The Role of FIP-2 (Optineurin) in Regulation of the Chemokines and Kinases

HEK293 (Human embryonic kidney cells) and U373 (Human astrocytoma cells) were obtained from American Type Culture Collection (ATCC) and grown in DMEM or a-MEM media accordingly, supplemented with 10% fetal calf serum, penicillin (50 units/ml) and streptomycin (50 µg/ml). pcDNA-T7-FIP-2 expression vector was constructed by cloning the FIP-2 coding region into T7-pcDNA3.1 as previously described (1, 10). IL-8/LUC reporter plasmid was a generous gift from Dr. Antonella Casola (Division of Child Health Research Center, Galveston, Texas) and was previously described in publications from this laboratory ^{11, 12}. Plasmid pCH110, which expresses b-galactosidase under the control of the SV40 promoter was received from Amersham Biosciences and was used for the normalization of the luciferase reporter assay. Plasmid pGreen-Lantern-1 (pGL), which expresses the Green Fluorescent Protein under the control of the CMV promoter was purchased from Life Technologies and was used to check efficiency of the transfection.

Total RNA (1µg) was isolated from FIP-2 transfected and mock transfected cells using RNAqueous kit and mRNA was further purified with Poly(A)Purist kit (Ambion). Purified RNA was converted to double-stranded cDNA with SuperScript kit (Invitrogen) using oligo-dT primers containing T7 RNA polymerase promoter (Genset). Biotinylated cRNA was prepared from cDNA by in vitro transcription with theT7 RNA polymerase (ENZO). The labeled cRNA was fragmented by incubation at 94^oC for 35 min. Hybridization, washing and staining were performed according to the Affymetrix technical manual using Affymetrix “human” chip (U95Av2). The chip was stained with streptavidin-phycoerythrin (Molecular Probes) and scanned with Hewlett-Packard Gene Array Scanner in the AECOM Micoarray facility. Data analysis was performed using Affymetrix GeneChip Analysis Suite software. All data were quantile normalized and the gene list was created with a minimum fold-change of 2. These genes were separated into functional groups according to Gene Ontology (http://www.geneontology.org).

Total RNA was purified with a High Pure RNA Isolation Kit (Roche). Single-strand cDNA was synthesized with SuperScript II DNA polymerase according to the manufacturer’s protocol (Invitrogen). Primers and TaqMan probes were chosen with Primer Express (version 2.0) software (Applied Biosystems) or used as pre-developed TaqMan Assay Reagents (PDAR) from Applied Biosystems. GAPDH probes from Applied Biosystems were used for normalization. qRT-PCR was performed using ABI Prism 7900HT (Applied Biosystems). Quantification of data was performed according to the Applied Biosystems recommendations based on the Delta-Delta Ct method.

Quantikine kit (R#D Systems, Minneapolis, MN) for IL-8 was used for the detection of secreted chemokine. The strips with adsorbed specific antibody were incubated with the 50ul prediluted cell culture supernatants and 100ul of the same antibody conjugated with horseradish peroxidase (HRP). After 3.0 h incubation at room temperature the strips were washed and incubated with a substrate in the dark until color appeared. The reaction was stopped with 2N sulfuric acid and readout was performed at 450 nm (substraction at 562 nm) using an ELISA reader (Molecular Dynamics). The amount of IL-8 was determined according to a standard curve and calculated with the GraphPad Prism 5.0 software.

The cells were lysed in PBS buffer containing 1% NP-40, 0.5% DOC, and protease inhibitors (Roche). Lysates were precleared and incubated with a specific antibody (anti-FIP-2 /home-made rabbit polyclonal antibody/; anti-PAK3 /rabbit polyclonal antibody from Pierce/) and protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4^oC each. The agarose beads were then washed 3-4 times with lysis buffer, and proteins were eluted by boiling in Laemmli buffer and separated by SDS-PAGE on a 10% polyacrylamide gel. Following electrophoresis, the proteins were transferred to nitrocellulose and detected with mouse anti-T7 antibody (Novagen) and anti-mouse IgG conjugated with HRP (Amersham Biosciences). The signals were developed using enhanced chemiluminescence (NEN).

IP-kinase assay was performed as previously described ¹³. Briefly, PAK3 was immunoprecipitated with anti-PAK3 antibody (Pierce) as described above and agarose beads were incubated in the kinase buffer (20mM HEPES, pH 7.5; 20mM b-glycerophosphate, 10mM MgCl₂, 10mM Na₃VO₄, 2mM DTT, and 1x complete protease inhibitor mixture (Roche)) with 2mg MBP, 20mM ATP and 2mCi [g-³²P]ATP for 20 min at 30^oC. The reaction was stopped by boiling in Laemmli buffer and SDS-PAGE was performed using 10% SDS-polyacrylamide gel.

HEK293 cells in 6-well plates were transfected using Lipofectamine (Invitrogen) with IL-8/LUC reporter plasmid (0.5µg), pcDNA-T7-FIP-2 (0.5µg), pCH110 (0.2µg), pGL(0.1µg), and pcDNA 3.1 to make DNA concentration equal. After overnight incubation the cells were lysed and luciferase activity was measured by a luciferase assay kit (Roche). Data were normalized according to the b-galactosidase activity.

Immunofluorescence was performed as described earlier (10). Briefly, HEK293 cells grown on the chamber slides (Nunc) were transfected (or not /Control/) with pcDNA-T7-FIP-2 by Lipofectamine (Life Technologies, Inc.) according to manufacturer’s protocol. After 24 h, the cells were fixed, permeabilized and incubated with mouse anti-T7 antibody (1 h at room temperature), followed by anti-mouse IgG conjugated with FITC (1 h at room temperature). Analysis was performed using an inverted microscope with an epi-fluorescent attachment.

For multiple comparisons, one-way ANOVA with Bonferoni post test was performed. For comparison of the two groups, Student’s unpaired t test was used. p value <0.05 was considered significant. All statistics were run using the GraphPad Prism 5.0 software.

HEK293 cells were transfected with the pcDNA 3.1 plasmid containing the Long splice variant of FIP-2 with T7-tag. The expression of FIP-2 was confirmed by immunofluorescence (Figure 1A) and Western blotting (Figure 1B). Transfected FIP-2 was diffusely distributed in the cytoplasm, although the endogenous protein is mostly associated with the Golgi network ^{not shown and Ref. 214}.

Figure 1.FIP-2 overexpression in HEK293 cells. HEK293 cells were transfected with T7-FIP-2 plasmid or empty vector (Control/Ctr) and tested by immunofluorescence (A) and Western blotting (B) with anti-T7 antibody as described in Materials and Methods. Scale bar = 200 mM.

Biotin-labeled cRNAs, which were in vitro transcribed from the mRNAs isolated from FIP-2 transfected (FIP-2 overexpressed) cells and mock transfected cells, were hybridized with a microarray representing over 12,000 transcripts. The microarray data were filtered and a 2 fold cut-off was applied in order to exclude high variability and reach statistical significance. Table 1 represents a list of genes that are up- and down-regulated by more than 2 fold by FIP-2. These genes were separated into functional groups using Gene Ontology (Figure 2). The most representative groups are: “Signal transduction” (18 members), “Transcription regulation” (9 members), “Oncogenesis” (7 members), and “Anti-Pathogen response” (7 members). Among the most changed genes were “Small inducible cytokine subfamily A (CC)” (172 fold up), “Complement factor H” (43 fold up), “Nedd-4 like ubiquitin-protein ligase WW P2 (9 fold up), “Rod photoreceptor CNG-channel beta subunit (RCNC2)” (14 fold down), “Myc-associated Zinc finger protein (10 fold down), and “Munc 13” (10 fold down) (Table 1). In total, the microarray analysis has shown that FIP-2 substantially altered the expression of the 75 genes.

Figure 2.Functional groups of genes which are up- and down-regulated by FIP-2. The genes were separated into groups using Gene Ontology and are presented as percentages for each functional group.

The genes with the highest differences between FIP-2 overexpressed and control (mock transfected) cells were validated by RT-PCR and qRT-PCR. RT-PCR data correlated with the microarray results (see Table 1 and Figure 3). The representative example of RT-PCR is shown in Figure 3A. At the top of the FIP-2 up-regulated genes was “Small inducible cytokine subfamily A”. This is a chemokine gene cluster on human chromosome 17q 11.2 containing five CC chemokine genes: MPIF-1 (CCL23), HCC-2 (MIP-1, CCL15), HCC-1 (CCL14), LEC (CCL16, HCC-4), and RANTES (CCL5) ¹⁵. The preliminary analysis showed that only MPIF-1 was significantly up-regulated with FIP-2 overexpression (Figure 3A and not shown). Next, we performed qRT-PCR to examine the changes of these proteins in FIP-2 transfected cells, but besides MPIF-1 we also included other chemokines, particularly MCP-1 (CCL2) and IL-8 (CXCL8). Although MPIF-1 and MCP-1 were significantly up-regulated in FIP-2 transfected cells, the most dramatic change was observed for IL-8 (Figure 3B). Then these data were analyzed in more details.

Figure 3.Validation of gene expression. Total RNA from the mock (Ctr) and FIP-2 transfected cells was tested by RT-PCR (A) and Q – RT-PCR (B) using specific primers as described in Materials and Methods. GAPDH was used as a control housekeeping gene. M – Markers (100bp DNA Ladder from Life Technologies/Invitrogen). MPIF-1 - Myeloid progenitor inhibitory factor 1, CF H – Complement Factor H, HLF – Hepatic Leukemia Factor, PAK3 - p21 protein (Cdc42/Rac)-activated kinase 3, GAPDH - Glyceraldehyde 3-phosphate dehydrogenase, MCP-1 - monocyte chemotactic protein-1, IL-8 – Interleukin-8 (CXCL8, chemokine (C-X-C motif) ligand 8).

FIP-2 Regulates the Expression of Interleukin 8

HEK293 cells as andhumanastrocytoma (U373) cells transfected with FIP-2 were tested for the production of IL-8 by ELISA. In both cell lines we determined high IL-8 in comparison with the low basal level in the control mock transfected cells (Figure 4A). This result was confirmed in the dose-response experiments. When using 1µg of DNA (FIP-2 plasmid) per 10⁶ cells we observed that the production of IL-8 increased by 25-30 fold. The cells treated with TNF were used as a positive control (Figure 4B).

In order to understand the mechanism of FIP-2 activity, we tested the ability of FIP-2 to change the IL-8 expression by binding with its promoter. We used the luciferase reporter assay and IL-8/LUC reporter plasmids: a) -1500/+44 IL-8/LUC plasmid, which contains the first 1500 bases of the IL-8 promoter and b) -162/+44 IL-8/LUC plasmid with minimal IL-8 promoter. This deletion does not affect the inducibility of the promoter ^{11, 12}. FIP-2 induced IL-8 promoter activity in both cases, which means that the minimal sequence of the promoter was enough for the FIP-2 binding (Figure 4C). Actinomycin D, a RNA synthesis inhibitor, completely blocked IL-8 gene transcription, which confirmed the specificity of this reaction (Figure 4C). To define the region of the IL-8 promoter involved in the regulation of gene expression with FIP-2 overexpression, HEK293 cells were transfected with -162/+44 IL-8/LUC plasmid with deletions in binding sites such as NF-kB and AP-1. As shown in Figure 4D, the mutation of the NF-kB site completely abolished the FIP-2 or TNF-induced promoter activity. In contrast, there was no effect when AP-1 site was deleted (Figure 4D). This result suggests that NF-kB site is involved in the regulation of IL-8 transcription by FIP-2.

Figure 4.FIP-2 regulates the expression of IL-8. (A, B) IL-8 production at the FIP-2 overexpression. HEK293 and U373 cells were transfected with FIP-2 (0.5 µg) for 24h and the protein production was estimated by ELISA as described in Materials and Methods. A: transfection of the HEK293 and U373 cells. Ctr - transfection with empty vector; B: HEK293 cells were transfected with different doses of FIP-2 as indicated and after 18 h incubation the IL-8 was determined in supernatants by ELISA. TNF (stimulation during 18 h with 10 ng/ml) was used as a positive control. The representative data performed in triplicate is shown. (C, D) Transcriptional induction of IL-8 by FIP-2. C: HEK293 cells were transiently transfected with minimal IL-8/LUC promoter -162/+44 (Pr-162bp) alone, with TNF stimulation, or FIP-2 overexpression, or with FIP-2 in the presence of actinomycin D (ActD) and full IL-8/LUC promoter -1.4/+44 (Pr-1.4kb) alone, with TNF stimulation, or FIP-2 overexpression, or with FIP-2 in the presence of actinomycin D (ActD). D: HEK293 cells were transiently transfected with site-mutated plasmids of the -162/+44 IL-8/LUC promoter: NF-kB or AP-1 and stimulated with TNFor with FIP-2. The representative data performed in triplicate is shown. RLU – relative luciferase units.

The results of microarray analysis (see Table 1) and RT-PCR (Figure 3A) indicated that the expression of PAK3 was significantly increased with FIP-2 overexpression. To study the relationship between these two proteins, we first performed a co-immunoprecipitation assay. We transfected HEK293 cells with FIP-2 (tagged with T7) and PAK3 separately and together and immunoprecipitated with antibody against the FIP-2 and PAK3, followed by Western blotting using the anti-T7 (FIP-2) antibody. Anti-PAK3 antibody did not precipitate FIP-2 alone but in the presence of PAK3 it precipitated FIP-2, which was then detected by Western blotting (Figure 5A). This result indicates the presence of protein-protein interaction between FIP-2 and PAK3.

Then we performed an in vitro kinase assay using MBP as a substrate to test the ability of FIP-2 to change PAK3 kinase activity. We demonstrated that FIP-2 could increase the PAK3 kinase activity by 3-4 folds, in comparison with PAK3 alone. No kinase activity was associated with FIP-2 alone (Figure 5B). Thus, FIP-2 could interact with PAK3 and increase its kinase activity.

Figure 5.FIP-2 regulates the PAK3 activity. (A) Co-immunoprecipitation. HEK293 cells were transfected with pcDNA3.1 plasmid containing FIP-2 or PAK3, or both and precipitated with anti-FIP-2 or anti-PAK3 antibody followed Western blotting with anti-T7 antibody. (B) In vitro kinase assay. Kinase assay was performed with the cell lysates from HEK293 cells alone (Ctr), cells treated with IFN, or transfected with FIP-2 or PAK3 or together. β-Actin was used as loading control.