DNA from WT splenic IgM+ B cells was used as positive control. understand the relationships between the signaling elements controlling cell cycle exit and transcription and relate these to those controlling E2A. Herein, we demonstrate that pre-BCR mediated Ras/MEK/ERK activation couples cell cycle exit to while reciprocal regulation of E2A and Id3 initiates transcription in pre-B cells. For these initial studies, we used cultured pre-B cells as our model of differentiation. In the presence of IL-7, these pre-BCR+ cells continually proliferate. However, upon attenuation of IL-7 signaling they exit cell cycle and initiate and encoding mRNAs (Fig. 1a) as well as the corresponding protein products (Fig. 1b). Initial pharmacological inhibitor studies failed to identify any negative regulators of transcription30. However, subsequent studies demonstrated that inhibiting either MEK or ERK abrogated IL-7 withdrawal-induced cyclin downregulation (Fig. 1a, 1b). The observed modulation of cyclin E was proportional to that observed for the D-type cyclins, consistent with cyclin D mediated regulation of cyclin E31. Open in a separate window Figure 1 MEK/ERK regulate both cyclins and pre-B cells were cultured in medium with (+ IL-7, 7.5ng/ml) or without (?IL-7, 0.1ng/ml) IL-7 for (aCb) 24 or (cCd) 48 hours. Cells were treated with either a MEK inhibitor PD98059 Nomegestrol acetate (MEK-i, 10M) or an ERK inhibitor (ERK-i, 5M) as indicated for the last six hours of culture. (a) Quantitative PCR (qPCR) for and expression; ?P 0.001 as compared with +IL-7 expression and *P 0.001 as compared with ?IL-7 expression; @P 0.001 as compared with +IL-7 expression and #P 0.001 as compared with ?IL-7 expression; $P 0.001 as compared with +IL-7 expression and &P 0.001 as compared with ?IL-7 expression. (b) Immunoblot for cyclin D3, cyclin D2 and cyclin E from pre-B total cell lysates. Cells were cultured according to the conditions described above. Actin immunoblot serves as an internal loading control. Analysis of three independent experiments indicated that expression of DN-Ras or DN-MEK increased cyclin D3 expression in ?IL-7 cultures 4-fold as compared to untransfected controls (P 0.001, data not shown). (c) qPCR for and expression; ?P 0.001 as compared with +IL-7 expression and *P 0.001 as compared with ?IL-7 expression; $P 0.001 as compared with +IL-7 expression and &P 0.001 as compared with ?IL-7 expression. (d) qPCR analysis for and (h) expression in large pre-B Nomegestrol acetate cells and ?P 0.001 compared with germline transcription expression in small pre-B cells. We next examined if the MEK/ERK pathway could regulate processes necessary for light chain recombination. Transcription of (Fig. 1c) and and and germline transcripts and low levels of pre-B cells and indicate that pre-BCR mediated MEK/ERK activation plays a central role in coordinating suppression with the induction of pre-B cells were infected with control retrovirus or retrovirus encoding either a dominant negative (DN) mutant of Ras (DN-Ras, N17-H-Ras) or MEK (DN-MEK, MKK1-E8-K97M). GFP expressing cells were isolated by FACS, expanded in IL-7, and then cultured in either the presence or absence of IL-7. Both pharmacological inhibition and expression of DN-Ras and DN-MEK attenuated ERK activation (Fig. 2a). Interestingly, IL-7 withdrawal did not significantly modulate ERK activation in cells and ERK phosphorylation was Nomegestrol acetate relatively low in pre-B Rabbit Polyclonal to TOB1 (phospho-Ser164) cells expressing mock MIGR1 alone, DN-Ras or DN-MEK were cultured in medium with or without IL-7 for 24 or 48 hours as indicated or untransfected pre-B cells were cultured with or Nomegestrol acetate without MEKCi Nomegestrol acetate for the last six hours of culture. (a) Immunoblot for phospho-ERK and total ERK using total cell lysates from pre-B cells cultured according to the conditions.