Major influence of cell differentiation status on characteristics of proteoglycans synthesized by cultured rabbit renal proximal tubule cells: role of insulin and dexamethasone.
To analyze the influence of epithelial cell differentiation and the effects of hormones on the characteristics of cell-associated and secreted proteoglycans (PGs), we studied their distribution, synthesis, and biochemical features in a model of renal proximal tubule cells in primary culture in which cell differentiation could be controlled by medium composition. In cells cultured in serum-free, hormonally defined medium supplemented with insulin and dexamethasone that exhibited a high degree of morphological and functional proximal differentiation (Ronco et al., 1990), cell-associated PGs were similar to those extracted in vivo by their size estimated by Sepharose CL-6B chromatography (Kav = 0.27, vs. 0.26), composition (heparan-sulfate), and localization in a continuous basal layer of extra-cellular matrix (ECM). In contrast, major quantitative and qualitative anomalies of cell-associated PGs were observed in poorly differentiated cells grown in 1% fetal calf serum-supplemented medium (FCS). PGs alterations included: (1) reduced and irregular expression of PGs at the cell basal pole, (2) a 2.8-fold decrease in [35S]-sulfate incorporation into cell-associated PGs, (3) a 3.1-fold increase in trypsin-releasable PGs, and (4) the emergence of a high MW PG composed exclusively of chondroitin-sulfate (CS) (Kav = 0.09 on Sepharose CL-6B) as well as of putative free CS-glycosaminoglycan (GAG) chains (Kav = 0.49 on Sepharose CL-6B). The same alterations were identified in the basal defined medium devoid of hormones but were partially or totally abolished by addition of insulin and dexamethasone, respectively. At variance with cell-associated PGs, production and biochemical features of secreted PGs were not influenced by cell differentiation status and medium composition.
Lelongt
B
B
INSERM U.64, Hôpital Tenon, Paris, France.
Vandewalle
A
A
Brenchley
P E
PE
Baudouin
B
B
Géniteau-Legendre
M
M
Verroust
P J
PJ
Ronco
P M
PM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007328:Insulin / Q000502:physiology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D011509:Proteoglycans / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D006025:Glycosaminoglycans; D007328:Insulin; D011509:Proteoglycans; D003907:Dexamethasone