Novel transcriptional regulation of VEGF in inflammatory processes

Abstract

Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and osteoarthritis). Recently, we showed that transcription factors LITAF and STAT6B affected the inflammatory response. This study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells cotransfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between -338 and -305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction in VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF(-/-) ), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF(-/-) as well as in STAT6B-silenced wild-type animals compared with control animals. The present data demonstrate that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signalling pathways and may be a useful target in the treatment of VEGF diseases.

Fig. 1

Effect of mSTAT6B and mLITAF on regulation of VEGF promoter activity. 1 x 10⁵ pre-cultured U2OS cells were cotransfected with DNA of 50 ng/ml mV-PWT (#s 2-5) plus 100 ng of pcDNA as control or plus different concentrations (10 ng, 25 ng or 100 ng) of mSTAT6BWT (A) or mLITAFWT (B) for 8 hrs. Lysate from each experimental cell group was analysed. Top Panel: Western blot assay (#s 1–5) using antibodies against actin as control or mSTAT6B (A) or mLITAF (B). Lower panel: luciferase assay of VEGF promoter activity. Triplicate assays were conducted. The relative promoter activities were analysed and graphed. Mean SEM.

Fig. 2

Detection of mLITAF- or mSTAT6B-mediated VEGF promoter activity by mutagenesis. Diagram of mouse WT VEGF promoter DNA and its deletions (A), of mouse WT LITAF cDNA and its deletions (C) and of mouse WT STAT6B cDNA and its deletions (E). (B) 1x10⁵ pre-cultured U2OS cells were cotransfected with 50 ng/ml mV-PWT DNA or its deletions plus pcDNA as control, mLITAF or mSTAT6B overnight. Lysate from each experimental cell group was analysed. Top Panel: Western blot assay using antibodies against actin as control, mSTAT6B or mLITAF. Lower panel: luciferase assay of VEGF promoter activity. (D) Cells were cotransfected with mV-PWT DNA plus pcDNA as control, mLITAFWT or its varied deletions. (F) Cells were cotransfected with mV-PWT DNA plus pcDNA as control, mSTAT6BWT or its varied deletions overnight. The lysate from each experimental cell group was analysed by luciferase assay. Triplicate assays were conducted. Mean SEM.

Fig. 3

Chromatin immunoprecipitation (ChIP) Assay. RAW 264.7 cells were treated with 0.1 μg/ml E. coli LPS for 2 hrs, washed with PBS, then cultured overnight. ChIP was carried out using a ChIP-IT Express Enzymatic Kit. Diagram arrows indicate the location of primer pairs in mV-PWT promoter DNA (A). The genomic template DNA from anti-mLITAF precipitated (B) or anti-mSTAT6B precipitated (C) nuclear extracts (NE) of cells was used to amplify VEGF promoter DNA with 3 VEGF primer pairs or GAPDH primer pairs as control by PCR. The PCR products are indicated with arrows. (D) Determination of DNA-protein binding site. RAW 264.7 cells were cotransfected with mV-PWT plus mLITAF (#s 1-3,5), mSTAT6B (#s 1,2,4, & 9), mLITAF deletions (#s 6-8), or mSTAT6B deletions (#s 10-14). The DNA from anti-HA- (#s 1 & 2, 5-14), or from anti-IgG- (as control, #s 3 & 4) precipitated protein extracts of cells or mV-PWT cDNA as the positive control (#15) was used to amplify VEGF promoter DNA with VEGF primer pairs (primer1+primer3, #s 2-15) or with GAPDH primer pairs as control (#1) by PCR. The PCR products (200 bp) are indicated with arrows.

Fig. 4

Analysis of VEGF promoter activity and gene expression after knockdown of mLITAF and/or mSTAT6B in cells. (A) U2OS cells were untreated as control (group 1), or cotransfected with 50 ng mV-PWT DNA (group 2–8) plus 0.5 μg pcDNA as control (group 2), 0.5 μg mLITAFWT (group 3), 0.5 μg mSTAT6BWT (group 4), or both 0.5 μg mLITAFWT and 0.5 μg mSTAT6BWT (groups 5–8), then combined with 20 nM m6BSsiRNA#1 (group 6), 40 nM m6BsiRNA#1 (group 7), or 40 nM m6BsiRNA#2 (group 8) overnight to test the efficacy of the siRNAs. Lysate from each experimental cell group was analysed. Top panel: Western blot assay using antibodies against actin as control, mSTAT6B or mLITAF. Lower panel: luciferase assay of VEGF promoter activity. (B) U2OS cells were untreated as control (group 1), or cotransfected with mV-PWT DNA (group 2–8), plus either 0.5 μg pcDNA as control (group 2), 0.5 μg mLITAFWT (group 3), mSTAT6BWT (group 4), or both mLITAFWT and mSTAT6BWT (groups 5–8), then combined with 20 nM mLFsiRNA#1 (group 6), 40 nM mLFsiRNA#1 (group 7), or 40 nM mLFsiRNA#2 (group 8) overnight. Lysate from each experimental cell group was analysed. Top Panel: Western blot assay using antibodies against actin as control, mSTAT6B or mLITAF. Lower panel: luciferase assay of VEGF promoter activity. (C) Mouse endothelial cells were untreated as control (group 1), or treated with siRNA (groups 2–4, 6–9) overnight and further treated with LPS (groups 5–9) for 3 hrs. ELISA immunoreactivity was quantified and graphed. Intensity in ELISA from LPS alone-treated cells as positive control was assigned to a base value (100%). Intensity of other treatments (LPS+siRNA) was calculated relative to this base value. (D) Mouse peritoneal macrophages (wild-type as control or macrophage-specific LITAF-deficient mouse, macLITAF^−/−) were untreated as control (white bars) or treated with 50 ng/ml VEGF164 for 2 (grey bars) or 8 hrs (black bars). Total mRNA from treated cells was assessed by RT-PCR and normalized with β-actin. Intensity of VEGF mRNA from VEGF164-treated cells for 8 hrs was assigned to a base value (100%). Intensity of VEGF mRNA from other treatments was calculated relative to this base value. Triplicate assays above were conducted. Mean SEM.

Fig. 5

Analysis of VEGF164-induced protein nuclear translocation and cell migration in the presence/absence of mLITAF/mSTAT6B. (A & B) Protein separated and purified from LPS- or VEGF164-treated mouse peritoneal macrophages (macrophage-specific LITAF-deficient mouse, macLITAF^−/− or wild-type cells as control, (A), or proteins from siRNA & VEGF164 cotreated wild-type mouse endothelial cells (5B) were detected by Western blotting with antibodies against mouse p38α, p-p-38α, LITAF, STAT6B, or actin, tubulin and TBP as control. (C) Mouse peritoneal macrophages were untreated as control (group 1), or cotransfected with 50 ng mV-PWT DNA (groups 2–4) plus either 0.5 μg pcDNA as control (group 2), or plus both 0.5 μg mLITAFWT and 0.5 μg mSTAT6BWT (group 3), or plus 50 ng/ml VEGF164 (group 4) overnight. Cells were analysed by luciferase assay. (D) Measurements of cell migration by in vitro chemotaxis assay. 1x10⁶ pre-cultured mouse endothelial cells were untreated as control (group 1), or treated with siRNA (groups 2, 3, 5 & 6) overnight and then treated with VEGF164 (groups 4–6) for 24 hrs. The treated cells were used for cell migration assay. Triplicate assays were done. The measurement was graphed.

Fig. 6

LITAF or STAT6 deficiency affects VEGF-induced angiogenesis in vivo. Photographs of VEGF-soaked Matrigel by surgical microscopy excised from wild-type (A), TamLITAF KO (C), mSTAT6B siRNA#2 (E) and mSTAT6B siRNA #1 (G) mice after 10 day implantation (x10). Corresponding plugs were prepared and stained with anti-CD31 for vessel staining: wild-type (B), mLITAF KO (D), mSTAT6B siRNA#2 (F) and mSTAT6B siRNA#1 (H). Arrows point at blood vessels stained on the Matrigel. This experiment is representative of two individual studies (3 mice per group). (I): Vessel density: 10 random high-power fields per 5 Matrigel sections were evaluated and stained capillary blood vessels were quantified by histomorphometric analysis (*, p < 0.01): M, matrigel; V, vessels. (K) Total mRNA from treated tissue was assessed by RT-PCR and normalized with β-actin. Intensity of VEGF mRNA from VEGF164-treated WT tissue was assigned to a base value (100%). Intensity of VEGF mRNA from other treatments was calculated relative to this base value. Triplicate assays were conducted. Mean SEM.