GPR35 prevents osmotic stress induced cell damage

Abstract

GPR35 is an orphan G-protein coupled receptor that has been implicated in the development of cancer. GPR35 regulates the Na⁺/K⁺-ATPase's pump and signalling function. Here we show GPR35's critical role in ion flux that in turn controls cellular osmotic pressure and Na⁺-dependent transport in HepG2 and SW480 cells. GPR35 deficiency results in increased levels of intracellular Na⁺, osmotic stress and changes in osmolytes leading to increased cells size and decreased glutamine import in vitro and in vivo. The GPR35-T108M risk variant, which increases risk for primary sclerosing cholangitis and inflammatory bowel disease, leads to lower intracellular Na⁺ levels, and enhanced glutamine uptake. High salt diet (HSD) in wildtype mice resembles the intestinal epithelial phenotype of their Gpr35^-/- littermates with decreased Goblet cell size and numbers. This indicates that GPR35's regulation of the Na⁺/K⁺-ATPase controls ion homeostasis, osmosis and Na⁺-dependent transporters.

Fig. 1. Lack of GPR35 results in changed cell size and ion homoeostasis.

A Knock down of GPR35 leads to ballooning and reduced growth of HepG2 cells. B Knock-down of GPR35 in human cancer cell lines leads to an increase of size. Bone marrow derived macrophages from Gpr35^−/− mice were increased compared to Gpr35^+/+ cells. Size was measured using flow cytometry. Size was calculated using forward scatter and size reference beads. N = 4 for cell lines. C Mice were on a low salt diet (LSD) or high salt diet (HSD). Cell diameters were measured and area was calculated from H&E stained intestinal samples. N = 42 (from 6 mice per group) D Intracellular Na⁺ levels measured by fluorescence (SBFI), ouabain used as control inhibits the Na⁺/K⁺-ATPase. Left panel SW480 intestinal epithelial cells, right panel HepG2 hepatic epithelial cells. N = 16. E Left panel. Measurement of intracellular Na⁺ (SFBI) with canonical GPR35 agonists or antagonists, ouabain as control. Right panel. Measurement of intracellular Na⁺ (SFBI) with adenyl cyclase inhibitors KH7 or SQ22536. N = 4. F Intracellular K⁺ levels measured by fluorescence (PBFI), ouabain used as control inhibits the Na⁺/K⁺-ATPase. Left panel SW480 intestinal epithelial cells, right panel HepG2 hepatic epithelial cells. N = 16. G Measurement of K⁺-flux by Tl assay over 5 min. N = 4. H Intracellular Na⁺ and K⁺ levels in hiPSC carrying the risk or non-risk variant of GPR35. Measured by fluorescence (SBFI for Na⁺ and PBFI for K⁺). N = 17 to 25. I Urine and blood levels of Na⁺ and K⁺ in Gpr35^fl/fl and Gpr35^DIEC mice. N = 9 –11. Data represented as mean ± s.e.m. Statistical significance was calculated using Mann Whitney U after Kruskal Wallis testing. *p < 0.05, **p < 0.01.

Fig. 2. GPR35 regulates intracellular osmotic pressure.

A Metabolomic screen of GPR35 silenced and control HepG2 cells. Data are shown as log fold change. Measured by mass spectrometry. N = 3. B Osmolytes were measured by mass spectrometry in control and GPR35 silenced HepG2 cells. N = 6. C Western blot analysis of p38MAPK activation with addition of 25 to 125 mM of NaCl. Western blot band density was calculated using ImageJ. N = 3 D The endogenous osmolyte GPC was measured by mass spectrometry in colonic biopsies of healthy control, UC or PSC patients. N = 7-8 E Right panel: GPR35 mRNA levels under normo- or hypersaline conditions with and without NFAT5 siRNA. Left panel: GPR35 mRNA levels under normo- or hypersaline conditions with and without p38 MAPK inhibitor. N = 3. FGPR35 mRNA levels in human macrophages under normo- or hypersaline conditions. N = 2. GNFAT5 mRNA levels under normo- or hypersaline conditions with and without GPR35 siRNA. N = 3. H Western blot analysis of NFAT5 in cell lysates and nuclear extracts. N = 5. Western blot band density was calculated using ImageJ. All data represented as mean ± s.e.m. Statistical significance was calculated using Mann Whitney U after Kruskal Wallis testing. *p < 0.05, **p < 0.01.

Fig. 3. Na⁺-dependent glutamine uptake is regulated by GPR35. A.

A Glutamine concentration in culture medium of SW480 cells at different time points of cell culture showing increased consumption of glutamine by control siRNA compared to GPR35 siRNA transfected cells. Measured by Promega glutamine GLO assay. N = 3. B [13C5] glutamine uptake over 60 s, measured by LC-MS, in SW480 cells 48 h following siRNA transfection. Left panel: Uptake under physiological K⁺ and Na⁺ conditions. right panel: Uptake under high K⁺ conditions (50 mM KCl). N = 3 C RNAseq of Gpr35^+/+ and Gpr35^−/− murine ileum. N = 6 per genotype. D Pathway analysis of RNAseq in murine ileum of Gpr35^+/+ and Gpr35^−/− mice. E RNAseq of Gpr35^+/+ and Gpr35^−/− murine ileum. Data are shown as % gene expression wildtype mice. N = 5 mice per genotype. FSLC6A14 mRNA expression in SW480 cells under physiological and high Na⁺ conditions. N = 6. G Glutamine uptake in control or GPR35 silenced SW480 cells with additional SLC6A14 knock down. N = 6. H Glutamine uptake in hiPSC carrying the 108 T norm or the 108 M risk variant of GPR35. Intracellular glutamine was measured by luminescence. N = 6. All data represented as mean ± s.e.m. Statistical significance was calculated using Mann Whitney U after Kruskal Wallis testing. *p < 0.05, **p < 0.01.

Fig. 4. GPR35 regulates Na⁺-dependent proliferation.

A SiRNA knock-down of GPR35 in several human cancer cell lines. Proliferation was measured fluorometrically by Cyquant and is shown as percent relative fluorescence units (RFU). N = 8 –18 per cell line in control and GPR35 silenced cells. B Canonical agonist lodoxamide, antagonist CID2745687 or ouabain added to control or GPR35 silenced HepG2. Proliferation was measured fluorometrically by Cyquant and shown as RFU. N = 6 C Left panel: Proliferation in GPR35 silenced hiPSC and control silenced cells. Right panel: Proliferation of hiPSC carrying the risk 108 M variant and cells carrying the non-risk 108 T variant. Proliferation was measured fluorometrically by Cyquant and shown as percent highest RFU. N = 9 D Proliferation of control or GPR35 silenced HepG2 cells with different concentrations of NaCl in the growth media, measured fluorometrically by Cyquant and shown as RFU. N = 6. E Proliferation of control or GPR35 silenced SW480 with different concentrations of NaCl in the growth media. Proliferation was measured fluorometrically by Cyquant and shown as RFU. N = 6. F Proliferation of HepG2 cells in normo- or hyperosmolality. Proliferation of control or GPR35 silenced HepG2 cells was measured fluorometrically by Cyquant and shown as RFU. N = 3. G Addition of mannitol (5 –50 mM/L) to DMEM media for 12 h in control and GPR35 silenced HepG2 cells. Proliferation was measured fluorometrically by Cyquant and shown as RFU. N = 3. H Proliferation of control or GPR35 silenced HepG2 cells in the presence of p38 MAPK inhibitor measured after 12 or 24 h. N = 6 All data represented as mean ± s.e.m. Statistical significance was calculated using Mann Whitney U after Kruskal Wallis testing. *p < 0.05, **p < 0.01.

Fig. 5. High salt diet induces inflammation and morphological changes in Gpr35^+/+ mice.

A p38 MAPK phosphorylation in Gpr35^+/+ and Gpr35^−/− mice after 7 days of either HSD or LSD. N = 7 mice per genotype per diet (n = 28) Left panel: representative immune-histochemistry. Right panel: phosphor p38MAPK semiquantitative analysis using Image J. B Intestinal epithelial cell proliferation after 7 days of HSD measured as BrdU intake. N = 7 mice per genotype per diet (n = 28) Left panel: representative immune-histochemistry. Right panel: BrdU positive cells per crypt. C Weights of Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD with 2 additional DSS cycles (2.5% for 4 days). Right panel: weight curve over 30 days. Left panel: weights on day 8 and day 30 of the experiment. N = 7 to 11 mice per genotype per diet. D Western blot analysis of p38 MAPK phosphorylation in Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD. Densitometry shown as ratio of phosphorylated p38 MAPK to total p38 MAPK. N = 3. E Left panel: CXCL1 levels in intestinal biopsies of Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD measured by ELISA after protein extraction. N = 6. Right panel: Infilterating inflammatory cells per high power field (HPF) in Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD. F Goblet cells morphology in Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD. Left panel: representative H&E stain. Right panel: diameter of goblet cells in µm. N = 6. G Periodic acid shift (PAS) stain of Goblet cells in Gpr35^fl/fl and Gpr35^ΔIEC mice under HSD or LSD. N = 9 per genotype and diet. H Expression levels of mucin-2 mRNA in Gpr35^fl/fl and Gpr35^ΔIEC mice. N = 3-4. All data represented as mean ± s.e.m. Statistical significance was calculated using Mann Whitney U after Kruskal Wallis testing. *p < 0.05, **p < 0.01, scale bars in (A–G): 100 µm.