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.
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.
Strachan
Gordon D
GD
Department of Pathology, University of Pennsylvania, 240 S. 40th St, Room 312 Levy Bldg, Philadelphia, PA 19104-6030, USA.
Kopp
Amanda S
AS
Koike
Maya A
MA
Morgan
Kathleen L
KL
Jordan-Sciutto
Kelly L
KL
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D018946:Chemokine CCL5 / Q000494:pharmacology; D018925:Chemokines / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004347:Drug Interactions; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004622:Embryo, Mammalian; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology*; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D019208:Brain-Derived Neurotrophic Factor; D018797:Cell Cycle Proteins; D018946:Chemokine CCL5; D018925:Chemokines; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493754:E2f1 protein, mouse; D005231:Fatty Acids, Unsaturated; D007211:Indoles; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009414:Nerve Growth Factors; D011500:Protein Synthesis Inhibitors; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C007293:DAPI; D004247:DNA; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D017239:Paclitaxel; C038753:leptomycin B