Abstract
Mouse F9 embryocarcinoma cells constitute a well established cell autonomous model system for investigating retinoic acid (RA) signaling in vitro. RA induces the differentiation of F9 cells grown as monolayers into endodermal-like cells and decreases their rate of proliferation. Knock-out of the retinoic X receptor alpha (RXRalpha) gene abolishes endodermal differentiation and the induction of several endogenous RA-responsive genes. RXRalpha null cells are also drastically impaired in their antiproliferative response to RA. The role of the RXRalpha phosphorylation site located in the N-terminal A region (Ser(22)) has been investigated here by establishing cell lines re-expressing RXRalpha either wild type or mutated at the phosphorylation site (RXRalphaS22A) in a RXRalpha-null background. We show that Ser(22) is dispensable for RA-induced endodermal differentiation but is crucial for the expression of several RA-responsive genes. Ser(22) is also indispensable for the antiproliferative effect of RA and necessary for the RA-induced down-regulation of p21(CIP) and p27(KIP) CKIs proteins that are known to be involved in the control of cell cycle progression.
Highlights
Retinoic acid (RA),1 the most potent biologically active metabolite of vitamin A, plays crucial roles in a wide variety of biological processes and influences the proliferation and differentiation of a variety of cell types
To investigate whether this phosphorylation of retinoid X receptors (RXRs)␣ is involved in primitive and parietal endodermal differentiation of F9 cells, as well as in their antiproliferative response to retinoic acid (RA), rescue lines re-expressing wild type RXR␣ (RXR␣WT line) or RXR␣ mutated at the phosphorylation site (RXR␣S22A line) were derived from RXR␣-null cells (Fig. 1A)
We investigated the ability of retinoic X receptor ␣ (RXR␣)WT and RXR␣S22A to rescue the expression of these RA target genes, using quantitative real time RT-PCR after treatment of the different cell lines with 100 nM RA for 24 h
Summary
Plasmid Constructs—The mouse full-length cDNA of RXR␣1 was cloned into the pD402A vector Antibodies—Rabbit polyclonal antibodies raised against the A region of RXR␣1, RPRX␣(A), were as described [34]. Those against p21CIP (C-19) and p27KIP (Ab-2) were purchased from Santa Cruz Biotechnology, Inc. Goat polyclonal antibodies raised against -actin were from Santa Cruz Biotechnology, Inc. Cell Extracts and Immunoblotting—Whole cell extracts (WCEs) were prepared as described previously [35]. Cell Growth Analysis—Cell counting experiments were performed in triplicate with untreated and RA-treated cells as follows. Subconfluent cultures of control or RA-treated cells were trypsinized and combined with their culture supernatants, pelleted, resuspended in 500 l of hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate) containing 50 g/ml propidium iodide, and incubated for 15 h in the dark at 4 °C. Statistical analysis was performed using the analysis of variance followed by 2 ϫ 2 comparisons based on the Newman-Keul’s test
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