after spontaneous dome formation. Since not only fatty acid composition of neutral GSL, sulfatide and phospholipid but also composition of long-chain bases (LCB) did not change, it was suggested that only C24 fatty acid of GM3 specifically increased in relation to dome formation. The spontaneous dome formation has been reported to be related with induction of cellular differentiation in many transporting epithelial cells. We thus assume that the change of fatty acid composition of GM3 is involved in cellular differentiation of SGE1 cells.

Rapid and severe proteinuria induced in rats by antibody against cultured rat glomerular epithelial cells.

The aim of this study was to investigate the role of glomerular visceral epithelial cell (GEC) antigens in the induction of proteinuria. An antibody (anti-GECIg) was produced by rabbit immunization with membrane extracts prepared from a clonal GEC line, SGE1, from the isolated rat renal glomeruli. A one-shot intraperitoneal injection of anti-GECIg (day 0) induced rapid and severe proteinuria in rats, reaching a peak on day 2 (mean value, 425 mg/24h), then gradually decreased to normal levels by day 10. By direct immunofluorescence, fine granular binding of anti-GECIg along the glomerular capillary walls was observed within 24 h. Electron microscopy and immunoelectron microscopy revealed GEC alterations consisting of extensive retraction of the foot processes, vacuolar changes in the cytoplasm, microvillous transformation of the cell surface, and binding of anti-GECIg to the surface of the foot process, mainly to its soles. Two major components with Mr of 108,000 and 39,000 were identified by immunoblotting assay using SGE1 cell membrane extracts and anti-GECIg. This model of rat nephropathy induced by anti-GECIg hopefully will lead to a greater understanding of the mechanism by which proteinuria is induced and developed.

Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats.

Injection of isologous monoclonal antibodies (SR2, SR3) caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY/NCrj rats. The antibodies were obtained from hybridoma cells derived from fusion of the spleen of a nephritic WKY/NCrj rat injected with rat solubilized renal basement membranes with adjuvant, and mouse SP2-myeloma cells. They belonged to the rat IgG2a subclass and bound to rat kidney in a linear pattern along the glomerular and tubular basement membranes. Histological changes in glomeruli were detected at day 1 after the injection; proteinuria with haematuria appeared on day 2; and proteinuria became severe and reached a plateau by day 5. These results demonstrate that anti-GBM nephritis can even be induced by an isologous monoclonal antibody and that the rat IgG2a subclass is at least nephritogenic. The experimental model of anti-GBM nephritis with isologous monoclonal antibodies makes it possible and easier to analyse further the mechanism of anti-GBM nephritis.