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. 2008 Jul 18;372(1):97-102.
doi: 10.1016/j.bbrc.2008.04.189. Epub 2008 May 15.

Genetic determination of the role of PU.1 in macrophage gene expression

Myungsoo Joo  1 , Minjae Kwon, Anser C Azim, Ruxana T Sadikot, Timothy S Blackwell, John W Christman
Affiliations

Genetic determination of the role of PU.1 in macrophage gene expression

Myungsoo Joo et al. Biochem Biophys Res Commun. .

Abstract

PU.1, an Ets family transcription factor, mediates macrophage effector function in inflammation by regulating gene expression. But, the extent and nature of PU.1 function in gene expression has not been genetically determined because ablation of PU.1 gene abolishes macrophage development. Here, we epigenetically suppressed PU.1 by stably expressing PU.1 specific siRNA in macrophages, and determined the effect of PU.1 deficiency on expressions of key inflammatory genes: Toll-like receptor 4 (TLR4), cyclooxygenase-2 (COX-2), and macrophage inflammatory protein-1alpha (MIP-1alpha). PU.1-silenced cell lines expressed lower TLR4 mRNA and COX-2 protein, but higher MIP-1alpha protein, than controls. Over-expression of PU.1 suppressed lipopolysaccharide-induced MIP-1alpha production. PU.1 occupied proximal and distal cognate sites in the endogenous MIP-1alpha promoter, but dissociated only from the distal sites in response to lipopolysaccharide, suggesting a novel negative regulatory mechanism by PU.1. Together, our results defined PU.1 function in differentially regulating expressions of TLR4, COX-2, and MIP-1alpha.

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Figures

Fig. 1
Fig. 1. PU.1 up-regulates TLR4 mRNA expression
(A) Silencing PU.1 expression in siRNA cell lines, PU 5.7 and PU 5.9, was determined by Western blot with α-PU.1 antibody (top panel) and α-actin antibody for internal controls (bottom panel). (B) Potential cross-silencing was examined in PU 5.7 by Western blot with α-ETS ½ antibodies. (C) TLR4 mRNA expression was examined by semi-quantitative RT-PCR of PU 5.7 (left panel) and PU 5.9 (right panel). Included was PCR for TLR4 without RT reaction (-RT) to exclude genomic DNA contamination. β-actin as internal controls was similarly analyzed.
Fig. 1
Fig. 1. PU.1 up-regulates TLR4 mRNA expression
(A) Silencing PU.1 expression in siRNA cell lines, PU 5.7 and PU 5.9, was determined by Western blot with α-PU.1 antibody (top panel) and α-actin antibody for internal controls (bottom panel). (B) Potential cross-silencing was examined in PU 5.7 by Western blot with α-ETS ½ antibodies. (C) TLR4 mRNA expression was examined by semi-quantitative RT-PCR of PU 5.7 (left panel) and PU 5.9 (right panel). Included was PCR for TLR4 without RT reaction (-RT) to exclude genomic DNA contamination. β-actin as internal controls was similarly analyzed.
Fig. 2
Fig. 2. Expression of cell surface TLR4 and NF-κB activation in the PU.1-silenced cell line
Surface expression of TLR4/MD2 was determined in RAW 264.7 cells (A) and PU 5.7 cells (B) by FACS analyses. The cells differentially treated with LPS (0.1μg/ml) were fixed and stained with α-TLR4/MD2 antibody. The dotted line represents staining with PE-conjugated isotypic IgG and solid line does for with PE-conjugated TLR4/MD2 antibody. (C) NF-κB activity of PU.1-silenced cell line was measured by a reporter assay. The cells transfected with an NF-κB-Luciferase reporter and tk-Renilla constructs were treated with LPS (1 μg/ml) for 4 h. The results were an average of triplicate settings, and experiment was performed three times independently.
Fig. 2
Fig. 2. Expression of cell surface TLR4 and NF-κB activation in the PU.1-silenced cell line
Surface expression of TLR4/MD2 was determined in RAW 264.7 cells (A) and PU 5.7 cells (B) by FACS analyses. The cells differentially treated with LPS (0.1μg/ml) were fixed and stained with α-TLR4/MD2 antibody. The dotted line represents staining with PE-conjugated isotypic IgG and solid line does for with PE-conjugated TLR4/MD2 antibody. (C) NF-κB activity of PU.1-silenced cell line was measured by a reporter assay. The cells transfected with an NF-κB-Luciferase reporter and tk-Renilla constructs were treated with LPS (1 μg/ml) for 4 h. The results were an average of triplicate settings, and experiment was performed three times independently.
Fig. 2
Fig. 2. Expression of cell surface TLR4 and NF-κB activation in the PU.1-silenced cell line
Surface expression of TLR4/MD2 was determined in RAW 264.7 cells (A) and PU 5.7 cells (B) by FACS analyses. The cells differentially treated with LPS (0.1μg/ml) were fixed and stained with α-TLR4/MD2 antibody. The dotted line represents staining with PE-conjugated isotypic IgG and solid line does for with PE-conjugated TLR4/MD2 antibody. (C) NF-κB activity of PU.1-silenced cell line was measured by a reporter assay. The cells transfected with an NF-κB-Luciferase reporter and tk-Renilla constructs were treated with LPS (1 μg/ml) for 4 h. The results were an average of triplicate settings, and experiment was performed three times independently.
Fig. 3
Fig. 3. PU.1 up-regulatesCOX-2 expression
COX-2 expression in PU.1-silenced cell lines was determined by Western blot analysis. The cells were treated with LPS (1 μg/ml) for up to 8 h, and total cell lysate was analyzed by immunoblotting for COX-2 (top panels) and actin (bottom panels).
Fig. 4
Fig. 4. PU.1 negatively regulates MIP-1α expression
(A) RAW 264.7 cells were transfected with pcDNA3.1 (lanes 1 and 2) or a plasmid encoding C/EBP-β (lanes 3 and 4) for 48h. Transfection was normalized with pcDNA3.1 to 4μg. Total cell lysate was analyzed by Western blot for MIP-1α (top panel) and actin (bottom panel). (B) MIP-1α expression in PU 5.7 following LPS treatment was determined by Western blot analysis. (C) RAW 264.7 cells were transfected with increasing amounts of a PU.1-expressing plasmid, and the transfected cells were treated with LPS for 2h. MIP-1α expression was determined by Western blot analysis. (D) PU.1 binding to the endogenous MIP-1α promoter was analyzed by ChIP assay. PU.1 bound to DNA was immunoprecipitated by α-PU.1 antibody (lanes 1 to 4), and co-precipitated DNA was analyzed by PCR for distal PU.1 sites (top two panels) and proximal site (bottom two panels). Included was isotypic IgG to exclude a nonspecific immunoprecipitation (lane 5).
Fig. 4
Fig. 4. PU.1 negatively regulates MIP-1α expression
(A) RAW 264.7 cells were transfected with pcDNA3.1 (lanes 1 and 2) or a plasmid encoding C/EBP-β (lanes 3 and 4) for 48h. Transfection was normalized with pcDNA3.1 to 4μg. Total cell lysate was analyzed by Western blot for MIP-1α (top panel) and actin (bottom panel). (B) MIP-1α expression in PU 5.7 following LPS treatment was determined by Western blot analysis. (C) RAW 264.7 cells were transfected with increasing amounts of a PU.1-expressing plasmid, and the transfected cells were treated with LPS for 2h. MIP-1α expression was determined by Western blot analysis. (D) PU.1 binding to the endogenous MIP-1α promoter was analyzed by ChIP assay. PU.1 bound to DNA was immunoprecipitated by α-PU.1 antibody (lanes 1 to 4), and co-precipitated DNA was analyzed by PCR for distal PU.1 sites (top two panels) and proximal site (bottom two panels). Included was isotypic IgG to exclude a nonspecific immunoprecipitation (lane 5).
Fig. 4
Fig. 4. PU.1 negatively regulates MIP-1α expression
(A) RAW 264.7 cells were transfected with pcDNA3.1 (lanes 1 and 2) or a plasmid encoding C/EBP-β (lanes 3 and 4) for 48h. Transfection was normalized with pcDNA3.1 to 4μg. Total cell lysate was analyzed by Western blot for MIP-1α (top panel) and actin (bottom panel). (B) MIP-1α expression in PU 5.7 following LPS treatment was determined by Western blot analysis. (C) RAW 264.7 cells were transfected with increasing amounts of a PU.1-expressing plasmid, and the transfected cells were treated with LPS for 2h. MIP-1α expression was determined by Western blot analysis. (D) PU.1 binding to the endogenous MIP-1α promoter was analyzed by ChIP assay. PU.1 bound to DNA was immunoprecipitated by α-PU.1 antibody (lanes 1 to 4), and co-precipitated DNA was analyzed by PCR for distal PU.1 sites (top two panels) and proximal site (bottom two panels). Included was isotypic IgG to exclude a nonspecific immunoprecipitation (lane 5).
Fig. 4
Fig. 4. PU.1 negatively regulates MIP-1α expression
(A) RAW 264.7 cells were transfected with pcDNA3.1 (lanes 1 and 2) or a plasmid encoding C/EBP-β (lanes 3 and 4) for 48h. Transfection was normalized with pcDNA3.1 to 4μg. Total cell lysate was analyzed by Western blot for MIP-1α (top panel) and actin (bottom panel). (B) MIP-1α expression in PU 5.7 following LPS treatment was determined by Western blot analysis. (C) RAW 264.7 cells were transfected with increasing amounts of a PU.1-expressing plasmid, and the transfected cells were treated with LPS for 2h. MIP-1α expression was determined by Western blot analysis. (D) PU.1 binding to the endogenous MIP-1α promoter was analyzed by ChIP assay. PU.1 bound to DNA was immunoprecipitated by α-PU.1 antibody (lanes 1 to 4), and co-precipitated DNA was analyzed by PCR for distal PU.1 sites (top two panels) and proximal site (bottom two panels). Included was isotypic IgG to exclude a nonspecific immunoprecipitation (lane 5).
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