Question:

PDZK1 is an important PDZ adaptor protein of the NHERF family that interacts with a variety of membrane proteins in the intestine, liver and kidney, thereby stabilizing the proteins in the membrane and facilitating interaction with signalling molecules. We previously reported a strong decrease in the NHERF-family PDZ adaptor protein PDZK1 in the inflamed murine and human intestine, so in this study, we searched for the molecular mechanisms of inflammation-associated PDZK1 downregulation, using an in vitro Caco-2bbe cell intestinal culture model.

Methods:

The PDZK1 promotor, as well as 5`deletion constructs, were cloned into the well-differentiating Caco2bbe colonic cell line. Reporter gene assays, real-time PCR and Western blots were performed to measure the PDZK1 promoter activity, mRNA and protein expression, respectively.

Results:

Treatment of Caco-2bbe cells with Interleukin-1b (IL-1b) dose-dependently decreased PDZK1 promoter activity, as well as PDZK1 mRNA and protein expression in Caco2bbe cells. TNF-α and IFN-g, alone, or in combination with IL-1b, didn't show an additional inhibitory effect compared to IL-1b alone. 5´-deletion analysis of PDZK1 promoter reporter constructs showed that the base pairs -4689 to -3995 are responsible for the basal and IL-1b mediated inhibitory effect in Caco-2bbe cells. In silico analysis of the PDZK1 promotor sequence showed that this regions harbours the major transcription factor binding elements, for NF-?B, AP-1, c-Jun and SP-1 proteins. The presence of BAY11-7082 (NF-?B inhibitor) and BIRB (p38MAPK inhibitor) could not prevent IL-1b mediated PDZK1 down regulation of mRNA and protein expression, which indicates the involvement of other signalling cascades for this inhibitory effect of IL-1b on PDZK1 expression. In addition to Caco-2bbe cells, treatment of the Huh-7 (hepatoma cells) cells with IL-1b also resulted in PDZK1 down regulation, suggesting that inflammation may cause PDZK1 downregulation.

Conclusion:

IL-1b decreased PDZK1 protein expression in Caco-2bbe cells through transcriptional inhibition of PDZK1 promoter. The region between base pairs -4689 to -3995 in the PDZK1 promoter is important for both its basal expression and IL-1b mediated inhibition. This effect is also seen in liver cells. PDZK1 downregulation may be an important factor that contributes to membrane transporter dysfunction in inflammation.