Regulation of mouse 4-1BB expression: multiple promoter usages and a splice variant

Abstract

The expression of 4-1BB has been known to be dependent on T cell activation. Recent studies have, however, revealed that 4-1BB expression is not restricted to T cells. We sought to determine the molecular basis for the differential gene expression. Here we report the expression pattern of two mouse 4-1BB transcripts, type I and type II. Whereas the type I transcript was specifically expressed on immune organ as previously reported, the type II transcript was ubiquitously expressed in tissues and various cell lines. However, both type I and type II transcript were highly induced on activated T cells. Primer extension assay of the two 4-1BB transcripts suggested that mouse 4-1BB had more than two transcripts. Using luciferase assay we have identified three promoter regions (PI, PII and PIII), which located on upstream region of second exon 1, first exon 1, and exon 2, respectively. In particular, the type I transcript was preferentially induced when naïve T cells are stimulated by anti-CD3 monoclonal antibody (mAb) since NF-κB specifically binds to the putative NF-κB element of PI. We have also shown that a splice variant, in which the transmembrane domain was deleted, could inhibit 4-1BB signaling. The splicing variant was highly induced by TCR stimulation. Our results reveal 4-1BB also has a negative regulation system through soluble 4-1BB produced from a splice variant induced under activation conditions.

Fig. 1.. 4-1BB expression in tissues, cell lines and T cells. (A) Organization of the mouse 4-1BB gene. Exons are shown as boxes; untranslational regions are open and the protein coding regions are grayed and indicated in Roman numerals. Primer regions for RT-PCR are marked by arrows and RPA probe regions are lined. Three distinct putative promoter regions named PI, PII, and PIII. Translational start site is designated +1. (B, C) RT-PCR analysis. Total RNA was extracted from tissues of mice, cell lines, and activated T cells by anti-CD3 (5 μg/ml) and then analyzed for the expression level of the 4-1BB mRNA transcripts. (D, E) RNase Protection Assay (RPA). 5 μg of toal RNA of CD4⁺ (D) and CD8⁺ T cells (E) stimulated by anti-CD3 subjected to RPA as described in “Materials and Methods”. The mRNA expression of the corresponding GAPDH was included to normalize for gel loading.

Fig. 2.. Primer extension analysis for determination of the 4-1BB mRNA start site. 5 μg total RNA of splenocytes (lane 1), CTLL-R8 (lane 2), and EL4 (lane 3) was used for extension analysis. Total mRNA was annealed with 2 × 10⁴ cpm of the end-labeled oligonucleotide using [α-³²P]dATP at 30℃, overnight, in a hybridization buffer. Synthesis of cDNA using AMV reverse transcriptase was then performed. The mixture was extracted with phenol-chloroform, and precipitated with ethanol. The precipitate was analyzed by gel sequencing. The nucleotide sequence ladder of the pGEM-T easy vector shown on the left is a size marker for the extension products. The specific extension products for 4-1BB mRNA are indicated by arrows with numbers.

Fig. 3.. Promoter analysis of the upstream regions (UTRs) of the mouse 4-1BB. (A) Upstream regions used for report analysis are indicated as PI, PII, and PIII. Translational start site is designated +1. Three distinct 1.8 kbp upstream regions of each exon were cloned into the pGL3-basic vector. (B) EL4 and CTLL-R8 cells were transiently transfected with UTR constructs and pCMVβ-gal as an internal control. PGL3-basic (Basic), promoterless luciferase report, was used as negative control. Cells were treated with anti-CD3 mAb (5 μg/ml), P/I (20 ng/ml and 1 ng/ml), or Con A (10 μg/ml), and luciferase activity was determined after 40 h. Anti-CD3 mAb was coated on culture plates for stimulation. (C) CTLL-R8 cells were transiently transfected with NF-κB mutant(PI-NF-κBmu), AP-1 mutant (PI-AP-1mu), and double mutant (PI-mu) constructs. The transfectants were stimulated with plate-bound anti-CD3 mAb for 40 h. Transfection efficiencies were normalized by β-gal activity and data are representative of three independent experiments. Values are presented as means ± s.e.

Fig. 4.. NF-κB binds to a putative NF-κB element of the PI region. (A) Nuclear extract prepared from CD4⁺ T cells was stimulated with anti-CD3 for 6 h, and EMSA was performed with [α-³²P] labeled probe. Cold competition assay (lane 4) was performed with a ten-fold excess of unlabelled probe. For antibody mediated supershift analysis, anti-p65 monoclonal antibody was used (lanes 3, 6). A commercial NF-κB probe was used as a positive control (lanes 5-7) (n.s., non-specific binding). (B) CHIP assay performed by using anti-p65, anti-AP-1, or IgG as a negative control. Immunoprecipitated DNA was amplified by PCR with PI, PII, and PIII upstreamspecific primers. (C) Relative intensity of amplified PCR products was quantified by Image J program and graphed. It is normalized by the intensity of input.

Fig. 5.. The splice variant without exon 8 was highly induced in activated T cells. (A) The splice variant, which did not contain exon 8, was amplified by PT-PCR using cDNA prepared from activated CD4⁺ and CD8⁺ T cells via anti- CD3 stimulation for the indicated time. For RTPCR analysis of full transcripts, each sense primer (F1, F2, and F3) for each transcript, and the identical full reverse primer (R2) were used (Fig. 1A). (B, C) RNase Protection Assay (RPA). Purified CD4⁺ (B) a nd CD8⁺ T cells (C) were harvested at the indicated time after stimulation with anti-CD3 and total RNA was extracted. 5 μg of total RNA was hybridized with [α-³²P] labeled probes and subjected to RPA analysis as described in the methods section, followed by autoradiography. The mRNA expression of the corresponding GAPDH was included to normalize for gel loading.

Fig. 6.. Inhibition effect of a splice variant on 4-1BB signaling. (A) EL4 cells were transiently transfected with the indicated expression plasmids of pCDNA3.1-4-1BB, pCDNA3.1-4-1BBL, or pCDNA3.1-ΔE8-4-1BB. pELAM-luc reporter plasmid and pCMV-β-gal were co-transfected. Luciferase activities were measured 24 h after transfection and normalized on the basis of β-galactosidase activity. Values are presented as means±s.e. Each experiment was performed in triplicate, and the data presented are representative of three different experiments. (B) The surface expression of 4-1BB on EL4 cells transfected with pCDNA3.1, pCDNA3.1-4-1BB, or pCDNA3.1-ΔE8-4-1BB was determined by staining with FITC conjugated 3E1 mAb and an isotype control. (C) RT-PCR performed with identical samples. (D) Soluble 4-1BB protein was measured by ELISA using the supernatant from transfected EL4 cells. A Wallac vector 1420 Multilabel Counter was used for measurement. Data are representative of three independent experiments. Values are presented as means ± s.e.