Induced expression of trimerized intracellular domains of the human tumor necrosis factor (TNF) p55 receptor elicits TNF effects

Abstract

The various biological activities of tumor necrosis factor (TNF) are mediated by two receptors, one of 55 kD (TNF-R55) and one of 75 kD (TNF-R75). Although the phenotypic and molecular responses elicited by TNF in different cell types are fairly well characterized, the signaling pathways leading to them are so far only partly understood. To further unravel these processes, we focused on TNF-R55, which is responsible for mediating most of the known TNF effects. Since several studies have demonstrated the importance of receptor clustering and consequently of close association of the intracellular domains for signaling, we addressed the question of whether clustering of the intracellular domains of TNF-R55 (TNF-R55i) needs to occur in structural association with the inner side of the cell membrane, where many signaling mediators are known to reside. Therefore, we investigated whether induced intracellular clustering of only TNF-R55i would be sufficient to initiate and generate a full TNF response, without the need for a full-length receptor molecule or a transmembrane region. Our results provide clear evidence that inducible forced trimerization of either TNF-R55i or only the death domain elicits an efficient TNF response, comprising activation of the nuclear factor kappaB, induction of interleukin-6, and cell killing.

Figure 1

cDNA sequence of CAT–R55i fusion constructs. (A) cDNA sequence of wild-type and mutated hTNF-R55. Ile 201 was mutated to Met 201 to provide a potential initiation codon. At the same time, an extra NcoI site was created. The last amino acid of the transmembrane region of hTNF-R55 is believed to be Leu 203 (Loetscher et al., 1990). (B) Schematic representation of the different CAT fusion proteins. The numbering of the amino acids is according to Loetscher et al. (1990). Gray boxes represent the DD of hTNF-R55.

Figure 2

Bacterial expression of hTNF-R55i. E. coli 2580/ pT7pol26 transformed with pEThTNFR55i were grown and induced as described in Materials and Methods. (a) Autoradiogram of a serial dilution of metabolically labeled, induced bacteria in the absence or presence of rifampicin. (b) Immunoprecipitation of metabolically labeled bacteria. Noninduced extracts were treated with 2 μg htr-13 (lane 1); induced extracts were treated with 2 and 4 μg htr-13, respectively (lanes 2 and 3).

Figure 3

hIFN-α–induced cytotoxicity in cells expressing CAT and CAT-R55i. CAT cl 15 (A and C) and CAT-R55i cl 2 (B and D) were treated with 1,000 IU/ml hIFN-α. Photographs were taken 8 h (A and B) or 24 h later (C and D). Bar, 100 μm.

Figure 4

Kinetics of cell death by trimerized TNF-R55i. Cytotoxicity on parental L929sA cells (D), CAT cl 15 (B), and CAT-R55i cl 2 (A, C, E, and F) was measured at different time points, following different induction conditions, by MTT conversion. Inductions were carried out using the following concentrations: 1,000 IU/ ml hIFN-α (▪), 1,000 IU/ml mIFN-γ (□), 10 mM LiCl (•), 10 mM LiCl + 1,000 IU/ ml hIFN-α (○), 1 μg/ml ActD (▴), 1 μg/ml ActD + 1,000 IU/ml hIFN-α (▵), 500 IU/ml mTNF (×), or 500 IU/ ml hTNF (*). The percentage of cell survival of treated cells was compared to the untreated cell population (♦).

Figure 5

Kinetics of cell death by trimerized TNF-R55i as followed by PI uptake. CAT-R55i cl 2 (A) and CAT cl 15 (B) were induced with 1,000 IU/ml hIFN-α (▪), and parental L929sA cells (C) were induced with 2,000 IU/ml mTNF (▴). At the indicated time points, cell samples were taken, stained with PI, and analyzed by flow cytometry as described. PI uptake of treated cells was compared to untreated cells (♦).

Figure 6

CAT-R55i mRNA and IL-6 mRNA expression levels. Total RNA of cells from CAT cl 15 and CAT-R55i cl 2, treated with 1,000 IU/ml hIFN-α or untreated, was isolated at different time points and used in Northern blot analysis as described. (a) CAT(stop)R55i mRNA expression level of CAT cl 15 untreated or treated with hIFN-α (left); expression level of CAT-R55i mRNA of CAT-R55i cl 2 cells under the same conditions (right). The ³²P-labeled probe consisted of the hTNF-R55i cDNA fragment. The membrane was stripped and reprobed with a labeled IL-6 cDNA fragment. (b) IL-6 mRNA levels in CAT cl 15 untreated or treated with hIFN-α (left); results with CAT-R55i cl 2 after the same treatments (right). (c) Control hybridization of the same filter with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Figure 7

NF-κB activation as measured by an electrophoretic mobility shift assay. (a) NF-κB activation in CAT-R55i cl 2 after treatment with 1,000 IU/ml mTNF (control) or hIFN-α for periods varying from 90 to 240 min. (b) Results of similar treatments on CAT cl 15. The lower, constitutively binding band has recently been characterized (Plaisance et al., 1997).

Figure 8

Characterization of the CAT-R55i protein on the basis of its enzymatic activity. (A) Parental L929sA, CAT cl 15, or CAT-R55i cl 2 cells were treated with hIFN-α (+) or untreated (−). Cell extracts were made as described. Lanes 1–3 represent serial dilutions of pure CAT enzyme; lanes 4 and 5 are total lysates of CAT cl 15 and CAT-R55i cl 2 treated with hIFN-α, respectively; lanes 6–10 represent htr-13 immunoprecipitates derived from L929sA, CAT cl 15, or CAT-R55i cl 2 cells, treated or not with hIFN-α as indicated. CAT enzymatic activity was measured by the synthesis of acetyl-[¹⁴C]1-deoxy-chloramphenicol as described in Materials and Methods. (B) CAT-R55i cl 2 cells were treated with hIFN-α or hIFN-α + PSI, and total lysates were used in the CAT assay.

Figure 9

Cytotoxicity and IL-6 induction in CAT-R55i and CATlinkDD-expressing cells. (A) CAT-R55i cl 2 (▪), CATlinkDD cl 4 (▴), and CATlinkDD cl 12 (•) cells were treated with 1,000 IU/ml hIFN-α. At the indicated time points, cell samples were taken, stained with PI, and analyzed by flow cytometry. PI uptake by treated cells was compared to untreated cells (□, CAT-R55i cl 2; ▵, CATlinkDD cl 4; and ○, CATlinkDD cl 12). (B) Cells were treated for 12 h either with 1,000 IU/ml hIFN-α (black bars) or with 2,000 IU/ml mTNF (gray bars), or left untreated (blank). IL-6 levels in the medium were measured as described.