Pancreatic islet β-cell mass is controlled by a dynamic balance between cell proliferation and cell death (apoptosis) (6
). Diabetes occurs when this balance is disrupted by autoimmune-mediated β-cell destruction (type 1 diabetes) or a failure of β-cell mass to compensate for metabolic demand (type 2 diabetes). Gaining a better understanding of molecular mechanisms that regulate β-cell replication and survival is therefore of great relevance for development of new diabetes therapies. In the present study, we provide evidence that the homeodomain transcription factor Nkx6.1 regulates adult β-cell proliferation via concerted upregulation of a host of cell cycle-regulatory genes. Moreover, unlike many prior studies in which stimulation of β-cell replication was accompanied by deterioration of β-cell function, the effects of Nkx6.1 on islet growth occurred with full retention of GSIS in human islets and enhancement of insulin secretion in rat islets.
Prior studies provide evidence that homeobox transcription factors can regulate proliferation of a variety of cell types (14
), although such a role has not been described previously for Nkx6.1. Both direct and indirect modes of regulation appear to be possible. For example, the homeobox protein Oct10 has been shown to regulate the cyclin D1 gene via direct interaction with its promoter (32
), whereas the degradation of cyclin A, B1, and E proteins is mediated through protein-protein interactions with the homeobox protein ESXR1 (36
). These studies provide a broad precedent for the idea that Nkx6.1, a homeobox protein, might influence β-cell proliferation via direct or indirect regulation of cell cycle factors, but our studies are the first to show that such mechanisms actually exist and are operative in adult β cells. Interestingly, targeted disruption of the Nkx6.1 gene resulted in mice with pancreatic islets that retained only 6% of the normal complement of β cells (41
). The decrease in β-cell mass was accompanied by a corresponding decrease in the number of BrdU-positive cells, suggesting that the loss of Nkx6.1 resulted in a decrease in the capacity of β-cell precursors to proliferate and differentiate into insulin-expressing cells. The present study helps to explain these prior results by demonstrating that Nkx6.1 can simultaneously enhance β-cell replication and function.
Despite the critical role of Nkx6.1 in β-cell development, little has been reported about its target genes prior to the present study. The microarray study reported herein reveals that in the adult β cell, Nkx6.1 functions as both an enhancer and a suppressor of gene expression to nearly equal degrees (187 genes suppressed by ≥50% and 156 genes upregulated ≥2-fold). Among the upregulated genes, 37 had ontology classifications related to proliferation, including genes encoding cyclins A2, B1, B2, and E1, Cdk1, Cdk2, Cdc6, Cdc25a, and PTTG1, whereas only seven of the suppressed genes fell into this category. Figure demonstrates schematically that Nkx6.1-upregulated genes have roles in all phases of the cell cycle, from G1 to M, and also summarizes our findings that Nkx6.1 can regulate these target genes by a diverse array of mechanisms, including direct interaction (those encoding cyclins A2 and B1) and alterations in protein levels independent of changes in mRNA (that encoding cyclin D2). The precise mechanisms by which Nkx6.1 regulates expression of other growth-related genes listed above and in Table S1 in the supplemental material remain to be investigated.
The foregoing findings were unanticipated in light of the broadly held view that Nkx6.1 functions primarily as a transcriptional repressor (34
) but are made more understandable by other recent structure/function studies. Nkx6.1 is a 364-residue polypeptide with four distinct domains. The homeodomain of Nkx6.1 binds to TAAT- or ATTA-containing promoter elements (27
), similar to core DNA-binding elements in other mammalian homeobox proteins (14
). Further specificity for Nkx6.1 binding to DNA is derived from flanking nucleotides, which extend the core motif to CATTTAATTACCCT (34
). The homeodomain fused to the VP16 activation domain activates reporter constructs containing multiple copies of the TAAT-containing consensus sequence (27
). However, in the same assay system, full-length Nkx6.1 is a potent transcriptional repressor (34
), demonstrating that the homeodomain is necessary for DNA recognition but that other domains regulate transcriptional activity, including the N-terminal repressor domain, the COOH-terminal binding interference domain region that acts to decrease the binding affinity of Nkx6.1 to its DNA target, and the COOH-terminal activation domain, which is required for Nkx6.1-mediated activation of gene expression (23
). The Nkx6.1-extrinsic factors, as opposed to the Nkx6.1-intrinsic factors, that allow repressor or activator functions to predominate in the context of specific target genes remain to be established.
Our studies appear to have defined a pathway for stimulating β-cell proliferation that is distinct from another prominent and recently emergent mechanism in which Akt1/protein kinase B stimulates islet β-cell proliferation via activation of the cyclin-dependent kinase Cdk4, which interacts with the D cyclins (5
). Other studies have confirmed that transgenic manipulation of D cyclins or Cdk4 affects islet growth and that cyclins D1 and D2 are essential for postnatal expansion of β-cell mass (17
). Moreover, adenovirus-mediated overexpression of hepatocyte growth factor, which causes upregulation of D cyclins, or overexpression of cyclin D1/Cdk4 in rodent islets increases β-cell replication while enhancing or maintaining the secretory function, respectively (12
). The growth-promoting effects of Nkx6.1 would appear to be mediated by a pathway that is largely distinct from that activated by hepatocyte growth factor or Akt1, since Nkx6.1 does not increase cyclin D1 or Cdk4 expression. The effect of Nkx6.1 instead seems to involve a broad array of effects on a distinct group of cell cycle-regulatory genes, including those encoding cyclins A, B, D2, and E and the ancillary cell cycle-regulatory genes encoding Cdk1, Cdk2, Cdc6, Cdc25a, and PTTG1. Time course studies revealed that cyclin E1 mRNA is upregulated in the first 24 h of Nkx6.1 overexpression, whereas cyclins A and B were upregulated at later time points (72 to 96 h) and cyclin D was never induced at the mRNA level. In addition, adenovirus-mediated overexpression of cyclin E was sufficient to cause a strong stimulation of [3
H]thymidine incorporation. Thus, the findings overall are consistent with a critical initiator role for cyclin E rather than D cyclins in mediating the effects of Nkx6.1. Interestingly, the cyclin E gene is not one of the genes that appear to interact with Nkx6.1 directly, based on ChIP studies performed to date. Explanations for this could include interaction of Nkx6.1 with regions of the cyclin E promoter not contained in genomic fragments studied to date, interactions only at earlier time points (the ChIP studies were performed 96 h after Nkx6.1 expression), or indirect effects of Nkx6.1 on other regulatory genes. The details of this mechanism remain to be defined.
An important finding of the present work was that Nkx6.1 overexpression is sufficient to stimulate cell division in both human and rodent islets. Interestingly, Nkx6.1 overexpression did not enhance GSIS in human islets as it did in rat islets, but neither did it cause impairment of insulin secretion. The lesser stimulation of islet cell replication and the lack of enhancement of GSIS by AdCMV-Nkx6.1 in human islets could be due to the efficiency of β-cell gene transfer in human islets being lower than that in rat islets, to subtle differences in the structure and function of hamster Nkx6.1 (the product of the gene contained in the AdCMV-Nkx6.1 adenovirus) compared to the those of the human protein, or to different levels of expression of cell cycle inhibitory or “pocket” proteins, such as p27kip1
or retinoblastoma protein (11
). Also, the genes involved in Nkx6.1-mediated enhancement of GSIS in rat islets remain to be identified. Given the strong suppression of GSIS observed for islets that were induced to grow by oncogene expression or application of certain growth factors or matrix manipulations (21
), the suppressor functions of Nkx6.1 may be used to maintain or enhance GSIS via control of genes that are normally induced in response to β-cell proliferation. These ideas remain to be investigated in future studies.
In closing, we have described a novel role for the transcription factor Nkx6.1 in the regulation of β-cell proliferation and function and demonstrated broad-scale gene repressor and activator functions of this transcription factor in the adult β cell. These findings suggest that modulation of Nkx6.1 expression or activity or alteration in Nkx6.1 target gene expression could play a role in the etiologies of both type 1 and type 2 diabetes, as dramatic changes in β-cell mass and function are at the heart of both diseases. Consistent with these ideas, Nkx6.1 expression is markedly decreased in islets of two models of β-cell dysfunction, the partially pancreatectomized rat and the Zucker diabetic fatty rat (26
). From the therapeutic perspective, development of methods for expansion of islet β-cell mass has been a long-sought-after but highly elusive goal (21
). A wide array of methods have been applied but have resulted almost universally in the loss of differentiated functions in inverse proportion to success in promoting replication (3
). Surprisingly, the Nkx6.1 gene seems to be a gene with growth-promoting properties that also contributes to maintenance of the mature β-cell phenotype, an ideal combination for enhancing β-cell function and mass in the context of both major forms of diabetes.