Transcriptional regulation of flotillins by the extracellularly regulated kinases and retinoid X receptor complexes.

Flotillin-1 and flotillin-2 are important regulators of signal transduction pathways such as growth factor signaling. Flotillin expression is increased under pathological conditions such as neurodegenerative disorders and cancer. Despite their importance for signal transduction, very little is known about the transcriptional regulation of flotillins. Here, we analyzed the expression of flotillins at transcriptional level and identified flotillins as downstream targets of the mitogen activated kinases ERK1/2. The promoter activity of flotillins was increased upon growth factor stimulation in a MAPK dependent manner. Overexpression of serum response factor or early growth response gene 1 resulted in increased flotillin mRNA and protein expression. Furthermore, both promoter activity and expression of endogenous flotillins were increased upon treatment with retinoic acid or by overexpression of the retinoid X receptor and its binding partners RARα and PPARγ. Our data indicate that the expression of flotillins, which can be detected in all cultured cells, is fine-tuned in response to various external stimuli. This regulation may be critical for the outcome of signaling cascades in which flotillins are known to be involved

Number of putative transcription factor binding sites in flotillin promoter regions.

<p>Approximately 1000 bp of each genomic sequence upstream of the ATG start codon were analyzed using the “Common TF” software (Genomatix software suite). Only transcription factor binding sites present in flotillin-1 and flotillin-2 sequences of the 5 species mentioned above are included in the table.</p

Murine flotillin promoters are activated in a similar manner as the human promoters.

<p>Murine flotillin-1 (A, C, E, G) or flotillin-2 (B, D, F, H) promoter constructs were transiently transfected into Hela cells, either alone or in combination with the expression constructs for transcription factors. One day post-transfection, the cells in A–D were stimulated with EGF (10 ng/ml), trans-RA (1 µM), PMA (10 ng/µl), or troglitazone (10 µM) in serum-free medium for 24 h. Cells in E–H were lysed 48 h post-transfection. Values are means ± standard deviation of at least 3 experiments measured in duplicates. ***, p<0.001; **, p<0.01; *, p<0.05 vs. control.</p

Flotillin-1 and flotillin-2 promoter activity is induced by Egr1, SRF and ERK2.

<p>Flotillin promoter constructs F1-1330 (A, C, E) or F2-2130 (B, D, F) were cotransfected into Hela cells together with expression plasmids for Egr1, SRF, empty PSV control plasmid or with the ERK2 expression constructs, and the cells were lysed 48 h post-transfection. Relative luciferase activity of the control sample was set as 1. Values are mean ± standard deviation of at least 4 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. control.</p

Flotillin promoter activity is induced by EGF, PMA, FGF and FCS.

<p>Flotillin promoter fragments of different length were transiently transfected into Hela cells. One day post transfection, the cells were stimulated with EGF (10 ng/ml), U0126 (10 µM) (A), PMA (10 ng/ml) (B), FGF (10 ng/ml) (C), or FCS (10%) (D) for 24 h in serum-free medium. Relative luciferase activity of the unstimulated longest promoter construct was set as 1. Values are means ± standard deviation of at least 3 experiments measured in duplicates. *** p<0.001; **p<0.01; *p<0.05 vs. unstimulated sample.</p

Mutagenesis of Egr1 or RXR binding sites prevents transcriptional activation of mouse flotillin-2.

<p>A: mouse flotillin-2 proximal promoter sequence. The three putative binding sites for the RXR family and the two sites for Egr1 are shown in bold and underlined. The sequence shown in italics was deleted to remove the Egr1 binding sites. Mouse flotillin-2 promoter activity after PMA stimulation (B) or retinoic acid stimulation (C). Transfection and stimulation were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045514#pone-0045514-g009" target="_blank">Figure 9</a>.</p

Induction of endogenous flotillin expression by Egr1 and SRF.

<p>Hela cells were transiently transfected with expression constructs for Egr1 or SRF. Empty PSV vector served as control. (A) Cell lysates were analyzed for flotillin-1 and -2 by Western blotting (left) and quantified (right). (B) RNA was isolated, transcribed into cDNA and analyzed by qPCR. Values are means ± standard deviation of 7 (Western Blot) or 4 (qPCR) experiments.*p<0.05, **, p<0.01 vs. control.</p

Pairwise similarities of promoter regions (shown as % identical nucleotides).

<p>Pairwise similarities of promoter regions (shown as % identical nucleotides).</p