The role of ankyrin repeat and SOCS box containing protein 4 (ASB4) in trophoblast differentiation and pseudovasculogenesis

Vascularization of the placenta is a critical developmental process that ensures fetal viability. The vascular health of the placenta affects both maternal and fetal well being; however, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic vascular lineage commitment and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates trophoblast differentiation during development and is a target of many ubiquitin ligases. Due to their contrasting effects during differentiation, we investigated whether ASB4 mediates vascular differentiation through its ligase activity on ID2 in the placenta. Placentas from Asb4-/- mice exhibited myriad vascular differentiation defects, including abnormal overexpression of ID2, and pregnant Asb4-/- mice phenocopied human pre-eclampsia. We determined that ASB4 directly interacted with ID2 in trophoblast cells, leading to ID2's ubiquitination and subsequent degradation. Further, ASB4 promoted placental cell differentiation and function, and co-expression of a degradation-resistant Id2 mutant abolished these effects. Together, these findings indicate that ASB4 regulates trophoblast cell differentiation into placental vasculature through the degradation of ID2 and that loss of Asb4 in the developing placenta contributes to placental disease.Doctor of Philosoph

Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem–loop rather than a poly(A) tail. The stem–loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3′ stem–loop is bound by the stem–loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP

The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease

Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease

MicroRNAs 1, 133, and 206: Critical factors of skeletal and cardiac muscle development, function, and disease

microRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. miRNAs are known to mediate myriad cell processes, including proliferation, differentiation, and apoptosis. With more than 600 miRNAs identified in humans, it is generally believed that many miRNAs function through simultaneously inhibiting multiple regulatory mRNA targets, suggesting that miRNAs participate in regulating the expression of many, if not all, genes. While many miRNAs are expressed ubiquitously, some are expressed in a tissue specific manner. The muscle specific miR-1, miR-133 and miR-206 are perhaps the most studied and best characterized miRNAs to date. Many studies demonstrate that these miRNAs are necessary for proper skeletal and cardiac muscle development and function, and have a profound influence on multiple myopathies, such as hypertrophy, dystrophy, and conduction defects

LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell ProliferationSignificance

We recently demonstrated that low-density lipoprotein receptor related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis

The Ubiquitin Ligase ASB4 Promotes Trophoblast Differentiation through the Degradation of ID2

Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that ASB4 mediates vascular differentiation in the placenta via its degradation of ID2

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt

INCREASING FOOD ACCESSIBILITY AND MENTAL HEALTH RESOURCES TO PROMOTE ADOLESCENT WELL-BEING IN A RURAL COMMUNITY

The Neighborhood and Built Environment is one of five Social Determinants of Health domains and is defined as where one lives, works, plays, and learns — all factors that affect one’s health. Cleveland County, North Carolina has a high percentage of school-age children who live in poverty, and face greater adversities including financial hardships, food insecurity, and adverse childhood experiences. The objective of this proposal is to increase the proportion of schools with policies and practices that promote health and safety. To address these issues, the nutrition-focused recommendation is to increase access to healthy foods by providing free meals to all participating County School students by expanding the Community Eligibility Provision of the National School Lunch Program. The policy-focused recommendation is to implement the “Require All Schools to Staff Mental Health Counselors” policy, encouraging a sustainable mental health support system in schools.Master of Public Healt