The evolutionary and functional significance of estrogen receptors in neuronal differentiation and cellular function
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Estrogen (E) and insulin-like growth factor I (IGF-I) promote neurite outgrowth in vivo and in cultured neurons. We examined the role of crosstalk between estrogen receptor alpha (ERalpha) and IGF-I receptor (IGF-IR) as a mediator of neurite outgrowth, specifically in control PC12 cells (PC12-C) and in PC12 cells stably transfected with ERalpha (PC12-ER cells). Both cell lines express IGF-IR. Cells were treated for one week with vehicle, 1 nM estradiol (E2) or 100 ng/ml IGF-I in the presence of either the IGF-IR antagonist JB1 or the ER antagonist ICI182,780. IGF-I significantly increased neurite outgrowth as measured both by the percentage of process-bearing cells and by absolute neurite length per cell in PC12-ER and PC12-C cells. E2 so increased neurite outgrowth only in PC12-ER cells. The IGF-I-induced increases in neurite length were blocked by either ICI182,780 or JB1 alone in both cell types. Either antagonist similarly blocked the E2-induced increase in neurite lengths in PC12-ER cells. Immunofluorescent analysis of phospho-neurofilament expression via the antibody SMI312, an axonal marker, verified that the processes extended by the PC12 cells were neurites. These data suggest that receptor crosstalk between IGF-IR and ERalpha has an important role in neurite formation and extension even in a single cell system.;The actions of IGF-I are mediated through the MAPK and PI3K signal cascades. In order to determine the further effects of IGF-I and ER crosstalk in a single cell system, PI3K and MAPK were blocked with antagonists. It was determined that blockade of both signal cascades were necessary to block neurite outgrowth and that the system forms a two armed positive feedback loop that maximally stimulates IGF-IR and ER. Akt levels have been determined to be elevated in these ER transfected PC12 cells, and that these cells undergo cell death when PI3K is inhibited.;A Genomic analysis of the ERbeta gene indicates four promoters in human, and three in rat and mouse. Of these 10 promoters only 3 have so far been identified. The mouse promoter has wrongly been assumed to have homology to the known promoter in rat. This mouse promoter actually has homology to a different promoter in rat.;Analysis of the ERbetacx promoter indicates that it has a role in hematopoiesis and tumor suppression. ERbetacx may be responsible for the blood cell defects in ERbeta knockout mice.;A complete analysis of the evolutionary precursors to human ER were analyzed in C. elegans, Drosophila, seasquirt (an early chordate), ray finned fish (pufferfish and zebrafish), rodent and human. It was determined that only a single ER related transcript exists in seasquirt and that this is likely the progenitor for both ERs and all three ERRs in man.;Whole or partial genome duplications are rare but critical events in the evolution of many organisms as a way to increase total gene number and gene diversity. It was determined that there has been at least one genome duplication in pufferfish and zebrafish supporting the tetraploid hypothesis of whole genome duplication. It is also likely that at least two duplication events occurred between early chordates and fish. Analysis of syntenic regions in fish indicates that there may be more than one duplication in zebrafish, at least for the ER genes.
- Theses and Dissertations