GLYCOSPHINGOLIPIDS ARE ESSENTIAL FOR THE CORRECT SEGREGATION OF PHOSPHOINOSITIDES MEDIATED BY PTEN IN RENAL EPITHELIAL CELLS

ROMERO DJ; PESCIO LG; FAVALE NO; HERRERA MP; STERIN SPEZIALE NB

Virtual

Congreso; SAIB - SAMIGE Joint meeting 2021 on line; 2021

Phosphoinositides act as critical regulators of cell polarization. PtdIns(4,5)P2 is enriched in the apical membrane, whereas PtdIns(3,4,5)P3 is basolateral, and this segregation is regulated by PTEN. The apical localization of PTEN allows the local synthesis of PtdIns(4,5)P2 and the consequent apical recruitment of the apical protein complex required for lumen development. We previously demonstrated that sphingolipid synthesis is essential for the correct localization of PTEN during the differentiation of MDCK cells induced by hypertonicity, and that the inhibition of PTEN impairs MDCK cell differentiation. In this study we transfected cells with biosensors for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and cultured them under hypertonicity (inductor of cell differentiation) in the presence of D-PDMP (a glucosylceramide inhibitor), SF1670 (a PTEN inhibitor) or siRNA PTEN; followed by immunofluorescence staining for gp135, an apical marker. MDCK cells transient transfected with PLCδ1-PH ? GFP, a PtdIns(4,5)P2 biosensor, showed a differentiated phenotype with PtdIns(4,5)P2 distributed at cell periphery, but accumulated at apical membrane in colocalization with gp135. Cells treated with SF1670 showed atypical gp135-containing lateral lumens with positive staining for PtdIns(4,5)P2. Similar results were observed in cells treated with siRNA PTEN, suggesting that the expression and the activity of PTEN are necessary for the correct localization of PtdIns(4,5)P2 and regulate the targeting of gp135 to induce the MDCK cell differentiation. To study the distribution of PtdIns(3,4,5)P3 we developed an MDCK cell line stably expressing the PH domain of Akt coupled to GFP (GFP-Akt-PH). Transfected cells cultured under hypertonicity 48 h post - confluence developed a differentiated phenotype with apical accumulation of gp135, as wild type cells. GFP-Akt-PH was mainly associated with lateral membranes. After treatment with D-PDMP, cells showed altered morphology with GFP-Akt-PH partially redistributed into apical membrane. The results shows that glycosphingolipid synthesis is necessary for the correct segregation of phospoinositides mediated by PTEN, suggesting an interplay between glycosphingolipids and phospoinositides that is essential for MDCK differentiation.