THE EFFECT OF TGF BETA ON THE Na+/K+ ATPASE IN CACO-2 CELLS

Abstract

Transforming growth factor- (TGF-) is a regulator of inflammatory responses. It was shown to be involved in the pathogenesis of inflammatory bowel disease (IBD), a chronic inflammatory disorder with a complex pathogenesis. Diarrhea is one of the symptoms of the disease and is usually the result of an intestinal hydro-electrolytic imbalance, due to aberrant sodium transport in the colon. The colon plays an important role in sodium transport processes which are driven by the sodium electro-chemical gradient established by the Na+/K+ ATPase or Na+/K+ pump. IBD patients were found to have a lower Na+/K+ ATPase activity and high TGF beta levels. This work was undertaken to examine if a cause-effect relationship exists between TGF beta and the colonic sodium potassium pump, using Caco-2 cells as a model. The activity of the pump was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the Na+/K+ ATPase. TGF beta (60ng/ml, 24 hours) was found to reduce the activity of the pump as well as its protein expression. The inhibitory effect of TGF beta did not appear in presence of a blocker of endocytosis or the proteasome but was still manifested in the presence of wortmannin (PI3K inhibitor), doramapimod (p38 inhibitor), (ERK inhibitor) and SP600125 (Jun-c Kinase inhibitor) ruling out any involvement of these kinases in its action. When sphingosine kinase one was inhibited, the effect of TGF beta disappeared, indicating an involvement of S1P as a signaling molecule mediating the action of TGF beta on the colonic ATPase. Treating the cells with FTY720P, an analogue of S1P, resulted in a similar inhibitory effect to that observed with TGF-beta. However, this inhibition was not observed in presence of a blocker of S1PR3 nor in presence of indomethacin, an inhibitor of COX enzyme implying that PGE2 is a downstream mediator. Treating the cells with exogenous PGE2 imitated the effect of TGF-beta and FTY720P, confirming thus its involvement in the signaling pathway. PGE2 was found to act via EP3 and did not exert any effect in the simultaneous presence of calphostin, a PKC inhibitor or dbcAMP, a PKA activator suggesting that the prostaglandin activates PKC and inhibits PKA. When cells were treated with PMA, an activator of PKC in the simultaneous presence of a PKA activator (dbcAMP) the inhibitory effect on the pump persisted indicating that PKA is upstream of PKC. It can be concluded that TGF beta exerts an inhibitory effect on the colonic Na+/K+ ATPase by decreasing its abundance in the cell membrane via endocytosis followed by proteasome degradation. The process is mediated through S1P which activates S1PR3 leading to PGE2 release and EP3 activation. The latter reduces the activity of the ATPase by inhibiting PKA leading to PKC activation.