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

Abstract

Background: sodium-dependent glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT-2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT-1 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 SGLT-1 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 SGLT-1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1a (IRE1a) 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 XBP1 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 SGLT-1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT-1 levels in these cellular model systems.

Conclusions: the present work establishes a causal relationship between ER stress and SGLT-1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

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

Figure 1.. SGLT-1 is upregulated in CF patient-derived airway lineage cells.

(A) Western blot of SGLT-1 and CFTR in the CFBE-WT cells and the CFBE-dF cells. (B) RT-qPCR of SLC5A1 in the CFBE-WT cells and the CFBE-dF cells. (C) Immunofluorescence staining of SGLT-1 in the CFBE-WT cells and the CFBE-dF cells. Scale bar: 20μm. (D) Western blot of SGLT-1 in CF patient-derived airway epithelial cells. HC-1 and −2: healthy control; CF-1 and −2: CF patients of homozygous dF508 mutation.

Figure 2.. Elevation of ER stress markers in CFBE cells.

(A) Western blot of GRP78, p-IRE1a, IRE1a, and XBP1s in the CFBE-WT cells and the CFBE-dF cells. (B) RT-qPCR of GRP78, IRE1a, and XBP1s in the CFBE-WT cells and the CFBE-dF cells.

Figure 3.. XBP1 upregulates the expression of SLC5A1.

(A) Left: western blot of SGLT-1 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 SGLT-1 XBP1s in CFBE-WT (WT) and CFBE-dF (dF) cells transduced with Ad-LacZ or AD-K907A (the dominant negative IRE1a) 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).

Figure 4.. Rapamycin attenuates SGLT-1 and ER stress in CFBE cells.

(A) Representative Western blots of SGLT-1, GRP78, p-IRE1a, IRE1a, XBP1s and CFTR in the CFBE-WT cells and the CFBE-dF cells treated with or without Rapamycin. (B) Quantification of Western blots of SGLT-1, GRP78, p-IRE1a, IRE1a and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Rapamycin.

Figure 5.. Sotagliflozin attenuates ER stress in CFBE cells.

(A) Western blot of SGLT-1, GRP78, p-IRE1a, IRE1a, and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Sota. (B) qRT-PCR of SLC5A1, GRP78, IRE1a, and XBP1s in the CFBE-WT cells and the CFBE-dF cells treated with or without Sota. (C) Immunofluorescence staining of SGLT-1 in the CFBE-WT cells and the CFBE-dF cells treated with or without Sota. Scale bar: 20μm.

Figure 6.. Effects of Sotagliflozin and Rapamycin on the CHIR-ALI culture.

(A) Western blot of SGLT-1, GRP78, p-IRE1a, IRE1a, and XBP1s in the CHIR-ALI cells treated with different combinations of Sota and Rapamycin (Rapa). (B) qRT-PCR of SLC5A1, GRP78, IRE1a, and XBP1s in 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.

Figure 7.

(A) Western blot of SGLT-1, GRP78, p-IRE1a, IRE1a, and XBP1s in the PA-Huh7 cells treated or without Sota. (B) qRT-PCR of SLC5A1, GRP78, IRE1a, 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 (50μg/mL) for 36 h. Control are Huh7 cells treated with BSA vehicle without any PA or Sota treatment.