Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors.

Abstract

Abstract. To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33°C) and restrictive (39.5°C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5°C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33°C respond only to isoproterenol (ISO, 10 -6 M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5°C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10 -7 M dDarginine vasopressin (dDAVP/ basal: 18.2, apparent Ka: 5 x 10 -9 M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (,050 fmol/10 ~ cells) which are undetectable when SV40 genome is activated (33°C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5°C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.

Matrix Metalloproteinase 2 (MMP2) and MMP9 Are Produced by Kidney Collecting Duct Principal Cells but Are Differentially Regulated by SV40 Large-T, Arginine Vasopressin, and Epidermal Growth Factor

“…Concentration-response experiments indicated that MMP9 induction or stimulation in the two populations of cells (33 and 39.5°C) occurred from 0.5 ng/ml (not shown). Cells cultured at the permissive temperature (33°C) did not express AVP receptors (16) and, as expected, the effect of AVP was only observed in differentiated cells (39.5°C) in which 10 Ϫ7 M AVP markedly inhibited MMP9 activity and antigen shown by zymography (Fig. 4A, lane 6) and Western blotting (Fig.…”

“…The RC.SVtsA58 renal collecting duct cell line was generated by infection of a primary culture of isolated rabbit renal cortical cells with the SV40 temperature-sensitive mutant tsA58 (22). Experiments were performed between the 30th and 60th passages following the protocol previously described (16). Briefly, cells were seeded in uncoated Petri dishes or in 12-well plastic trays at a concentration of 2 ϫ 10 4 cells/cm 2 and cultured to confluency (day 5) at the permissive temperature (33°C) to allow functional expression of the large-T oncogene.…”

“…4, A, lanes 2 and 5, and B, lanes 2 and 4) or by 10 Ϫ7 M AVP (Fig. 4A, lanes 3 and 6), which induces a peak cyclic AMP response (16).…”

“…When cultured at the permissive temperature of 33°C (functional large-T antigen), cells display characteristics of transformed cells. After shifting the cells to the restrictive temperature of 39.5°C (nonfunctional large-T antigen), cells stop dividing and acquire a differentiated phenotype characteristic of collecting duct principal cells including expression of functional type-2 arginine-vasopressin (AVP) receptors (16,17). We had also shown that cells shifted to 39.5°C synthetized a well organized basement membrane.…”

“…We had previously established a rabbit collecting duct epithelial cell line (RC.SVtsA58) infected with the temperaturesensitive SV40 strain tsA58 (16). When cultured at the permissive temperature of 33°C (functional large-T antigen), cells display characteristics of transformed cells.…”