Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells

Abstract

Acute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation.

Figure 1. Expression of proton sensing GPCRs in EndoC-βH2, SKPC and human islets.

Transcript levels of proton sensing GPCRs (GPR68, GPR4, GPR65 and GPR132) determined by RT-qPCR in (a) EndoC-βH2 cells compared with ductal carcinoma SKPC cell-line; (b) human islet preparation. Data represented as mean values ± SEM of at least 3 independent experiments. ***p < 0.001; (one-way ANOVA, followed by a Tukey’s multiple comparisons post-test).

Figure 2. RFX6 regulates GPR68 expression in human β-cells.

EndoC-βH2 cells (a) and adult human islets (b) were transfected with control non-target siRNA (siNT) or siRNA targeting RFX6 (siRFX6). GPR68 expression was analyzed 72 h post transfection by RT-qPCR. Data are expressed as percentage of siNT transfected cells. (c–e) wtRFX6 and Mut506RFX6 (one-way ANOVA) (c), transactivation domain VP16- conjugated RFX6 (d) and transcriptional repression domain KRAB- conjugated RFX6 (e) were expressed in EndoC-βH2 cells using bicistronic constructs with IRES-EGFP. GFP^+ve cells were FACS isolated 48 h post-transfection and GPR68 expression was analyzed by RT-qPCR. Data are mean ± SEM of 3–5 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 and ns, not significant.

Figure 3. Extracellular pH modulates IP production but not cAMP accumulation in EndoC-βH2 cells.

(a) IP formation was determined in EndoC-βH2 cells incubated at pH 7.4 and 6.4. The G_q/11-selective inhibitor compound YM-254890 (100 nM) was also tested. Results are expressed as fold change over the IP values at pH 7.4. (b) For cAMP formation assay, EndoC-βH2 cells were incubated for 30 min with and without Forskolin (25 μM) at pH 7.4 or 6.4. Results are expressed as cAMP accumulation (nM) per 5,000 cells. (c) EndoC-βH2 cells were transfected with control siRNA (siNT), siRNA targeting either GPR68 (siGPR68) or RFX6 (siRFX6). 72 h post transfection, cells were incubated at pH 7.4 or 6.4 for 30 min and analyzed for the IP formation. Results are expressed as fold change over the IP values at pH 7.4. Data are mean ± SEM of 3–5 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 and ns, not significant (one-way ANOVA, followed by a Tukey’s multiple comparisons post-test).

Figure 4. Acidic extracellular pH induces the expression and secretion of IL-8 by EndoC-βH2 cells.

(a) EndoC-βH2 cells were cultured at pH 7.4 or 6.4 for 24 h and screened by RT-qPCR for the expression of selected pro/anti-inflammatory cytokines. (b) IL-8 expression was determined by RT-qPCR in EndoC-βH2 cells cultured at different pH for 24 h (one-way ANOVA, post test for linear trend (p = 0.0008)). (c) IL-8 expression was determined by RT-qPCR in EndoC-βH2 cells cultured at pH 7.4 or 6.4 for the indicated time points (one-way ANOVA, post test for linear trend (p = 0.067)). (d) EndoC-βH2 cells were cultured at pH 7.4 or 6.4 for 24 h/48 h/72 h or with PMA (100 ng/ml for 12 h) (one-way ANOVA). Secreted IL-8 was quantified by ELISA. (e) Immunostaining for IL-8 in EndoC-βH2 cells cultured at pH 7.4 or 6.4 for 72 h or with PMA (100 ng/ml) for 12 h. Scale bar 10 μm. Data are mean ± SEM of 3–5 experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. IL-8 induction upon extracellular acidification is RFX6- and GPR68-dependent.

(a) EndoC-βH2 cells were cultured for 24 h at pH 7.4 or 6.4 with or without YM-254890 (100 nM), a G_q/11-selective inhibitor compound. IL-8 expression was analyzed by RT-qPCR. (b) EndoC-βH2 cells were transfected with siRNA (siNT), siGPR68 or siRFX6. After 48 h, cells were cultured for an additional 24 h period at pH 7.4 or 6.4 and next analyzed for the expression of IL-8 by RT-qPCR. Data are mean ± SEM of 3–5 experiments. **p < 0.01; ***p < 0.001 (one-way ANOVA, followed by a Tukey’s multiple comparisons post-test).

Figure 6. IL-8 induction in EndoC-βH2 cells by extracellular acidification is NF-кB dependent.

(a) EndoC-βH2 cells were cultured at pH 7.4 or 6.4 for 12 h or with PMA (100 ng/ml for 8 h) and analyzed by immunofluoresence for the nuclear accumulation of RELA (p65) component of NF-кB complex. Scale bar 10 μm. (b) EndoC-βH2 cells were cultured at pH 7.4 for 8 h, at pH 6.4 for 1, 2 and 8 h or with PMA (100 ng/ml for 8 h). Proteins were extracted and used for Electrophoretic mobility shift assay (EMSA) analysis using a [³²P] radiolabeled NF-кB probe. (c) EndoC-βH2 cells were treated with 1 μM NF-кB Activation inhibitor-II, JSH-23 for 24 h and analyzed for IL-8 expression by RT-qPCR (one-way ANOVA). (d) EndoC-βH2 cells were transfected with either control siRNA (siNT) or siRNA targeted RELA (siRELA). After 48 h, siNT or siRELA transfected cells were cultured for 24 h at pH 7.4 or 6.4. Efficient RELA knock-down was verified by RT-qPCR and immunoblot analysis (left panel). The effect of pH on IL-8 expression following RELA was quantified by RT-qPCR (right panel) (one-way ANOVA). Data are mean ± SEM of 3–5 experiments. **p < 0.01 and ***p < 0.001.

Figure 7. IL-8 secreted by EndoC-βH2 cells in acidic conditions attracts neutrophils.

(a) Neutrophils were isolated from whole blood using MACSxpress kit, analyzed for the expression of CD16 and used for in-vitro transwell migration assay. (b) Neutrophil chemotaxis was tested using conditioned media from EndoC-βH2 cells cultured at pH 7.4 or pH 6.4 for 72 h. Acidic pH 6.4 conditioned medium pre-treated for 10 min with Anti-human IL-8 (1 μg/ml) was also used as well as pH 7.4 conditioned medium supplemented with recombinant human-IL-8 (50 ng/ml). Data are represented as migration index, calculated by assigning a value of 1 to the number of migrating neutrophils towards pH 7.4-conditioned medium. Data are mean ± SEM of 4 blood donors. *p < 0.05; **p < 0.01 (one-way ANOVA, followed by a Tukey’s multiple comparisons post-test).