pRb and p107 regulate E2F activity during lens fiber cell differentiation

Abstract

During growth arrest and di erentiation, 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 di erentiate into ®ber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and ®bers. RT/PCR detected all ®ve known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in ®ber cells. Proteins extracted from lens epithelia of newborn rats formed at least two speci®c complexes with an E2F consensus oligonucleotide. Proteins from lens ®ber cells formed three speci®c complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and ®ber cell extracts with an antibody speci®c for p107 demonstrated that two ®ber cell complexes and one epithelial cell complex contained p107. Although the remaining ®ber 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 con®rmed that pRb protein is present in the nuclei of both epithelial cells and ®ber 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 ®ber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identi®ed p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the ®ber cells. Thus, although p130/E2F complexes are implicated in the terminal di erentiation of many cell types, in di erentiating lens ®ber cells pRb and p107 seem to be the primary regulators of E2F activity.

Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases

“…This finding is similar to lens development in vivo, for which the lens epithelial cells must first pass through a distinct zone of proliferation before they withdraw from the cell cycle and commence their differentiation program (Persons and Modak, 1970). In past studies, the embryonic lens has proven to be an important model in deciphering the molecular elements of cell cycle control (Harris et al, 1992;Rinaudo and Zelenka, 1992;Rampalli et al, 1998;Lovicu and McAvoy, 1999). Similarly, the lens cell cultures provided us with a valuable model in which we have determined how SFK signaling regulates a cell's decision to proliferate or differentiate.…”

“…Their switch from proliferation to differentiation occurs in the transition zone, which is located within the equatorial region of the developing lens. The presence of distinct zones of proliferation and differentiation has made the embryonic lens a valuable model in the study of molecular changes associated with cell proliferation and cell cycle withdrawal (Harris et al, 1992;Rinaudo and Zelenka, 1992;Rampalli et al, 1998;Lovicu and McAvoy, 1999). Similar to lens development in vivo, lens epithelial cells grown in culture first proliferate and then withdraw from the cell cycle before they begin their differentiation (Menko et al, 1984).…”

Developmental defects and tumor predisposition in Rb mutant mice

“…Mice carrying mutations in p107 and p130 (Cobrinik et al, 1996) do not show any lens defects. Biochemical analysis of lens extracts showed that p107 but not p130 is found in complexes with E2Fs in lens epithelial cells (Rampalli et al, 1998). Functional overlap between pRb and p107 in lens development can now be studied in chimeric mice carrying cells both mutant for Rb and p107 (RobanusMaandag et al, 1998).…”

The retinoblastoma gene family in differentiation and development

“…pRB is present in both the precursor cells as well as in fully dierentiated tissues. Its levels can either slightly increase (for example in muscle, erythroid and mouse embryonic carcinoma P19 cells dierentiating into neuroectoderm) (Condorelli et al, 1995;Kiess et al, 1995;Martelli et al, 1994;Slack et al, 1993), remain constant (adipose, dierentiating rat lens ®ber cells) (Rampalli et al, 1998;Richon et al, 1997) or even decline (most neuronal dierentiation systems) (Callaghan et al, 1999;Kastner et al, 1998). In all cases, however, pRB is found hypophosphorylated at the time of cell cycle exit.…”