FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

Abstract

L-selectin plays important roles in lymphocyte homing and leukocyte rolling. Mounting evidence shows that it is involved in many disease entities including diabetes, ischemia/reperfusion injuries, inflammatory diseases, and tumor metastasis. Regulation of L-selectin at protein level has been well characterized. However, the regulation of human L-selectin transcription remains largely unknown. To address transcriptional regulation of L-selectin, we cloned 1088 bp 5' of the start codon ATG. Luciferase analysis of the serial 5' deletion mutants located the core promoter region at -288/-1. A major transcription initiation site was mapped at -115 by 5'RACE. Transcription factors Sp1, Ets1, Mzf1, Klf2, and Irf1 bind to and transactivate the L-selectin promoter. Significantly, FOXO1 binds to a FOXO1 motif, CCCTTTGG, at -87/-80, and transactivates the L-selectin promoter in a dose-dependent manner. Over-expression of a constitutive-active FOXO1 increased the endogenous L-selectin expression in Jurkat cells. We conclude that FOXO1 regulates L-selectin expression through targeting its promoter.

Keywords: FOXO1; L-selectin; promoter; transcriptional regulation.

Figure 1

Nucleotide sequence of the 5′ flanking sequence of human Sell gene. Notes: Annotated sequence is the first ∼317 bp of 5′-flanking region of human Sell. The ‘A’ in the start codon ATG (in bold) was defined as +1 and nucleotides upstream were numbered negative. Arrowheads indicate the position of the nucleotides relative to +1. Small arrow indicates the major TIS mapped by 5′ RACE. Putative transcription factor binding sites are underlined and labeled underneath. Abbreviations: Mzf1, Myeloid Zinc Finger Protein 1; Klf2, Kruppel-Like Factor 2; Ets1, E26 transformation specific sequence; Sp1, Specificity protein 1; Irf1, Interferon Response Factor 1; FOXO1, Forkhead box protein O1.

Figure 2

Mapping the core promoter region of human Sell gene. (A) 5′ serial deletion mutants shown on the left side were transiently transfected into Jurkat (stripped bars), EL4 (open bars), or HeLa cells (solid bars) and Luciferase activity shown on the right side was analyzed 30 hours after transfection. (B) Jurkat cells were co-transfected with core promoter construct, hSell288, with plasmids expressing Sp1, Mzf1, Klf2, Irf1, Ets1. Notes: Luciferase activity was analyzed 30 hours after the co-transfection. Luciferase activity was expressed as percentage of that of pGL3-Promoter in 2A and as fold changes relative to that of pGL3 vector transfected Jurkat cells in 2B. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times.

Figure 3

Constitutive active FOXO1 up-regulates endogenous human Sell expression. Jurkat cells were transiently transfected with pcDNA3 (white bar), pcDNA3-FOXO1 (striped bar), or pcDNA3-FOXO1-3A (solid bar) for 30 hours. Of the two sets of transfected cells, one set was lysed with SDS buffer and equal amount of lysate from each treatment was analyzed for expression of FOXO1 by Western blot, where β-actin was used as loading control (A); the other set was used to analyze the expression of human Sell by real time PCR, which was normalized to that of GAPDH (B). Notes: Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

Figure 4

Constitutive active FOXO1 up-regulates human Sell core promoter activity in a dose-dependent manner. Notes: Jurkat cells were transiently co-transfected with the combination of plasmids as labeled in the figure. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

Figure 5

Locating the FOXO1 motif. Notes: Jurkat cells were transiently co-transfected with either pcDNA3 (open bars) or FOXO1-3A (solid bars) and one of the 5′ serial deletion mutants as labeled. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after the co-transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 co-transfected Jurkat cells, which was set as 1.

Figure 6

FOXO1 transactivates human Sell promoter through binding to the motif. (A) Alignment of the IRE with the FOXO1 motifs in the Sell promoter of both human and chimpanzee. (B) Jurkat cells were transiently co-transfected with the combination of plasmids, pcDNA3 and hSell108, FOXO1-3A and hSell108, or FOXO1-3A and hSellFmut, as labeled on the bottom. Notes: Luciferase activity was analyzed 30 hours after transfection. Luciferase activity expressed as fold increase relative to that of pcDNA3 transfected cells. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times in triplicates.

Figure 7

FOXO1 binds to FOXO1 motif in vitro. Notes: DNA probe containing IRE from APOC3 gene was labeled with γ-³²P-ATP (lane 1), which was then incubated with nuclear extract from HeLa cells over-expressing FOXO1-3A (lane 2). The protein-DNA complex was then competed with either 10× and 100× cold APOC3 probe (lane 3 and 4, labeled as APOC3), or with 10× and 100× cold probe containing FOXO1 motif from human Sell (lane 5 and 6, labeled as FOXO1), or with 10× and 100× cold probe containing mutated FOXO1 motif from human Sell (lane 7 and 8, labeled as FOXO1m). Free probe was indicated with arrowhead and specific DNA-protein complex was indicated with arrow on the side.