(577) SGLT1 is associated with ER stress response in CF and transcriptionally regulated by XBP1s

Background: The misfolded CFTR protein is retained in the endoplasmic reticulum and trigger the unfolded protein response (UPR). Our previous work revealed that the marked increase of ER stress markers IRE1α and spliced XBP1 (XBP1s) in CF rabbit tissues and in CFTR-F508del (dF) human airway epithelial cells, in correlation with the elevated expression level of SGLT1, a sodium glucose cotransporter encoded by the gene SLC5A1. We reported that a potent SGLT1 inhibitor drug, Sotagliflozin, effectively attenuates ER stress in CF rabbits [1, 2]. However, the mechanism of the elevated SGLT1 in CF remains unclear.

Methods: Western blotting, real-time PCR analyses and immunofluorescent staining were performed to examine the expressions of SGLT1 and ER stress markers including GRP-78, IRE1a, XBP1s in CF patient derived bronchial epithelial (CFBE) cells. Chromatin immunoprecipitation (ChIP) assay was performed to determine if the ER stress transducer XBP1 binds to the promoter region of SLC5A1. A luciferase reporter assay was next employed to confirm that binding motif at the -590 bp proximal position of the SLC5A1 promoter is necessary for the XBP1 binding.

Results: The levels of the SGLT1 protein and the SLC5A1 transcripts are higher in CFBE-dF cells compared to that in the CFBE-WT cells. The ER stress markers including GRP-78, IRE1a, and XBP1s are also transcriptionally and translationally upregulated in the CFBE-dF cells than in the CFBE-WT cells. Interesting, an ER stress inhibitor Rapamycin effectively reduced the levels of SGLT1 and ER stress markers in the CFBE-dF cells. We thus hypothesized that ER stress upregulation transcriptionally upregulate SLC5A1. To test this, we overexpressed XBP1s by adenorvirus-XBP1s (Ad-XBP1s) to CFBE-dF cells and CFBE-WT cells. In both cells, the infection led to upregulation of SLC5A1 at the transcription level and SGLT1 at the protein level. ChIP assay revealed that XBP1s binds to the promoter of SLC5A1. We then constructed a luciferase reporter by fusing the SLC5A1 promoter with that of the coding sequence of luciferase. Overexpression of Ad-XBP1s led to a significant increase of SLC5A1 promoter-driven luciferase activity in CFBE cells. The putative binding motif on SGLT1 promoter by XBP1s is predicated to be “5’-CCACCCACCCACC-3’” at the -595 to -582 bp position, which is confirmed by the observation that mutation of this motif led to significantly reduced luciferase signals after Ad-XBP1s transduction in the luciferase reporter assay.

Conclusions: Our data demonstrate that the elevated SGLT1 are accompanied with the elevated ER stress response in CFBE-dF cells. XBP1s is a transcription factor of SLC5A1 to transcriptionally regulates SGLT1 expression in CFBE-dF cells.

Acknowledgements: The work was supported by NIH (HL133162) to J-P Jin and J Xu; part of the work was also supported by NIH grant DK090313 (to K Zhang).

References: [1]. Sun Y, Liang X, Hou X, Bouhamdan M, Song J, Chen Y, Jin J, Xu J. SGLT1/2 inhibitor sotagliflozin attenuates pancreatic endoplasmic reticulum stress through IRE1α/XBP1 pathway in cystic fibrosis rabbits. J Cystic Fibrosis. 2021;20S2:S4
[2]. Liang X, Hou X, Song Z, Bouhamdan M, Song J, Sun Y, Chen Y, Jin J, Zhang K, Xu J. Protective effects of SGLT1/2 inhibitor sotagliflozin on CF-related liver disease in CF rabbits. J Cystic Fibrosis.2021;20S2: S97