Glucosylceramide Synthase is essential in MDCK cell Differentiation

PESCIO, L.G.; IRIBARREN, P.A.; STERIN-SPEZIALE, N.B.

Tucumán, Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2009

Sociedad Argentina de investigación Bioquímica y Biología Molecular

GLUCOSYLCERAMIDE SYNTHASE IS ESSENTIAL IN MDCK CELL DIFFERENTIATION In a previous study we observed that the synthesis of glycosphingolipids (GSLs) was increased under hypertonic conditions. This increase was inhibited by PDMP, an inhibitor of glucosylceramide synthase (GCS); but neither cycloserine (CS), a serine palmitoyltransferase inhibitor, nor fumonisin B1 (FB1), a ceramide synthase inhibitor, were able to reduce GSL levels. We now demonstrate by molecular analysis (RT-PCR and Immunoblot) that GCS is up-regulated by hypertonicity at the transcriptional level. Moreover, the distribution analysis of differentiation markers by immunofluorescence showed that MDCK cells could develop a hypertonicity- induced differentiated phenotype even in the presence of CS or FB1. Since FB1 is able to inhibit both the de novo and salvage pathways, we used other metabolic strategy to determine the source and mechanism involved in the hypertonicityinduced glucosylceramide (GC) increase. MDCK cells were labeled with 14C palmitic acid for 1 or 24h. 14C GC was significantly increased under hypertonic conditions in long-term labeling but not in pulse conditions. All together, these results indicate that hypertonicity induces an increase in the synthesis of GSLs by the regulation of GCS expression and the activation of the salvage pathway of GC formation, and that such mechanism is specific and essential to MDCK cell differentiation. COMUNICACIÓN ORAL