Diabetes Metab J.  2019 Oct;43(5):649-658. 10.4093/dmj.2018.0232.

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3

• Lee K
• Kim H
• Lee J
• Oh CM
• Song H
• Kim H
• Koo SH
• Lee J
• Lim A
• Kim H

• KMID: 2460958
• DOI: http://doi.org/10.4093/dmj.2018.0232

Abstract

BACKGROUND
Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated.
METHODS
Pancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells.
RESULTS
Prmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3.
CONCLUSION
PRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Keyword

Diabetes mellitus; Islets of Langerhans; Neurog3 protein, mouse; Pancreas; Prmt1 protein, mouse

MeSH Terms

Animals
Diabetes Mellitus
Endocrine Cells
Gene Expression
Islets of Langerhans
Mice
Pancreas*
Phenotype
Protein Stability*
Protein-Arginine N-Methyltransferases*
Proteolysis
Stem Cells
Protein-Arginine N-Methyltransferases

Figure

• 

  Fig. 1 Pancreas-specific protein arginine methyltransferase 1 (Prmt1) knock-out (KO) (Prmt1 PKO) mice exhibit a diabetic phenotype. (A) Representative images obtained by immunofluorescence (IF) staining of PRMT1 (green), pancreatic and duodenal homeobox 1 (PDX1, red), and 4′,6-diamidino-2-phenylindole (DAPI, blue) from wild type (WT) littermates and Prmt1 PKO mouse embryos at embryonic day 10.5 (E10.5). White scale bar, 50 µm. (B) Representative images of WT, Prmt1 heterozygous PKO (Het) littermates and Prmt1 PKO (KO) mice at postnatal day 7 (P7). (C) Body weights and (D) random blood glucose levels of WT, Prmt1 heterozygous PKO (Het) littermates and Prmt1 PKO (KO) mice at 4 weeks of age. (C, D) Each dot represents an individual data set from a given group. Lines and error bars indicate mean±standard error of mean (n=16 per group). aP<0.001 by one-way analysis of variance (ANOVA) with post hoc Bonferroni's test.

• 

  Fig. 2 Pancreas-specific protein arginine methyltransferase 1 (Prmt1) knock-out (KO) (Prmt1 PKO) mice show hypoplastic pancreas with reduced β-cell mass. (A) Representative images of gastrointestinal tracts from wild type (WT) littermates and Prmt1 PKO mice at postnatal day 7 (P7). (B) Representative images obtained by hematoxylin and eosin (H&E) staining of pancreatic samples from WT littermates and Prmt1 PKO mice at P0 (black scale bar, 50 µm). (C) Representative images obtained by immunofluorescent staining of insulin (INS, green), amylase (AMY, red), mucin 1 (MUC1, white), and 4′,6-diamidino-2-phenylindole (DAPI, blue) from WT littermates and Prmt1 PKO mice at P0 (white scale bar, 50 µm). (D) Quantification of insulin-positive β-cell areas from WT littermates and Prmt1 PKO mice at P0. Each dot represents an individual data sets from a given group. Lines and error bars indicate mean±standard error of mean (n=4 per group). aP<0.001 by Student's t-test.

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  Fig. 3 Protein arginine methyltransferase 1 (PRMT1) is required for endocrine cell commitment during the secondary transition. (A) Representative images obtained by immunofluorescent staining of SRY-box 9 (SOX9) or pancreatic and duodenal homeobox 1 (PDX1, green), NK2 homeobox 2 (NKX2.2), glucagon (GCG) or neurogenin 3 (NGN3, red), mucin 1 (MUC1, white), and 4′,6-diamidino-2-phenylindole (DAPI, blue) from wild type (WT) littermates and Prmt1 PKO mouse embryos at embryonic day 13.5 (E13.5) (white scale bar, 50 µm). (B) Representative images obtained by immunofluorescent staining of SOX9 (green), NKX2.2, NKX6.1 or ISL LIM homeobox 1 (ISL1, red) and DAPI (blue) from WT littermates and Prmt1 PKO mouse embryos at E14.5 (white scale bar, 50 µm).

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  Fig. 4 Pancreas-specific protein arginine methyltransferase 1 (Prmt1) knock-out (KO) (Prmt1 PKO) mice exhibit increased numbers of neurogenin 3 (NGN3)-expressing cells during the secondary transition. (A) Representative images obtained by immunofluorescent staining of SRY-box 9 (SOX9, green), NGN3 (red), mucin 1 (MUC1, white) and 4′,6-diamidino-2-phenylindole (DAPI, blue) from wild type (WT) littermates and Prmt1 PKO mouse embryos at embryonic day 14.5 (E14.5) (white scale bars, 50 µm). (B) Representative images obtained by immunofluorescent staining of pancreatic and duodenal homeobox 1 (PDX1, green), phosphorylated histone H3 (PHH3, red) and MUC1 (white) from WT littermates and Prmt1 PKO mouse embryos at E14.5 (white scale bar, 50 µm). (C) Quantification of PHH3-positive proliferative cells relative to PDX1-positive pancreatic progenitor cells from WT littermates and Prmt1 PKO (KO) mouse embryos at E14.5. Each dot represents an individual data set from a given group. Lines and error bars indicate mean±standard error of mean (n=3 per group). aP<0.05 by Student's t-test.

• 

  Fig. 5 Protein arginine methyltransferase 1 (PRMT1) is necessary for rapid degradation of the neurogenin 3 (NGN3) protein. (A) Representative images obtained by immunofluorescent staining of NGN3 (red) and mucin 1 (MUC1, white) from wild type (WT) littermates and Prmt1 PKO mouse embryos at embryonic day 14.5 (E14.5), E16.5, and E18.5 (white scale bar, 50 µm). (B) Relative Neurog3 expression levels were assessed by quantitative reverse transcription polymerase chain reaction of pancreatic samples from WT littermates and Prmt1 PKO mouse embryos at E13.5, E14.5, E16.5, E17.5, E18.5, and postnatal day 0 (P0). Data are expressed as the mean±standard error of mean (n=5 or 6 per group). (C) Immunoblot analysis detecting NGN3 protein levels in the pancreas of WT, Prmt1 heterozygous PKO littermates and Prmt1 PKO mouse embryos at E14.5. PRMT1 and α-tubulin were included in the analysis as reference proteins. (D) mPAC cells were transduced with adenovirus expressing nonspecific RNAi or Prmt1 RNAi and transfected with a vector encoding hemagglutinin (HA)-NGN3. Cells were then treated with cycloheximide (CHX, 100 µg/mL) for the indicated durations. NGN3 protein levels were determined by immunoblot analysis. α-Tubulin was included as a reference protein. KD, knockdown. aP<0.05, bP<0.01 by Student's t-test.

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