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pRb and p107 regulate E2F activity during lens fiber cell differentiation
A M Rampalli1, C Y Gao, V M Chauthaiwale
1Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, Maryland 20892, USA.
Abstract:
During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.
Insights
In differentiating lens cells, retinoblastoma protein (pRb) and p107, not p130, primarily regulate E2F transcription factor activity. This study identifies key proteins involved in lens cell growth arrest and differentiation.
Area of Science:
- Molecular Biology
- Cellular Differentiation
- Ophthalmology
Background:
- E2F transcription factors are crucial for cell cycle regulation.
- Interactions with pRb family proteins (pRb, p107, p130) inhibit E2F activity during differentiation and growth arrest.
- Lens epithelial cells differentiate into fiber cells, a process involving regulated growth arrest.
Purpose of the Study:
- To investigate which E2F and pRb family members regulate growth arrest during lens epithelial cell differentiation.
- To characterize E2F protein complexes in rat lens epithelia and fiber cells.
Main Methods:
- Reverse Transcription Polymerase Chain Reaction (RT/PCR) to detect E2F family gene expression.
- Electrophoretic Mobility Shift Assay (EMSA) to analyze E2F protein complexes.
- Immunoblotting (Western blot) and Immunocytochemistry to identify specific proteins (pRb, p107, p130) within complexes and their cellular localization.
Main Results:
- Lens epithelial cells express all five E2F family members; fiber cells express E2F-1, E2F-3, and E2F-5.
- Epithelial cells form two E2F complexes; fiber cells form three, with one shared complex.
- p107 is present in one epithelial and two fiber cell E2F complexes. pRb is found in a fiber cell complex (detected by shift/Western assay) and is hypophosphorylated in fiber cells compared to epithelial cells.
- p130/E2F complexes are absent in fiber cells, correlated with ubiquitinated p130 forms.
Conclusions:
- pRb and p107 are the main regulators of E2F activity during rat lens fiber cell differentiation.
- The absence of p130/E2F complexes suggests a distinct regulatory mechanism in lens fiber cell terminal differentiation compared to other cell types.
- Understanding these molecular interactions is key to comprehending lens development and potential pathologies.