No abstract

Erythropoietin (EPO) and its receptor are highly expressed in the developing nervous system, and exogenous EPO therapy is potentially neuroprotective, however the epigenetic and transcriptional changes downstream of EPO signaling in neural cells are not well understood. To delineate epigenetic changes associated with EPO signaling, we compared histone H3 lysine 4 dimethylation (H3K4me2) in EPO treated and control fetal neural progenitor cells, identifying 1,150 differentially bound regions. These regions were highly enriched near protein coding genes and had significant overlap with H4Acetylation, a mark of active regulatory elements. Motif analyses and co-occupancy studies revealed a complex regulatory network underlying the differentially bound regions, including previously identified mediators of EPO signaling (STAT5, STAT3), and novel factors such as REST, an epigenetic modifier central to neural differentiation and plasticity, and NRF1, a key regulator of antioxidant response and mitochondrial biogenesis. Global transcriptome analyses on neural tubes isolated from E9.0 EpoR-null and littermate control embryos validated our in vitro findings, further suggesting a role for REST and NRF1 downstream of EPO signaling. These data support a role for EPO in regulating the survival, proliferation, and differentiation of neural progenitor cells, and suggest a basis for its function in neural development and neuroprotection.

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.

The observed declines in nitrofen-associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition.