PTEN knockdown or inhibition induces aberrant lumens at the lateral domain of renal epithelial cells

PESCIO, LUCILA GISELE; LABROUSSE, MARIANO ELISEO; KRIVOCAPICH, CLARA; D´ANDREIZ, MARÍA JOSEFINA; FAVALE, NICOLÁS OCTAVIO; STERIN-SPEZIALE, NORMA BEATRIZ

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Epithelial cell differentiation, characterized by apicobasal polarization and asymmetric expression of the proteins and lipids of the membrane, is essential for the development of functioning epithelial renal tubules. MDCK cells are a prototype of renal epithelial cell line widely used to study the polarized trafficking machinery used by epithelial cells to distribute their plasma membrane proteins into apical and basolateral domains. In these cells, the apical accumulation of the marker gp135 is necessary for a correct lumen formation. We have previously demonstrated that the correct localization of PTEN, a key enzyme in the metabolism of polyphosphoinositides, depends on glycosphingolipid metabolism. In this study, we used immunofluorescence and phalloidin-FITC stain to analyze by confocal microscopy the distribution of gp135 and f-actin in cells cultured under hypertonicity (inductor of cell differentiation) in the presence of SF1670, a selective PTEN inhibitor, or siRNA specific for PTEN. MDCK cells cultured under hypertonicity 48 h post-confluence developed a differentiated phenotype with typical cobblestone-like morphology defined by cortical actin fibers and apical accumulation of gp135. Cells cultured under hypertonicity treated with SF1670 revealed aberrant lumen formation between neighboring cells. Instead of at the apical membrane, gp135 was found localized to these uncharacteristic lateral lumens, that were also enriched with F-actin. Similar results were observed in PTEN - knockdown cells. Confocal images showed that SF1670 - treated and PTEN - knockdown cells displayed elongated morphology and dissipation of actin cortex with evident stress fibers at the lower plane. Normally, MDCK cells organize their lumen at the apical surface, in contrast to hepatocytes, which form lateral lumen known as bile canaliculi. Either PTEN inhibition or depletion induces the mislocalization of gp135 at the lateral lumens, thus shifting the kidney epithelial phenotype of MDCK cells to hepatic polarity and denoting an important role of PTEN activity in MDCK cell differentiation. Due to GSLs synthesis being essential for the correct localization of PTEN, our study suggests that sphingolipid polarity and protein polarity are highly interconnected to accomplish apical-basal polarization of epithelial cells.