PKC-epsilon-dependent cytosol-to-membrane translocation of pendrin in rat thyroid PC Cl3 cells.
We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC Cl3 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. (125)I pre-loaded cells showed an (125)I efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin (1 microg/ml; 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I(-) content in (125)I pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon translocation inhibitor peptide and also by PKC-epsilon downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon-dependent intracellular pathway.
Muscella
A
A
Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), Università del Salento, Italy.
Marsigliante
S
S
Verri
T
T
Urso
L
L
Dimitri
C
C
Bottà
G
G
Paulmichl
M
M
Beck-Peccoz
P
P
Fugazzola
L
L
Storelli
C
C
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007454:Iodides / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D034741:RNA, Small Interfering; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076988:Sulfate Transporters; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*
D027963:Chloride-Bicarbonate Antiporters; D007004:Hypoglycemic Agents; D007328:Insulin; D007454:Iodides; D034741:RNA, Small Interfering; C494615:Slc26A4 protein, rat; D000076988:Sulfate Transporters; D051744:Protein Kinase C-epsilon