The data presented in this study suggest a critical role for Nkx2.2 in maintaining mature β-cell function and forming correct islet architecture. Consistent with our previous findings that Nkx2.2 functions as a repressor during embryonic islet cell specification (10
), embryonic pancreas development and differentiation proceed normally in the Nkx2.2-repressor mice until birth, when the Nkx2.2-repressor derivative interferes with the expression and function of endogenous Nkx2.2, leading to a reduction in MafA, Glut2, and insulin gene expression in mature β-cells. Furthermore, the islets of the Nkx2.2-repressor mice fail to cluster properly, and disrupted islet architecture persists throughout adulthood. In adult Nkx2.2-repressor mice, β-cells display impaired insulin secretion, suggesting that Nkx2.2 is also an important regulator of mature β-cell function. We are currently exploring whether the defects observed in the perinatal islets contribute to the functional defects that are manifest later in the adult β-cell.
In this analysis we used a dominant repressor derivative of Nkx2.2 to assess the function of Nkx2.2 in the mature islet. Although there are limitations associated with this approach and the transgene does not contain regions of the Nkx2.2 protein that may contribute to the specificity of DNA binding, we have been able to demonstrate that the Nkx2.2hdEnR transgene is able to functionally substitute for endogenous Nkx2.2 in the embryo (10
). Furthermore, we have determined that the observed adult β-cell phenotypes are not caused by inordinately high levels of transgene expression that could lead to transcriptional squelching effects or misregulation of gene targets of the closely related pancreatic transcription factor Nkx6.1; however, we cannot definitively rule out the possibility that other nonspecific off-target binding of the transgene is occurring. Finally, we have shown that the Nkx2.2 repressor is able to interfere with the expression of two known endogenous Nkx2.2 targets, Nkx2.2 itself and MafA, suggesting that the dominant transgene is functioning to block endogenous Nkx2.2 activity in the adult islet. It is possible that this reduction of Nkx2.2 in the islet is a major contributing factor to the disruption of normal β-cell function.
We observed two β-cell defects related to the expression of the Nkx2.2 repressor: a reduction of insulin content and a defect in insulin secretion. Insulin mRNA levels are moderately reduced, and insulin protein content is decreased in the adult islet of Nkx2.2-repressor mice by ~2-to 3-fold. These findings are consistent with previous in vitro studies, which suggested that Nkx2.2 regulates insulin transcription directly (16
); however, we also observed a reduction of MafA, which could contribute to the decrease in insulin gene expression. We did not detect changes in other known regulators of insulin transcription, including Pdx1, NeuroD, and Islet1 (), suggesting that the defect is primarily associated with transcriptional targets of Nkx2.2. Interestingly the decrease in circulating insulin upon glucose challenge in the Nkx2.2-repressor males is much more severe than was expected from the moderate reduction of islet insulin content; therefore, there may be additional defects associated with the insulin secretion pathway. The decreased expression of Glut2 probably contributes to the insulin secretion defects.
Our discovery that the Nkx2.2hdEnR transgene does not disrupt embryonic pancreas development but does interfere with adult β-cell functions strengthens our hypothesis that Nkx2.2 functions normally as a repressor during embryonic development but as an activator in the mature β-cell. This observation is consistent with our previous findings that the Nkx2.2 repressor can replace endogenous Nkx2.2 in the embryonic islet to rescue the immature β-cell population but is not sufficient to fully rescue the mature β-cell. An activator function for Nkx2.2 in the adult islet also supports findings by others that Nkx2.2 directly activates both its own promoter and the recently discovered insulin regulatory protein MafA (12
). It is not surprising that Nkx2.2 has complex regulatory activities in the development and maintenance of the different islet cell types. The Drosophila
ortholog of Nkx2.2, vnd, functions as either an activator or repressor, depending on its interaction with regulatory cofactors (22
). The identification of Nkx2.2 islet cofactors and additional downstream targets will be instrumental in understanding how Nkx2.2 carries out its essential roles in islet cell development and function.
A novel finding of this study is that the Nkx2.2-repressor mice display altered islet morphology, and this defect is manifest at the onset of islet formation. We have been unable to identify the underlying cause of the altered islet architecture; islet cell ratios are normal, and there are no significant alterations in expression of many of the islet cell adhesion molecules, including the integrins (β1, α3, α5, or α6), cadherins 1 and 2, Ncam, α- and β-catenins, occludin, or members of matrix metalloproteinase family (data not shown). Of note, islet architecture is also disrupted in several other transcription factor mutation models, including MafA, and in each case the contributing cause of this phenotype has yet to be ascertained (4
). At this point, we cannot distinguish whether the defects in β-cell function are due directly to inappropriate Nkx2.2-repressor activity or are a consequence of the disrupted islet architecture.
We have demonstrated that Nkx2.2 functions in the mature islet to influence the formation of the characteristic islet architecture and to maintain normal β-cell functions, including optimal expression of the insulin genes and glucose-stimulated insulin secretion. Interestingly, the β-cell defects observed in the Nkx2.2-repressor transgenic mice are similar to defects associated with several maturity-onset diabetes of the young transcription factors. Additional studies with hypomorphic and conditional Nkx2.2 alleles are ongoing to determine the mechanism by which Nkx2.2 may contribute to β-cell dysfunction.