A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions

Abstract

Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.

Figure 1. CUL4B deficiency in pancreatic δ cells impairs glucose metabolism.

(A and B) Western blots and quantitative data for CUL4A and CUL4B protein levels in islets from 12-week-old diabetic db/db mice and their heterozygous littermates (db/+). n = 6 mice per group. Representative Western blots from at least 3 independent experiments are shown. (C) Immunostaining for CUL4B (green) and somatostatin (SST, red) in pancreatic sections from db/db and db/+ mice. Scale bar: 100 μm. n = 6 mice per group; 4–7 random areas were selected from each islet section, and 10 sections were randomly selected from each mouse. (D) Confirmation of pancreatic δ cell–specific CUL4B deficiency (Sst-Cre^+/– Cul4b^fl/Y) through immunofluorescence. The colocalization of somatostatin (red) and CUL4B (green) in δ cells of WT mice was absent in Sst-Cre^+/– Cul4b^fl/Y mice. Scale bar: 100 μm. (E) Immunostaining for insulin (red) and somatostatin (green) in WT and Sst-Cre^+/– Cul4b^fl/Y mice. Scale bar: 50 μm. (F) Quantitative data for islet density, pancreatic δ cell number, and β cell mass. n = 6 mice per group; 4–10 random areas were selected from each section, and 12 sections were randomly selected from each mouse. (G) The fasting and fed blood glucose levels of Sst-Cre^+/– Cul4b^fl/Y mice and their WT littermates. n = 8 mice per group. (H) Glucose tolerance test for Sst-Cre^+/– Cul4b^fl/Y mice and their WT littermates (n = 11–12). (I) Insulin tolerance test for Sst-Cre^+/– Cul4b^fl/Y mice and their WT littermates. Insulin-induced decreases in blood glucose levels were significantly lower in Sst-Cre^+/– Cul4b^fl/Y mice than in their WT littermates, and they did not return to baseline levels at the 2-hour time point, whereas the levels of their WT littermates did (n = 9–11). *P < 0.05; **P < 0.01; ***P < 0.001. db/db mice were compared with their db/+ littermates, and Sst-Cre^+/– Cul4b^fl/Y mice were compared with their WT littermates. Error bars in F represent mean ± SD; other bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 2. Impaired glucose tolerance in Sst-Cre^+/– Cul4b^fl/Y mice is due to increased somatostatin paracrine signaling.

(A and B) Plasma insulin levels (A) and somatostatin levels (B) of WT and Sst-Cre^+/– Cul4b^fl/Y mice in the fasted and fed states. n = 6–7 mice per group. (C and D) 1 mM, 5.5 mM, and 20 mM glucose-induced insulin (C) and somatostatin (D) secretion in islets isolated from Sst-Cre^+/– Cul4b^fl/Y mice and WT littermates after 5 minutes. n = 5 mice per group. (E and F) Glucose- and high KCl–induced insulin secretion (E) or somatostatin secretion (F) of islets isolated from WT and Sst-Cre^+/– Cul4b^fl/Y mice after 5 minutes. n = 4 mice per group. (G and H) Short- and long-term glucose-induced insulin secretion (G) and somatostatin secretion (H) of islets isolated from WT mice and Sst-Cre^+/– Cul4b^fl/Y mice. n = 4 mice per group. (I) Effect of the somatostatin receptor antagonist cyclosomatostatin (12 μM) on glucose-induced insulin secretion of islets isolated from Sst-Cre^+/– Cul4b^fl/Y mice and WT littermates after 5 minutes. n = 4 mice per group. (J) Glucose- or high KCl–induced somatostatin secretion from CUL4B knockdown and control TGP52 cells after 1 hour (n = 5). A–I, *P < 0.05; **P < 0.01. Sst-Cre^+/– Cul4b^fl/Y mice were compared with their WT littermates. J, *P < 0.05. CUL4B knockdown cells were compared with control shRNA knockdown cells. Bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 3. L-type calcium channels play key roles in enhanced somatostatin secretion from CUL4B-deficient δ cells.

(A) Effect of diazoxide (250 μM), a KATP channel opener, on glucose-induced somatostatin secretion in islets isolated from Sst-Cre^+/– Cul4b^fl/Y mice and WT littermates after 5 minutes. (n = 4). (B) Effect of diazoxide on glucose-induced insulin secretion in islets isolated from Sst-Cre^+/– Cul4b^fl/Y or WT littermates after 5 minutes (n = 4). (C) Glucose-induced somatostatin secretion from islets in the absence or presence of the R-type channel blocker SNX482 (100 nM) and the L-type channel blockers isradipine (10 μM) and nicardipine (10 μM) after 5 minutes (n = 4). (D) Glucose-induced insulin secretion from islets in the absence or presence of an R-type channel blocker or L-type channel blockers after 5 minutes (n = 4). (E and F) Representative curves and statistical analysis of a high glucose–induced calcium signal in isolated pancreatic δ cells from Sst-Cre^+/– GFP^fl/+ Cul4b^fl/Y mice (E, lower panel) and WT littermates (E, upper panel) (n = 8). (G and H) Representative curves and statistical analysis of high KCl–induced calcium signal in δ cells from Sst-Cre^+/– GFP^fl/+ Cul4b^fl/Y mice (G, lower panel) or WT littermates (G, upper panel) (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001. Sst-Cre^+/– GFP^fl/+ Cul4b^fl/Y mice were compared with their WT littermates. Bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 4. CRHR2 activity and the cAMP signaling pathway are important in increased somatostatin secretion.

(A) Glucose-induced somatostatin secretion in islets isolated from WT and Sst-Cre^+/– Cul4b^fl/Y mice treated with inhibitors for PKA (H89: 10 μM), cAMP (Rp-cAMP: 100 μM), ERK (U0126: 10 μM), PLC (U73122: 10 μM), or AC (DDA: 100 μM) after 5 minutes (n = 3). (B) Glucose-induced cAMP signals in CUL4B knockdown and control TGP52 cells (n = 4). (C–D) Effects of Ast2B on glucose-induced insulin (D) and somatostatin (C) secretion in islets isolated from Sst-Cre^+/– Cul4b^fl/Y or WT littermates after 5 minutes (n = 4). A, C, D, *P < 0.05; ***P < 0.001. Sst-Cre^+/– Cul4b^fl/Y mice were compared with their WT littermates. B, *P < 0.05; ***P < 0.001. CUL4B knockdown cells were compared with control shRNA knockdown cells. Bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 5. Transcriptional upregulation of Cav1.2 and AC6 contributes to the enhanced somatostatin secretion in Cul4b-deficient pancreatic δ cells.

(A) The mRNA levels of L-type calcium channels, adenylyl cyclases, G proteins, PKA and transcription factors in CUL4B knockdown and control TGP52 cells, as determined by quantitative reverse-transcriptase PCR (qRT-PCR) (n = 3). (B–E) Cav1.1, Cav1.2, Cav1.3, AC6, AC7 and Gs protein levels in CUL4B knockdown and control TGP52 cells. B and D, representative Western blots from at least 3 experiments are shown. C and E, bar graph representation and statistical analyses of B and D, respectively. (F) qRT-PCR was used to analyze the mRNA levels of L-type calcium channel α subunits and adenylyl cyclases in primary δ cells from Sst-Cre^+/– GFP^fl/+ Cul4b^fl/Y and WT littermates (n = 7). (G) Effects of Cav1.2 or Cav1.3 knockdown on somatostatin secretion from CUL4B knockdown and control TGP52 cells (n = 4). (H) Effects of AC6 or AC7 knockdown on somatostatin secretion from CUL4B knockdown and control TGP52 cells. (I) Glucose-induced cAMP levels in AC6 or AC7 knockdown and control TGP52 cells, with or without CUL4B knockdown. Cells were incubated with 20 mM glucose for 15 minutes.*P < 0.05; **P < 0.01; ***P < 0.001. Sst-Cre^+/– GFP^fl/+ Cul4b^fl/Y mice were compared with their WT littermates, and CUL4B knockdown cells were compared with control shRNA-treated cells. The bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 6. CUL4B negatively regulates Cav1.2 and AC6 expression in pancreatic δ cells through an epigenetic mechanism.

(A–C) Levels of ubiquitinated Cav1.2 and AC6 in CUL4B overexpression and control TGP52 cells. HA-tagged ubiquitin was transiently transfected into WT and CUL4B overexpression TGP52 cell lines in the presence or absence of 10 nM MG-132. The ubiquitinated proteins were pulled down with anti-HA beads, and ubiquitinated Cav1.2 and AC6 were detected with specific antibodies. Representative Western blots from at least 3 independent experiments are shown. (D–I) qChIP analysis of the recruitment of the indicated proteins on specific regions of the Cav1.2 promoter in CUL4B knockdown and control TGP52 cells. Whereas CUL4B, DDB1, K119-ubiquitinated-H2A, EZH2, and K27-3–methylated H3 protein bound to the –1500 to –1219 region of the Cav1.2 promoter, K4-3–methylated H3 bound to the –1060 to –900 region (n = 3). (J) qChIP analysis of the recruitment of CUL4B on specific regions of the Cav1.2 and AC6 promoter in HHEX knockdown and control TGP52 cells (n = 4). (K and L) qChIP analysis of HHEX binding patterns of the Cav1.2 and AC6 promoters in TGP52 cells (n = 4). (M) Coimmunoprecipitation of HHEX and the CRL4B-PRC2 component in TGP52 cell lines. TGP52 cells were lysed and incubated with HHEX antibody or normal rabbit/mouse IgG on a rotator at 4°C overnight, followed by the addition of protein A/G beads for 2 hours at 4°C. Beads were then washed 5 times with lysis buffer. *P < 0.05; **P < 0.01; ***P < 0.001. CUL4B knockdown cells were compared with control shRNA-treated cells. Bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 7. CUL4B and DDB1 regulate somatostatin secretion by recruiting EZH2 to constitute a functional CRL4B-PRC2 complex at the promoter regions of Cav1.2 and AC6.

(A and B) ChIP and Re-ChIP (sequential ChIP) experiments revealed that CUL4B, DDB1, and EZH2 form multiple protein complexes in the promoter regions of Cav1.2 (A) and AC6 (B) in TGP52 pancreatic δ cells. (C and D) qChIP analysis of the Cav1.2 (C) and AC6 (D) promoters in CUL4B, DDB1, or EZH2 knockdown TGP52 cells using the indicated antibodies. The results indicate that CRL4B promotes the recruitment of PRC2 to the promoters of Cav1.2 and AC6 (n = 3). (E and F) Western blot analysis demonstrated increased expression of Cav1.2 and AC6 in DDB1 (E) and EZH2 (F) knockdown TGP52 cells. A representative Western blot from at least 3 experiments is shown. (G) High glucose–induced somatostatin secretion in CUL4B, DDB1, and EZH2 knockdown TGP52 cells after 15 minutes of stimulation (n = 3). (H) Schematic of the sequential IP strategy and a model of CUL4B/DDB1/EZH2 assembly at the promoters of Cav1.2 and A6. *P < 0.05; **P < 0.01. CUL4B, DDB1, and EZH2 knockdown cells were compared with control shRNA- or siRNA-treated cells. Bars represent mean ± SEM. All data were analyzed using 1-way ANOVA.

Figure 8. Reciprocal regulation of the CUL4B/PRC2 complex, Cav1.2, and AC6 by glucose and UCN3 in pancreatic δ cells.

(A and B) Protein-level changes in CUL4B, EZH2, and DDB1 expression in TGP52 cells stimulated with 20 mM glucose for the indicated time intervals. (C, D, I, and J) Protein-level changes in Cav1.2 and AC6 expression in CUL4B knockdown and control TGP52 cells stimulated with 20 mM glucose for the indicated time intervals. (E and F) Protein level changes in CUL4B, EZH2, DDB1, Cav1.2, and AC6 expression in TGP52 cells stimulated with 100 nM UCN3. (G and H). The mRNA levels of Cul4b, Cav1.2, and AC6 in primary isolated islet δ cells treated with 20 mM glucose (G) or 100 nM UCN3 (H) for the indicated number of hours. (K and L) Protein level changes in CUL4B, EZH2, DDB1, Cav1.2, and AC6 expression in TGP52 cells stimulated with 100 nM UCN3 and CRHR2-selective antagonist Ast2B for the indicated time intervals. (A, C, E, I, and K) Representative Western blots from at least 3 independent experiments are shown. (B, D, F, J, and L) Statistical analyses of A, C, E, I and K, respectively. (M and N) AC inhibitor DDA (100 μM) and L-type channel blocker isradipine (10 μM) completely blocked the enhanced somatostatin secretion from human islets incubated with UCN3 (100 nM) (M) or MLN4924 (200 nM) (N) compared with their control islets. *P < 0.05; **P < 0.01; ***P < 0.001. Glucose- and UCN3-stimulated cells were compared with nonstimulated cells, and human islets incubated with UCN3 were compared with control islets. Bars represent mean ± SEM. All data were analyzed through 1-way ANOVA.

Figure 9. Graphic model of the CRL4B-PRC2 complex–mediated regulation of pancreatic somatostatin paracrine signaling and of adjustments in glucose homeostasis in response to high glucose or UCN3 stimulation.

As demonstrated by previous studies, paracrine factor UCN3 and insulin were coreleased from the same vesicles from pancreatic β cells (5). The secreted UCN3 increases somatostatin secretion in response to high glucose stimulation in pancreatic islets, and L-type channel activity is required for the potentiation effects of UCN3 (5). Here, we found that in response to sustained UCN3 stimulation, the expression levels of CUL4B and EZH2, the core components of CRL4B-PRC2, decreased in pancreatic δ cells. Under the control of HHEX, the CRL4B-PRC2 complex epigenetically regulates the expression of calcium channel Cav1.2 and adenylyl cyclase AC6 through the modulation of methylation and ubiquitination states of the histone proteins that bind to the promoters of these 2 proteins in pancreatic δ cells. The decreased expression of CUL4B and EZH2 releases the repressive role of the CRL4B-PRC2 complex in the expression of Cav1.2 and AC6, thereby increasing levels for second messengers Ca²⁺ and cAMP in pancreatic δ cells and ultimately leading to increased somatostatin secretion levels. Consistent with a previous study (5), this feedback loop is essential for avoiding excessive sustained insulin secretion in a timely manner and for coordinating normal glucose homeostasis. In contrast, the protein levels of CUL4B and EZH2 increase in response to sustained high glucose stimulation. The upregulated CRL4B-PRC2 complex function decreases the expression of Cav1.2 and AC6, which in turn decreases somatostatin secretion and thus ensures sufficient insulin secretion for maintaining glucose homeostasis. The epigenetic regulation of somatostatin secretion by the CRL4B-PRC2 complex is important for maintaining glucose homeostasis.