Chemokine- and neurotrophic factor-induced changes in E2F1 localization and phosphorylation of the retinoblastoma susceptibility gene product (pRb) occur by distinct mechanisms in murine cortical cultures

Autor: Amanda S. Kopp, Gordon D. Strachan, Maya A. Koike, Kathleen L. Morgan, Kelly L. Jordan-Sciutto
Rok vydání: 2004
Předmět:
Indoles
Fluorescent Antibody Technique
Cell Cycle Proteins
Retinoblastoma Protein
Mice
Neurotrophic factors
Pregnancy
E2F1
Drug Interactions
Cycloheximide
Phosphorylation
Chemokine CCL5
Cells
Cultured
Cerebral Cortex
Protein Synthesis Inhibitors
Mice
Inbred BALB C
Microscopy
Confocal
Neurodegeneration
Cell cycle
Cell biology
DNA-Binding Proteins
Neurology
Fatty Acids
Unsaturated
Immunohistochemistry
Female
biological phenomena
cell phenomena
and immunity
Chemokines
Microtubule-Associated Proteins
endocrine system
medicine.medical_specialty
Paclitaxel
Blotting
Western
Biology
Developmental Neuroscience
Internal medicine
medicine
Animals
Nerve Growth Factors
Nuclear export signal
Brain-Derived Neurotrophic Factor
DNA
Hydrogen Peroxide
medicine.disease
Embryo
Mammalian
E2F Transcription Factors
Endocrinology
Gene Expression Regulation
Cytoplasm
Immunostaining
E2F1 Transcription Factor
Transcription Factors
Zdroj: Experimental neurology. 193(2)
ISSN: 0014-4886
Popis: The retinoblastoma susceptibility gene product (pRb) and E2F1 have been found to exhibit altered localization and increased staining in several neurodegenerative diseases. We have observed similar localization in primary murine cortical cultures treated with neurotrophic factors (NTF) or chemokines. In untreated cultures, E2F1 exhibited minimal immunostaining using the KH95 antibody, which recognizes the pRb interaction domain. In primary E16 murine cortical cultures, NTF- or chemokine-treated neurons, KH95 E2F1 staining was increased in the cytoplasm. However, an antibody recognizing the amino-terminus of E2F1 (KH20) stained the cytoplasm of both untreated and treated neurons. Taken together these results suggest that the change seen in E2F1 using the KH95 antibody is due to antigen unmasking of a carboxy-terminal epitope in response to NTF and chemokines. When we assessed staining for the hyperphosphorylated, inactive form of pRb (ppRb) in untreated cultures, ppRb was predominantly cytoplasmic. In response to NTF or chemokine treatment, staining for ppRb was observed predominantly in nuclei of neurons indicating a change in subcellular distribution. Immunoblot analysis demonstrated increased levels of ppRb in response to NTF and chemokines. Inhibitors of translation, nuclear export, and phoshpatidylinositol-3-kinase blocked NTF- and chemokine-induced nuclear ppRb localization while having no effect on E2F1 staining. Instead increased cytoplasmic KH95 E2F1 staining was dependent on cytoskeletal destabilization which did not influence ppRb localization. These findings demonstrate that alterations in ppRb distribution and E2F1 antigen availability by NTF and chemokines occur by distinct mechanisms suggesting that E2F1 function may be independent of pRb regulation in post-mitotic neurons.
Databáze: OpenAIRE
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