Nkx6.1 regulates islet β-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors

Abstract

Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

Fig. 1.

Overexpression of Nkx6.1 induces expression of Nr4a1 and Nr4a3. Rat islets were treated with AdCMV-GFP or AdCMV-Nkx6.1. (A and B) Nr4a1 (A) and Nr4a3 (B) mRNA levels. (C) Immunoblot analysis of whole-islet lysates 48 h after viral transduction. Data in A--C represent the means ± SEM of three independent experiments. (D and E) ChIP analysis of endogenous Nkx6.1 binding to the Nr4a1 (D) and Nr4a3 (E) promoter regions relative to a nonbound amplicon in INS-1–derived 832/13 cells. Data represent means ± SEM of six independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. P values represent the comparison between Nkx6.1- and GFP-treated islets for RT-PCR and immunoblots and the comparison between anti-Nkx6.1 and IgG-precipitated samples in ChIP analyses.

Fig. 2.

Overexpression of Nr4a1 or Nr4a3 is sufficient to induce β-cell proliferation. (A) Immunoblot analysis demonstrating adenovirus-mediated overexpression of Nkx6.1, Nr4a1, or Nr4a3. (B) Incorporation of [³H-methyl]thymidine in rat islets. (C) Islets were treated with lentiviruses expressing GFP or Nkx6.1 or left untreated, and [³H-methyl]thymidine incorporation was measured after 136 h. (D) Percentage of EdU⁺insulin⁺ islet cells cultured with EdU for 96 h. (E) Percentage of EdU⁺insulin⁺ islet cells that are also PHH3⁺. (F) Representative images (100× magnification) of islets labeled with EdU. Solid arrows indicate insulin⁺EdU⁺ nuclei, and broken arrows indicate insulin⁻EdU⁺ nuclei. DAPI is blue, insulin is green, and EdU is pink. (G and H) Treatment of human islets with AdCMV-Nkx6.1 increases Nr4a1 (G) and Nr4a3 (H) mRNA levels. (I) [³H-methyl]thymidine incorporation into human islets measured 96 h after indicated treatments. Data represent means ± SEM of a minimum of three independent experiments; human islet data represent eight independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. All comparisons with AdCMV-GFP–treated islets.

Fig. 3.

Nr4a1 and Nr4a3 are necessary for Nkx6.1-mediated proliferation. Rat islets were treated with AdCMV-Nkx6.1 and either Ad-siNr4a1, Ad-siNr4a3, or Ad-siControl (siCTRL) for 96 h. (A) Immunoblot assay of whole-islet lysates. (B) [³H-methyl]thymidine incorporation. Rat islets were treated with adenoviruses expressing GFP, Nkx6.1, or the dominant-negative Nr4a construct Nr4a1-M1 (M1), followed by immunoblot assay of whole-islet lysates (C). (D) [³H-methyl]thymidine incorporation. Data represent means ± SEM of a minimum of three independent experiments. **P ≤ 0.01; ***P ≤ 0.001 compared with AdCMV-Nkx6.1 plus Ad-siControl (B) or Nkx6.1-treated islets (D).

Fig. 4.

Mice deficient for Nr4a1 have decreased β-cell area and replication. (A) β-Cell area in pancreas sections from 11-d-old wild-type (WT) and Nr4a1 knockout (KO) mice, expressed as β-cell area relative to total pancreas area. (B) β-Cell area in pancreas sections from 2-mo-old WT or Nr4a1 KO mice. (C) Representative images at 20× magnification of pancreata from WT or Nr4a1 KO mice at 11 d of age. Insulin is red, amylase is green, and DAPI is blue. The percentage of Ki-67⁺insulin⁺ (D) and PHH3⁺insulin⁺ (E) cells was calculated at 11 d of age. *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 5.

Nkx6.1 up-regulates APC components, leading to degradation of p21. (A) Overexpression of Nr4a1 or Nr4a3 up-regulates expression of Ube2c mRNA at 48 h. (B) Nkx6.1-mediated up-regulation of Ube2c at 96 h is dependent on the expression of Nr4a1 and Nr4a3. (C) Overexpression of Nkx6.1 induces expression of p21 mRNA. (D) Immunoblot analyses demonstrate that p21 protein levels decrease between 48 and 72 h after Nkx6.1 overexpression. Data represent means ± SEM of three independent experiments. For A and D, P values represent the comparison with GFP-treated islets. For B, P values represent comparison with AdCMV-Nkx6.1 plus Ad-siControl islets. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Fig. 6.

Loss of p21 stimulates β-cell proliferation (A–E) and summary model (F). (A) p21 protein levels are maintained at baseline levels 48 h after Nr4a1 or Nr4a3 overexpression. (B) Overexpression of Ube2c for 72 h in INS-1 832/13 cells decreases p21 protein levels. (C) Treatment of islets with Ad-siUbe2c decreases [³H-methyl]thymidine incorporation in response to Nkx6.1 overexpression. (D) Percentage of insulin⁺Edu⁺ and insulin⁻Edu⁺ islet cells in the presence and absence of p21 knockdown. (E) Percentage of EdU⁺insulin⁺ cells that are also PHH3⁺. Data represent means ± SEM of a minimum of three independent experiments. For A, B, and E, P values represent comparison with GFP-treated islets. For C, the P value is for the comparison with AdCMV-Nkx6.1 plus Ad-siControl islets. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. (F) Schematic summary of bipartite mechanism of Nkx6.1-activated β-cell proliferation.