Effect of TPA on aquaporin 4 mRNA expression in cultured rat astrocytes

Abstract

Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, localized in the astrocyte plasma membrane. The regulation of AQP4 is believed to be important for the homeostasis of water in the brain, but the AQP4 regulatory mechanisms are not yet known. In this study, we investigated the effect of a protein kinase C (PKC) activator on the expression of AQP4 mRNA in cultured rat astrocytes. Cultured rat astrocytes constitutively expressed AQP4 mRNA. Treatment of the cells with 0.1 microM of phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, caused a rapid decrease in AQP4 mRNA. This effect was time- and dose-dependent. The TPA-induced decrease in AQP4 mRNA was inhibited by a relatively specific PKC inhibitor, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) in a dose-dependent manner. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decrease in AQP4 mRNA by TPA. These results strongly suggest that the TPA-induced decrease in AQP4 mRNA is mediated by PKC activation. To test whether the effect of TPA requires protein synthesis, astrocytes were pretreated with cycloheximide, an inhibitor of protein synthesis. Pretreatment of the cells with cycloheximide did not inhibit the decrease in AQP4 mRNA induced by TPA. To test whether the TPA-induced decrease in AQP4 was due to a decrease in the mRNA stability, we examined the effect of actinomycin D, an inhibitor of transcription, on TPA-treated cells. The stability of AQP4 mRNA was not decreased by the pretreatment of the cells with actinomycin D. The results suggest that AQP4 mRNA is inhibited by TPA via PKC activation without de novo protein synthesis, and that the inhibition of AQP4 mRNA could be at the transcriptional level.

The Protein Kinase C Activator Phorbol Myristate Acetate Decreases Brain Edema by Aquaporin 4 Downregulation after Middle Cerebral Artery Occlusion in the Rat

“…Our findings are consistent with those of in-vitro studies demonstrating that PKC activators inhibit AQP4 activity , and also to downregulates the expression of AQP4 protein (Yamamoto et al, 2001), and AQP4 mRNA (Nakahama et al, 1999). However, the experimental model used may also affect the changes seen in AQP4 expression.…”

“…PKC serves as a second messenger for G-protein receptors, which are coupled to the phosphoinositide pathway, causing either a transient rise in intracellular Ca 2þ through inositol triphoshate, or activation of PKC through diacylglycerol. The phorbol ester and known PKC activator 12-Otetradecanoylphorbol 13-acetate has been found to decrease AQP4 mRNA in cultured rat astrocytes, but prolonged treatment eliminated the subsequent decrease in AQP4 mRNA (Nakahama et al, 1999;Yamamoto et al, 2001). Since pre-treatment with cycloheximide did not eliminate the reduction of AQP4 mRNA, PKC may be involved in AQP4 regulation on the transcriptional level, rather than affecting de novo protein synthesis.…”

“…However, direct and indirect evidence suggests that protein kinase C (PKC) is involved in AQP4 regulation (Nakahama et al, 1999;Neely et al, 1999;Yamamoto et al, 2001). PKC serves as a second messenger for G-protein receptors, which are coupled to the phosphoinositide pathway, causing either a transient rise in intracellular Ca 2þ through inositol triphoshate, or activation of PKC through diacylglycerol.…”

“…Although PKC activation has been found to decrease AQP4 mRNA in cultured astrocytes (Yamamoto et al, 2001), prolonged treatment eliminated the subsequent decrease in AQP4 mRNA (Nakahama et al, 1999). Since PMA activates PKC by binding PKC to the cell membrane with a very high affinity, there may be a relative depletion of PKC with administration of PMA for a extended time period (Mosior and Newton, 1995).…”

Role of aquaporin-4 in the regulation of migration and invasion of human glioma cells

“…in addition to applying aQP4 rna interference to reduce the aQP4 protein level, we also used the aQP4 inhibitor phorbol 12-myristate 13-acetate (PMa) which activates protein kinase c (PKc) and inhibits aQP4 expression (22). the results of Western blotting showed that the expression of AQP4 was significantly reduced with treatment of PMA (1 and 5 µM) compared to the control cells (Fig.…”