ISL1 promotes pancreatic islet cell proliferation

Abstract

Background: Islet 1 (ISL1), a LIM-homeodomain transcription factor is essential for promoting pancreatic islets proliferation and maintaining endocrine cells survival in embryonic and postnatal pancreatic islets. However, how ISL1 exerts the role in adult islets is, to date, not clear.

Methodology/principal findings: Our results show that ISL1 expression was up-regulated at the mRNA level both in cultured pancreatic cells undergoing glucose oxidase stimulation as well in type 1 and type 2 diabetes mouse models. The knockdown of ISL1 expression increased the apoptosis level of HIT-T15 pancreatic islet cells. Using HIT-T15 and primary adult islet cells as cell models, we show that ISL1 promoted adult pancreatic islet cell proliferation with increased c-Myc and CyclinD1 transcription, while knockdown of ISL1 increased the proportion of cells in G(1) phase and decreased the proportion of cells in G(2)/M and S phases. Further investigation shows that ISL1 activated both c-Myc and CyclinD1 transcription through direct binding on their promoters.

Conclusions/significance: ISL1 promoted adult pancreatic islet cell proliferation and probably by activating c-Myc and CyclinD1 transcription through direct binding on their promoters. Our findings extend the knowledge about the crucial role of ISL1 in maintaining mature islet cells homeostasis. Our results also provide insights into the new regulation relationships between ISL1 and other growth factors.

Figure 1. ISL1 was highly expressed in adult pancreatic islets and could reduce apoptosis in HIT-T15 cells.

(A) Relative mRNA expression level of insulin in pancreatic islets from STZ mice (n = 6), Akita mice (n = 5) and db/db mice (n = 6) were measured by real-time RT-PCR. C57BL/6 mice (n = 6) were used as controls to STZ mice and Akita mice, db/w mice (n = 6) were as controls to db/db mice. Each bar represents mean ± SD (**p<0.01, *p<0.05, vs. the controls). (B) Level of ROS production was measured by flow cytometry analysis in HIT-T15 cells treated with glucose oxidase (GO) at various concentrations (0–100 mU/mL) for 4 h. (C) Level of apoptosis rate was measured by flow cytometry analysis in HIT-T15 treated with GO at various concentrations (0–100 mU/mL) for 4 h. (D) Relative level of ISL1 mRNA expression in HIT-T15 cells treated with different GO concentrations was examined by real-time RT-PCR. (E) Level of apoptosis rate was measured by flow cytometry analysis in stable ISL1 knockdown HIT-T15 cells treated with or without 5 mU/mL GO. Each bar represents mean ± SD from three samples (**p<0.01, vs. the control).

Figure 2. The expression of ISL1 was altered in adult islet mass by ISL1 overexpression or knockdown.

(A) Infection efficiency (as indicated by the percentage of GFP positive cells in gated cells) of ISL1 overexpression lentivirus or ISL1-siRNA lentivirus were detected by flow cytometry analysis after 72 h infection. Real-time RT-PCR (B, D) and Western blotting (C, E) results showed the expression level of ISL1 in ISL1 overexpressed (B and C) islet cells and in ISL1 knockdown (D and E) islet cells. Data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD (**p<0.01, vs. the control). Lentivirus without any insert was used as a control.

Figure 3. ISL1 promoted proliferation of adult pancreatic cells.

Adult pancreatic islet cells infected with ISL1 lentivirus (A) or ISL1-siRNA lentivirus (B) were subjected to cell cycle analysis by flow cytometry. The data represent 3 independent experiments. The representative cytometric results from these experiments are shown. EdU incorporation assay was analyzed by confocal microscopy (scale bar, 50 µm; scale bar in magnified field, 10 µm) in adult islets infected with ISL1 lentivirus (C) or ISL1-siRNA lentivirus (D). (E) The EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst33342 positive cells. Each bar represents mean ± SD from 3 samples (**p<0.01, vs. the control).

Figure 4. ISL1 promoted proliferation of HIT-T15 cells.

The expression of ISL1 in stable overexpression (A) or knockdown (B) HIT-T15 cell lines was examined by RT-PCR and Western blotting. The cell proliferation was determined by CCK-8 analysis. A total of 1×10³ cells (either stably overexpressing (C) ISL1 or stably knockdown (D) ISL1) per well were seeded in 96-well plate and measured for their proliferation after 12 h, 24 h, 48 h, and 72 h. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD (**p<0.01, *p<0.05, vs. the control). The cell cycle profile was analyzed by flow cytometry in ISL1 (E) and ISL1-siRNA (F) stable HIT-T15 cell lines. EdU incorporation was detected by confocal microscopy (scale bar, 50 µm) in ISL1 (G) and ISL1-siRNA (H) stable cells. (I) Stable cells were maintained in G418 or puromycin-containing medium for 21 days before staining with crystal violet and counting for colony numbers. Each bar represents mean ± SD from 3 samples (**p<0.01, *p<0.05, vs. the control).

Figure 5. ISL1 promoted the expressions of c-Myc and CyclinD1.

The mRNA level (A, C) and protein level (B, D) of c-Myc and CyclinD1, Cyclin A, p53 were analyzed by real-time RT-PCR and Western blotting in ISL1 stable overexpression HIT-T15 cells (A and B) and ISL1 knockdown HIT-T15 cells (C and D). The transcriptional activity of ISL1 was analyzed by luciferase reporter assay. ISL1 activated the promoter of c-Myc (E) or CyclinD1 (F) in a dose-dependent manner. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD (**p<0.01, *p<0.05, vs. the control).

Figure 6. ISL1 band on the c-Myc or Cyclin D1 promoter directly.

Consensus binding site (TAAT, box) for ISL1 on the c-Myc (A) or CyclinD1 (B) promoter was analyzed by Matinspector software. (C and D) Nuclear extracts were subjected to EMSA for the ISL1 proteins binding ability to the ³²P-labeled oligonucleotides containing the consensus sequence of the c-Myc or CyclinD1 promoter. (C) Lane 1, free probe. Lane 2, nuclear extracts from HIT-T15 cell without transfection. Lanes 3–9, nuclear extracts from HIT-T15 cells transfected with ISL1 expression construct. Lane 3 shows the direct binding of ISL1. Lanes 4 and 5 show wild-type unlabeled oligonucleotide competition. Lanes 6 and 7 show mutant unlabeled probe competition. Lane 8, the mouse normal IgG was used as a negative control. Lane 9, 1 µg anti-ISL1 antibody (H00003670-M05, Abnova) was added. (D) Lanes 1–7, same as C, Lane 8, rabbit normal IgG was used as a negative control. Lane 9, 1 µg unrelated rabbit anti-GATA4 was added. Lane 10, mouse normal IgG was used as a negative control. Lane 11, 1 µg mouse anti-ISL1 polyclonal antibody was added. (E and F): ISL1 recruited on the c-Myc or Cyclin D1 promoter was analyzed by ChIP assay. Soluble chromatin was prepared from HIT-T15 cells stably transfected with 2 µg of pcDNA3.1-ISL1 plasmid followed by immunoprecipitation with antibodies against ISL1. The DNA extractions were amplified using the primers that cover the ISL1 binding sites on the c-Myc or Cyclin D1 promoter by PCR (E) or real-time PCR (F) with normal IgG as a control. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD (**p<0.01, *p<0.05, vs. the control).