Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP.

MDCK cells stably transfected with betaine/GABA transporter tagged with EGFP (EGFP-BGT) were used to study plasma membrane insertion of EGFP-BGT. Adaptive response to hypertonicity requires nuclear migration of TonEBP. Confocal microscopy showed that after 6 h hypertonicity, the nuclear/cytoplasmic ratio of TonEBP fluorescence was increased to 2.4 compared to 1.4 in isotonic controls (P<0.001). The ratio in hypertonic cells was reduced by the proteasome inhibitor MG-132 in a dose-dependent way. Inhibition was 50% at 3 microM. After 6 h, hypertonicity expressed EGFP-BGT was localized in the plasma membrane, but there was no change in total EGFP-BGT abundance compared to isotonic controls. In contrast, EGFP-BGT remained mostly intracellular when 3 microM MG-132 was included in the hypertonic medium. The transport function of EGFP-BGT was studied as Na(+)-dependent uptake of [(3)H]GABA. This was not changed by MG-132 in isotonic controls, but MG-132 produced dose-dependent inhibition of hypertonic upregulation of Na(+)/GABA cotransport. Inhibition was 80% at 3 muM MG-132. Transport likely reflects membrane insertion of EGFP-BGT and there was a positive correlation (P<0.05) between Na(+)/GABA cotransport and the N/C ratio of TonEBP. Results are consistent with a role for TonEBP-mediated transcription in synthesis of additional proteins required for membrane insertion of EGFP-BGT protein.

Hypertonic upregulation of betaine transport in renal cells is blocked by a proteasome inhibitor.

The renal betaine transporter (BGT1) protects cells in the hypertonic medulla by mediating uptake and accumulation of the osmolyte betaine. Transcription plays an essential role in upregulating BGT1 transport in MDCK cells subjected to hypertonic stress. During hypertonic stress, the abundance of the transcription factor TonEBP increases and it shifts from the cytoplasm to the nucleus where it activates transcription of the BGT1 gene. Little is known about post-transcriptional regulation of BGT1 protein. In the presence of the proteasome inhibitor MG-132, which blocked nuclear translocation of TonEBP, the hypertonic upregulation of BGT1 protein and transport was prevented and cell viability in hypertonic medium was impaired over 24 h. Urea also prevented the hypertonic upregulation of BGT1 protein and transport, but did not interfere with TonEBP translocation and cell viability. Shorter treatments of hypertonic cells with MG-132 avoided viability problems and produced dose-dependent inhibition of translocation and transport. When stably transfected MDCK cells that over-expressed BGT1 were treated for 6 h with hypertonic medium containing 3 microM MG-132, there was 43% inhibition of nuclear translocation, 83% inhibition of BGT1 transport, and no change in viability. While other proteasome functions may be involved, these data are consistent with a critical role for nuclear translocation of TonEBP in upregulation and membrane insertion of BGT1 protein.