XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions

Abstract

Background: Sodium-Glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT1 is expressed in the kidneys but also in other organs throughout the body. Many SGLT inhibitor drugs have been developed based on the mechanism of blocking glucose (re)absorption mediated by SGLT1/2, and several have gained major regulatory agencies' approval for treating diabetes. Intriguingly these drugs are also effective in treating diseases beyond diabetes, for example heart failure and chronic kidney disease. We recently discovered that SGLT1 is upregulated in the airway epithelial cells derived from patients of cystic fibrosis (CF), a devastating genetic disease affecting greater than 70,000 worldwide.

Results: In the present work, we show that the SGLT1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1α (IRE1α) and the ER stress-induced transcription factor X-box binding protein 1 (XBP1), in CF epithelial cells, and in epithelial cells of other stress conditions. Through biochemistry experiments, we demonstrated that the spliced form of XBP1 (XBP1s) acts as a transcription factor for SLC5A1 by directly binding to its promoter region. Targeting this ER stress → SLC5A1 axis by either the ER stress inhibitor Rapamycin or the SGLT1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT1 level in these cellular model systems.

Conclusions: The present work establishes a causal relationship between ER stress and SGLT1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

Keywords: ER stress; Epithelial cells; SGLT1; SLC5A1; XBP1.

Fig. 1

SGLT1 is upregulated in CF patient-derived airway lineage cells. (A) Representative gel of Western blot (WB) of SGLT1 and CFTR in the CFBE-WT cells and the CFBE-dF cells. (B) Quantification of WB data for the protein levels of SGLT1 in CFBE-WT cells and the CFBE-dF cells. (C) RT-qPCR of SLC5A1 in the CFBE-WT cells and the CFBE-dF cells. (D) Western blot of SGLT1 in CF patient-derived airway epithelial cells. HC-1 and − 2: healthy control; CF-1 and − 2: CF patients of homozygous dF508 mutation. (E) Immunofluorescence staining of SGLT1 in the CFBE-WT cells and the CFBE-dF cells. Scale bar: 20 μm

Fig. 2

Elevation of ER stress markers in CFBE cells. (A) Representative Western blot (WB) gel of BiP, p-IRE1α, IRE1α, and XBP1s in the CFBE-WT cells and the CFBE-dF cells. (B) Quantification of WB data for the protein levels of BiP, p-IRE1α, IRE1α and XBP1s in CFBE-WT cells and the CFBE-dF cells. (C) RT-qPCR of HSPA5, ERN1, and XBP1 in the CFBE-WT cells and the CFBE-dF cells

Fig. 3

XBP1 upregulates the expression of SLC5A1. (A) Left: western blot of SGLT1 and XBP1s in CFBE-WT (WT) and CFBE-dF (dF) cells transduced with Ad-LacZ or Ad-XBP1s or without any viral infection (Con). Right: SLC5A1 mRNA levels in WT and dF cells transduced with Ad-LacZ or Ad-XBP1. (B) Western blot of SGLT1 and XBP1s in CFBE-WT (WT) and CFBE-dF (dF) cells transduced with Ad-LacZ or AD-K907A (the dominant negative IRE1α) or without any viral infection (Con). (C) ChIP assay of CFBE-WT (WT) and CFBE-dF (dF) cells transduced with Ad-LacZ or Ad-XBP1s. The binding of XBP1 to the SLC5A1 promoter was determined by qPCR. (D) Top: illustration of the putative wild-type (wt) binding sequence (from − 595 to -583) or the mutated binding sequence (mut) of the reporter plasmid. Red colored letters indicated mutated sequences. Bottom: relative firefly/renilla luciferase signal levels in CFBE-WT (WT) and CFBE-dF (dF) cells transfected with different combinations of the reporter plasmids (wt or mut) and the Ad viruses (Ad-LacZ or Ad-XBP1s).

Fig. 4

Rapamycin attenuates SGLT1 and ER stress in CFBE cells. (A) Representative Western blots of SGLT1, BiP, p-IRE1α, IRE1α, XBP1s and CFTR in the CFBE-WT cells and the CFBE-dF cells treated with or without Rapamycin. (B) Quantification of Western blots of SGLT1, BiP, p-IRE1α, IRE1α and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Rapamycin

Fig. 5

Sotagliflozin attenuates ER stress in CFBE cells. (A) Representative Western blot gel of SGLT1, BiP, p-IRE1α, IRE1α, and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Sotagliflozin (Sota). (B) Quantification of WB data of SGLT1, BiP, p-IRE1α, IRE1α and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Sotagliflozin. (C) RT-PCR of SLC5A1, HSPA5, ERN1, and XBP1 in the CFBE-WT cells and the CFBE-dF cells treated with or without Sota. (D) Immunofluorescence staining of SGLT1 in the CFBE-WT cells and the CFBE-dF cells treated with or without Sota. Scale bar: 20 μm

Fig. 6

Effects of Sotagliflozin and Rapamycin on the CHIR-ALI culture. (A) Representative Western blot (WB) gel of SGLT1, BiP, p-IRE1α, IRE1α and XBP1s in the CHIR-ALI cells treated with different combinations of Sota and Rapamycin (Rapa). (B) Quantification of WB data of SGLT1, BiP, IRE1α and XBP1s in the CHIR-ALI cells treated with different combinations of Sota and Rapamycin (Rapa). (C) qRT-PCR of SLC5A1, HSPA5, ERN1 and XBP1 in the CHIR-ALI cells treated with different combinations of Sota and Rapamycin (Rapa). Control (the leftmost bar in each panel) are ALI-no-CHIR cells without Sota or Rapa supplementation to the culture medium. ns: not statistically different among these three groups

Fig. 7

(A) Representative Western blot gel of SGLT1, BiP, p-IRE1α, IRE1α and XBP1s in the PA-Huh7 cells treated or without Sota. (B) Quantification of WB data of SGLT1, BiP, IRE1α and XBP1s in the PA-Huh7 cells treated or without Sota. Huh-7 cells were treated with BSA-complexed PA (10 µg/mL) in the presence or absence of Sota (50 µg/mL) for 36 h. Control are Huh7 cells treated with BSA vehicle without any PA or Sota treatment. (C) qRT-PCR of SLC5A1, HSPA5, ERN1 and XBP1 in the PA-Huh7 cells treated or without Sota