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

Chandra V, Karamitri A, Richards P, Cormier F, Ramond C, Jockers R, Armanet M, Albagli-Curiel O, Scharfmann R - Sci Rep (2016)

Bottom Line: However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1016, Institut Cochin, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, 75014, France.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2: 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.

Mentions: RFX6 is a key transcription factor highly expressed in β-cells and required for their function. Our previous transcriptomic analyses indicated that siRNA-mediated RFX6 knock-down decreased GPR68 expression in EndoC-βH2 cells [FC, −3.85; p = 7.78.10−5] (GEO No: GSE59049) without effecting the expression of other proton sensing receptors23. Further validation by RT-qPCR showed that decreased expression level of RFX6 mRNA (63.92 ± 10.5%) was consistently accompanied by decrease in the level of GPR68 mRNA (59.73 ± 15%) in EndoC-βH2 cells (Fig. 2a). Similar results were obtained in human islets where decreased RFX6 expression (79.34 ± 13%) resulted in a 42.15 ± 10% decrease of GPR68 transcripts (Fig. 2b). Additionally, overexpression of wtRFX6 but not p.V506G mutant RFX623, increased the expression of GPR68 transcripts (Fig. 2c). GPR68 expression was also enhanced following transfection of EndoC-βH2 cells with a trans-activation domain VP16 conjugated RFX6 (Fig. 2d), while transfection of a trans-repression domain conjugated KRAB-RFX6 in EndoC-βH2 cells resulted in a decreased expression of GPR68 (Fig. 2e). GPR68 is thus the major proton sensor expressed in β-cells, its expression being tightly controlled by RFX6.


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

Chandra V, Karamitri A, Richards P, Cormier F, Ramond C, Jockers R, Armanet M, Albagli-Curiel O, Scharfmann R - Sci Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4863151&req=5

f2: 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.
Mentions: RFX6 is a key transcription factor highly expressed in β-cells and required for their function. Our previous transcriptomic analyses indicated that siRNA-mediated RFX6 knock-down decreased GPR68 expression in EndoC-βH2 cells [FC, −3.85; p = 7.78.10−5] (GEO No: GSE59049) without effecting the expression of other proton sensing receptors23. Further validation by RT-qPCR showed that decreased expression level of RFX6 mRNA (63.92 ± 10.5%) was consistently accompanied by decrease in the level of GPR68 mRNA (59.73 ± 15%) in EndoC-βH2 cells (Fig. 2a). Similar results were obtained in human islets where decreased RFX6 expression (79.34 ± 13%) resulted in a 42.15 ± 10% decrease of GPR68 transcripts (Fig. 2b). Additionally, overexpression of wtRFX6 but not p.V506G mutant RFX623, increased the expression of GPR68 transcripts (Fig. 2c). GPR68 expression was also enhanced following transfection of EndoC-βH2 cells with a trans-activation domain VP16 conjugated RFX6 (Fig. 2d), while transfection of a trans-repression domain conjugated KRAB-RFX6 in EndoC-βH2 cells resulted in a decreased expression of GPR68 (Fig. 2e). GPR68 is thus the major proton sensor expressed in β-cells, its expression being tightly controlled by RFX6.

Bottom Line: However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive.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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1016, Institut Cochin, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, 75014, France.

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.

No MeSH data available.


Related in: MedlinePlus