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The transcription factor NFATC2 induces β cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced β cell proliferation, suggesting the FOXP family works to regulate β cell proliferation in concert with NFATC2. NFATC2 induced β cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β cell proliferation.

The homeobox transcription factor Nkx6.1 is sufficient to increase functional β-cell mass, where functional β-cell mass refers to the combination of β-cell proliferation, glucose-stimulated insulin secretion (GSIS) and β-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional β-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase β-cell proliferation in primary rat islets and the INS-1 832/13 β-cell line. The increase in HDAC1-mediated proliferation occurs while maintaining GSIS and increasing β-cell survival in response to apoptotic stimuli. We demonstrate that HDAC1 overexpression results in decreased expression of the cell cycle inhibitor Cdkn1b/p27 which is essential for inhibiting the G1 to S phase transition of the cell cycle. This corresponds with increased expression of key cell cycle activators, such as Cyclin A2, Cyclin B1 and E2F1, which are activated by activation of the Cdk4/Cdk6/Cyclin D holoenzymes due to down-regulation of Cdkn1b/p27. Finally, we demonstrate that overexpression of Cdkn1b/p27 inhibits HDAC1-mediated β-cell proliferation. Our data suggest that HDAC1 is critical for the Nkx6.1-mediated pathway that enhances functional β-cell mass., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Decreased β-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional β-cell mass through transplantation or by enhanced growth of endogenous β-cells. We have shown that the transcription factor Nkx6.1 induces β-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce β-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat β-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in β-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated β-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce β-cell proliferation while maintaining glucose stimulated insulin secretion.

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.