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ISL1 Promotes Pancreatic Islet Cell Proliferation

  • Ting Guo ,

    Contributed equally to this work with: Ting Guo, Weiping Wang

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Weiping Wang ,

    Contributed equally to this work with: Ting Guo, Weiping Wang

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Hui Zhang,

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Yinan Liu,

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Ping Chen,

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Kangtao Ma,

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

  • Chunyan Zhou

    chunyanzhou@bjmu.edu.cn

    Affiliation Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China

ISL1 Promotes Pancreatic Islet Cell Proliferation

  • Ting Guo, 
  • Weiping Wang, 
  • Hui Zhang, 
  • Yinan Liu, 
  • Ping Chen, 
  • Kangtao Ma, 
  • Chunyan Zhou
PLOS
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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 G1 phase and decreased the proportion of cells in G2/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.

Introduction

Pancreatic islets in mouse embryos are developed between embryonic day (E) 13.5 and 15.5 [1], [2]. After birth, the number of islet cells is determined by the balance of cell renewal and cell loss [3]. The dynamic change of islet cells number is essential in maintaining euglycemia and thus it is important to understand how islet cells balance in the adult pancreas is achieved, specifically the mechanisms involved in stimulating pancreatic islet cells growth and preventing pancreatic islet cells from apoptosis. The growth rate of pancreatic islet cells is normally low but changes in response to different stimuli. The cell death or apoptosis is also an important factor in maintaining the appropriate number of islet cells. Increasing reactive oxygen species (ROS) concentration is one of the major causes to induce apoptosis of cells. It is continuously derived from glucose metabolism and cannot be effectively eliminated by endogenous antioxidant enzymes. Pancreatic islet cells undergo apoptosis in either physiological or pathological conditions. There is also an evidence that pre-existing β-cells are the major source of new β-cells during adult life span and after pancreatectomy in mice [4]. However, the exact mechanisms involved in the regulation of these processes are not yet clarified. In particular, the factors involved in these important physiological or pathological conditions are not fully identified.

Insulin gene enhancer binding protein-1 (ISL1), which belongs to LIM homeobox gene family, was first discovered and cloned in 1990 [5]. ISL1 is mainly expressed in adult islet endocrine cells (α, β, γ, ε) as well in the central nervous system [6], [7]. As a key transcription factor, the functions of ISL1 involve cell fate specification and embryonic development. In pancreas its function is twofold: 1) control the four endocrine islet cell lineages development and 2) control of dorsal pancreas mesenchyme development. Complete loss of dorsal pancreatic mesenchyme and endocrine islet cells was found in ISL1 knock-out mice embryos. It has also been shown that ISL1 could regulate the expression of several islet specific genes, such as proglucagon/glucagon (α cells), somatostatin (γ cells), amylin (δ cells) and insulin (β cells), although it is not the master regulator for these genes [8], [9], [10], [11]. However, it is not clear whether ISL1 plays more important roles rather than the regulation of these endocrine hormones secretion in postnatal pancreatic islets. Recent studies demonstrate that ISL1 is required for proliferation, migration and survival of cardiac progenitor cells [12]. It also promotes proliferation and repairing of injured motor neurons [13], [14]. Overexpressing ISL1 in endothelial cells and mesenchymal stem cells can promote blood vessel formation [15]. ISL1 is also involved in the establishment of pancreatic endocrine cells during the secondary transition (E13.5–E15.5) and controls the proliferation and survival of endocrine cells during embryonic islet developmental stage [16]. However, the roles of ISL1 in adult islets are yet not clear.

Based on reports that ISL1 can promote some types of cell proliferation as mentioned above, we designed this study in order to investigate whether ISL1 plays roles in maintaining the balance of islet cell renewal and cell loss. We show that ISL1 can promote adult pancreatic islet cells proliferation and attenuate cell apoptosis against oxidative stress. The mechanism involving ISL1 in promoting adult pancreatic islet cells proliferation includes the direct activation the cell autonomous factors c-Myc and CyclinD1. Our findings advance our understanding of the roles of ISL1 in adult pancreas and provide insights into the regulation of adult pancreatic cell proliferation.

Results

ISL1 was highly expressed in adult pancreatic islets

The important role of ISL1 in pancreatic islet development has been well established. However, its role in adult pancreatic islets remains unclear. The high level of ISL1 expression in adult pancreatic islets indicates that ISL1 must play important tissue specific roles. We previously reported that ISL1 enhances the transcriptional activation of the insulin gene in vitro [17]. In order to explore the biological functions of ISL1 in adult pancreatic islets, we detected the expression of ISL1 in different diabetes animal models. To our surprise, the increasing expression of ISL1, compared to normal control mice, was detected in all diabetes models, which is not parallel to the expression of insulin (Fig. 1A). The results indicate that ISL1 may play other roles in diabetic mice other than regulating insulin.

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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).

https://doi.org/10.1371/journal.pone.0022387.g001

ISL1 reduced apoptosis in pancreatic HIT-T15 cells

It has been reported that ISL-1 controls the proliferation and survival of endocrine cells in postnatal pancreas [16]. Considering that pancreatic islet cells are challenged by continuous oxidative stress derived from the glucose metabolism throughout life, we suspected that ISL1 plays a role in anti-oxidative stress mechanism. We used glucose oxidase (GO) to generate H2O2 from glucose, mimicking oxidative stress in HIT-T15 (a pancreatic β-cell line) cells. The flow cytometry (FCM) results showed that GO strongly promoted the production of ROS (Fig. 1B and Fig. S1) and increased the level of apoptosis (Fig. 1C and Fig. S2), in a dose-dependent manner (0–100 mU/mL). Real-time RT-PCR results showed that GO also dynamically changed the level of ISL1 expression, with a peak at 5 mU/mL of GO (Fig. 1D).

To further confirm the anti-apoptosis role of ISL1, ISL1-specific siRNA (ISL1-siRNA) was designed and transferred into HIT-T15 cells. The expression of ISL1 at both mRNA and protein levels was significantly reduced by ISL1-siRNA compared to non-silence siRNA (data not shown). The FCM result showed that knockdown of ISL1 could increase HIT-T15 cells apoptosis three folds regardless GO stimulation (Fig. 1E). These results implied that ISL1 could protect cells against apoptosis under physiological or oxidative stress conditions.

ISL1 promoted pancreatic islet cells proliferation

To further define whether ISL1 plays a role in adult islets, islet mass were isolated from adult Sprague-Dawley (SD) rats and infected with ISL1 overexpressing lentivirus or ISL1-siRNA lentivirus (multiplicity of infection, MOI = 10). The infection efficiency reached approximately 53% and 66%, respectively (Fig. 2A). Real-time PCR and Western blotting results showed that the expression level of ISL1 was ameliorated ten folds in ISL1 overexpressed islet cells (Fig. 2B, 2C) and was attenuated to 30% (Fig. 2D, 2E) in ISL1 knockdown islet cells, which provided a model for further study.

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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.

https://doi.org/10.1371/journal.pone.0022387.g002

To examine the impact of ISL1 on the proliferation of adult pancreatic cells, the cell cycle profile was analyzed using propidium iodide staining and flow cytometry. Compared with the control (infected with control lentivirus), ISL1 overexpression was associated with a decreased cell population in G0/G1 phases (from 76.27±1.17% to 66.72±1.62%) and an increased cell population in the G2/M and S phases (Fig. 3A). Conversely, adult islet mass exposed to ISL1-siRNA lentivirus exhibited an increase in the proportion of cells in G1 phase (from 76.76±0.67% to 82.74±0.92%) and a decrease in the proportion of cells in G2/M and S phases (Fig. 3B). These data indicate that ISL1 plays a role in promoting adult pancreatic cells proliferation.

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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).

https://doi.org/10.1371/journal.pone.0022387.g003

Subsequently, we employed the EdU incorporation assay, a more sensitive and specific method [18], [19], to further define the function of ISL1 in promoting cell proliferation. The number of EdU positive cells was increased by 2.5 folds in ISL1 overexpressing adult islets cells relative to control cells (Fig. 3C, 3E). More importantly, the number of EdU positive cells in ISL1-siRNA lentivirus-infected cells was reduced by 40% relative to that of the cells infected with non-silencer siRNA lentivirus (Fig. 3D, 3E). These results indicate that the knockdown of ISL1 could inhibit the proliferation of adult islets in vivo, while the overexpression of ISL1 promotes adult pancreatic islet cells proliferation.

We also constructed a stable ISL1 overexpressing HIT-T15 cell line with the pcDNA3.1-ISL1 expression plasmid and a stable ISL1 knockdown HIT-T15 cell line with ISL1-siRNA. RT-PCR and Western blotting results showed that both overexpression (Fig. 4A) and knockdown (Fig. 4B) were established successfully in cell lines. Then, cell proliferation was determined by CCK-8 analysis. As shown in Fig. 4C, stable transfection of pcDNA3.1-ISL1 expression plasmid promoted the proliferation of HIT-T15 cells three folds relative to that of with pcDNA3.1 (control) after 72 h culture. As expected, the knockdown of ISL1 inhibited cells growth by 20% compared to inhibition with non-silencer siRNA after 48 h culture (Fig. 4D). EdU assay also showed more EdU positive cells in ISL1 overexpressing cells (Fig. 4G) and less EdU positive cells in ISL1 knockdown (Fig. 4H) cells, indicating that ISL1 promoted cell proliferation. These results are well in agreement with cell-cycle analysis that showed a decrease (from 75.94±1.45% to 63.78±1.76%) of the percentage of cells in G0/G1 phase and an increase (from 24.05±1.45% to 36.21±1.76% ) in the G2/M and S phases in ISL1 stable HIT-T15 cells (Fig. 4E). In contrast, ISL1 knockdown increased cell population in G0/G1 phase from 76.54±0.28% to 86.27±0.56% and decreased cell population in the G2/M and S phases from 23.45±0.28% to 13.73±0.56% (Fig. 4F). Colony formation assays revealed that ISL1 overexpressing cells resulted in a significant increase in colony number compared with the control cells; while ISL1 knockdown resulted in a significant decrease in colony number (Fig. 4I). These results further confirm that ISL1 promotes pancreatic islet cells proliferation.

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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,