The Role Of Distal-Less (Dlx) 3 In The Pathophysiology Of Placental Development

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Proper placentation is critical for normal fetal growth, development, and fitness throughout gestation. This research examines the central hypothesis that Dlx3 and the gene targets of Dlx3 help to direct normal placental labyrinth morphogenesis in the mouse placenta. Further, misregulation of key features of this gene network appears to be correlated with changes in gene expression associated with the pathophysiology of preeclampsia (PE). Yeast-two hybrid screening of a placental cDNA library identified Smad6 as a binding partner of Dlx3. Investigation using immunoprecipitation assays mapped the Smad6 interaction domain to Dlx3 residues 80-163. Further, studies indicate Smad6 has the ability to repress Dlx3 transcriptional activity on the human glycoprotein hormone [alpha] subunit promoter in JEG3 cells likely through allosteric interference. Gene profiling using Affymetrix microarray was performed on RNA from wildtype and Dlx3-/- placentas and identified 401 genes that were differentially expressed following the loss of Dlx3, including expression of matrix metalloproteinase (MMP-) 9, an extracellular matrix (ECM) remodeler. Analysis of cultured media using Western blot and zymography from Dlx3 +/+, +/-, and -/- placental explants corroborated these findings. Using luciferase reporter gene analysis and DNA binding studies, Dlx3 was shown to bind and transactivate a 1.9kb portion of the mouse MMP9 gene promoter in a dose dependent manner in JEG3 cells. Further siRNA-mediated reduction of Dlx3 resulted in decreased human MMP-9 protein levels and activity. Consistent with this result, chromatin immunoprecipitation (ChIP) assays revealed Dlx3 is capable of binding to the human MMP-9 gene promoter. Lastly, Dlx3 and MMP-9 were both coordinately misregulated in a mouse model of preeclampsia. These studies indicate the potential role for the interaction between Dlx3 and MMP-9 during placental development and potentially the causal pathophysiology of PE. Analysis of fetal weights during gestation revealed a potential intrauterine growth retardation (IUGR) phenotype in Dlx3+/- animals. Analysis of Dlx3+/placentas throughout gestation found evidence for an accumulation of reactive oxygen species (ROS) likely leading to a high apoptotic index in these placentas relative to controls. Antioxidant therapy using the superoxide dismutase mimetic, Tempol, resulted in amelioration of ROS and a rescue of the IUGR phenotype. Tempol treatment also rescued a subset of Dlx3-/- embryos 3 days beyond their normally identified embryonic demise. Additionally, micro computer tomography (micro-CT) and histological analyses of Dlx3+/+ and +/- placentas identified important changes in maternal spiral arteries associated with Dlx3+/- which were reversed by antioxidant supplementation. These findings support the conclusion that loss of a single Dlx3 allele is detrimental to fetal growth in utero. Antioxidant therapy rescued fetal growth abnormalities due to oxidative stress suggesting potential treatment for pregnancy complications associated with placental insufficiency. Together, these studies provide evidence for the novel role of Dlx3 in the regulation of an important gene network during placental development. This includes Dlx3-dependent regulation of a key MMP linked to placental ECM remodeling. Both Dlx3 and MMP-9 are misregulated in a mouse model of PE indicating involvement in pregnancy complications. Additionally, these studies characterize a new IUGR model due to haploinsufficiency at the Dlx3 locus. Studies also provide support for the possible benefits of gestational antioxidant therapy in controlling ROS accumulation that can occur during abnormal placentation as observed in the Dlx3 mouse model.
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