The development of Na(+)-dependent glucose transport during differentiation of an intestinal epithelial cell clone is regulated by protein kinase C.
The sodium-dependent glucose transporter SGLT1 is expressed on the apical plasma membrane of fully differentiated enterocytes. Recently, we have found that the cotransport function appears gradually during the process of differentiation of the human intestinal epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected in both undifferentiated and differentiated HT-29-D4 cells suggesting that sodium-glucose cotransport was dependent on post-translational events controlling the efficient targeting of the protein in the plasma membrane. In the present study, we have analyzed the molecular mechanisms controlling the functional expression of the SGLT1 protein during the course of HT-29-D4 differentiation. We show that the appearance of the cotransport function in the apical membrane is blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7 treatment was associated with an inability of HT-29-D4 cells to organize into a polarized monolayer of differentiated cells. Reciprocally, short term treatment (15 min) of undifferentiated cells by 0.1 microM phorbol myristyl acetate resulted in the appearance of the cotransport function. In contrast, inhibition of cAMP and cGMP-dependent protein kinases by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not prevent the development of sodium-glucose cotransport during the differentiation of HT-29-D4 cells. In addition, stimulation of cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the cotransport function in undifferentiated HT-29-D4 cells. By using immunogold labeling at the electron microscopy level, we demonstrated that phorbol myristyl acetate induced the redistribution of SGLT1 protein from intracellular sites to the plasma membrane. In conclusion, our data show that the appearance of a functional sodium-glucose cotransporter in HT-29-D4 cells is controlled, at least in part, by intracellular pathways regulated by the activity of protein kinase C.
Delézay
O
O
INSERM U270, Faculté de Médicine Nord, Marseille, France.
Baghdiguian
S
S
Fantini
J
J
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002272:Carcinoembryonic Antigen / Q000302:isolation & purification; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002451:Cell Compartmentation; D002454:Cell Differentiation; D016764:Cell Polarity; D002999:Clone Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000201:enzymology; D007546:Isoquinolines / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002272:Carcinoembryonic Antigen; D002352:Carrier Proteins; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010879:Piperazines; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; C027020:methylglucoside; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate