LPS induces the interaction of a transcription factor, LPS-induced TNF-alpha factor, and STAT6(B) with effects on multiple cytokines

Abstract

TNF-alpha is a pivotal cytokine whose overproduction can be lethal. Previously, we identified a transcription factor, LPS-induced TNF-alpha factor (LITAF), that regulates TNF-alpha transcription. We now report the discovery and characterization of a regulatory cofactor that we call signal transducer and activator of transcription (STAT) 6(B) because of its considerable homology to STAT6 [here referred to as STAT6(A)]. The STAT6(B) gene expression was found to be activated by LPS. Furthermore, we show that cotransfection of STAT6(B) and LITAF induces an interaction between the two proteins, consequently forming a complex that subsequently translocates into the nucleus and up-regulates the transcription of cytokines. The effect of the complex on a panel of cytokines was tested. In addition, the specific role of LITAF in this complex was established with experiments, including RNA interference technology. Overall, these findings describe roles for LITAF, STAT6(B), and the LITAF-STAT6(B) complex in the regulation of inflammatory cytokines in response to LPS stimulation in mammalian cells.

Fig. 1.

Detection of the transcripts of STAT6(A) and STAT6(B) by Northern blot. Filters (Clontech) containing preblotted mRNA (2 μg of each) from different adult human tissues were separately hybridized with an α-³²P-dCTP-labeled DNA probe of STAT6(A) (full-length) or STAT6(B) (amino acids 1–150) and then autoradiographed with Biomax MR film (Kodak).

Fig. 2.

Immunoprecipitation of murine macrophages. The cell lysates from untreated (lanes 1, 3, and 5) or LPS-treated (100 ng/ml LPS) (lanes 2, 4, and 6) macrophages were immunoprecipitated with anti-LITAF (lanes 3 and 4) or anti-STAT6 (lanes 5 and 6), and then protein A/G beads (SC-2003, Santa Cruz Biotechnology) were added. The precipitates were subjected to Western blotting with the antibodies indicated by arrows.

Fig. 3.

Detection of LPS-induced LITAF, STAT6(A), or STAT6(B) production in THP-1 cells. Cells were untreated (lane 1) or pretreated with LPS at 5 (lane 2), 25 (lane 3), 50 (lane 4), 100 (lane 5), or 200 (lane 6) ng/ml for 3 h, then washed with PBS and maintained overnight. Cell protein lysates were extracted and subjected to Western blot analyses.

Fig. 4.

EMSA of the protein–DNA interaction. Protein (30 μg) extracted from each culture of transfected U2OS cells was added to the appropriate reaction buffer with the oligonucleotide DNA probe, ³²P-ATP-labeled hTNF-α promoter DNA as described (9). Protein from untransfected cells was applied in lanes 1 and 2, and a 50-fold excess of unlabeled competitor was added (lane 2). The proteins from each condition of cells treated with 1 μg of DNA of empty vector alone (lane 3), full-length LITAF alone (lane 4), both full-length LITAF and STAT6(A) (lane 5), full-length LITAF plus a fragment of STAT6(B) (amino acids 1–150, lane 6), full-length LITAF plus a fragment of STAT6(B) (amino acids 151–404, lane 7), and full-length LITAF plus full-length STAT6(B) (amino acids 1–404, lane 8) were assessed by EMSA. The shifted DNA bands are indicated by arrows.

Fig. 5.

Detection of translocation of LITAF-STAT6(B) complex in THP-1 cells. Protein extracts were collected from whole cells, cytoplasm, or nuclei after overexpression of both LITAF and STAT6(B). The alterations in the protein levels of exogenous LITAF or STAT6(B) and endogenous STAT6(A) over time were measured by Western blot with the following antibodies: LITAF (611615, BD Biosciences), Stat6 (S-20, Santa Cruz Biotechnology), or β-tubulin (C-20, Santa Cruz Biotechnology) as control.

Fig. 6.

Cytokine antibody array and Western blot of DNA-transfected THP-1 cells. (A) Human 44 cytokines were blotted onto a membrane and arrayed three times following the manufacturer's protocol. The intensities of the relative expression levels of cytokines were quantified by densitometry (VersaDoc imaging system, Bio-Rad). The β-gal gene was included in all transfections. The density value of each test sample was normalized to β-gal from the same lysates as described (15) and graphed. (B) Detection of LITAF/STAT6(B) complex-regulated gene expression in THP-1 cells. Proteins from THP-1 whole cells without DNA transfection (lane 1) or after DNA transfection with empty vector (lane 2), overexpression of LITAF alone (lane 3), STAT6(A) alone (lane 4), STAT6(B) alone (lane 5), and both LITAF and STAT6(B) (lane 6) were extracted and subjected to Western blot analyses. A total of 80 μg of protein extract was loaded per lane. Blots were probed with the following antibodies: Stat6 (S-20, Santa Cruz Biotechnology), LITAF (611615, BD Biosciences), IL-1α (R-20, Santa Cruz Biotechnology), IL-10 (M-18, Santa Cruz Biotechnology), MCP-2 (C-17, Santa Cruz Biotechnology), RANTES (C-19, Santa Cruz Biotechnology), or actin (C-11, Santa Cruz Biotechnology) as control.

Fig. 7.

Detection of shRNA-silenced LITAF gene expression. (A) Ten micrograms of total RNA extracts from LPS-stimulated THP-1 cells after transfection of 0 (lanes 1 and 2), 0.25 (lane 3), or 0.5 (lane 4) μg of pSHAG-LITAF-DNA to induce shRNA were isolated and subjected to Northern blot analysis with an α-³²P-dCTP-labeled DNA probe of LITAF (full-length) or GAPDH (Clontech). (B) The protein extracts from the treated cells were probed with the following antibodies: LITAF (611615, BD Biosciences) or actin (C-11, Santa Cruz Biotechnology) as a constitutive control. (C) Transcriptional activity of a series of deletion constructs of TNF-α promoter DNA. U2OS cells were transiently transfected with LITAF alone, STAT6(B) alone, or empty vector (mock) as control or were cotransfected with LITAF plus pSHAG-LITAF, LITAF plus STAT6(B), or LITAF plus STAT6(B) plus pSHAG-LITAF. Subsequent luciferase production was measured as described (9) in triplicate assays. The β-gal gene was included in all transfections. The promoter activity of each test sample was normalized to β-gal in the same lysates as described (15) and graphed. (D) ELISA in THP-1 cells. Cells were transiently transfected with LITAF alone, STAT6(A) alone, STAT6(B) alone, or empty vector as control and cotransfected with LITAF plus STAT6(A), LITAF plus STAT6(B), LITAF plus pSHAG-LITAF, or LITAF plus STAT6(B) plus pSHAG-LITAF. After 16 h of incubation, supernatants from treated cells were collected, and TNF-α secreted from each was measured by ELISA, in triplicate assays. The β-gal gene was included in all transfections. The density value of each test sample was normalized to β-gal in the same lysates and graphed.