The pleitropic actions of tumour necrosis factor-α (TNF) are transmitted by the type I 55kDa TNF receptor (TNFR1) and type II 75kDa TNF receptor (TNFR2), but the signalling mechanisms elicited by these two receptors are not fully understood. In the present study, we report for the first time subtype-specific differential kinase activation in cell models that respond to TNF by undergoing apoptotic cell death. KYM-1human rhabdomyosarcoma cells and HeLa human cervical epithelial cells, engineered to overexpress TNFR2, displayed c-Jun N-terminal kinase (JNK) activation by wild-type TNF, a TNFR1-specific TNF mutant and a TNFR2-specific mutant TNF in combination with an agonistic TNFR2-specific monoclonal antiserum. A combination of the TNFR2-specific mutant and agonistic antiserum elicited maximal endogenous or exogenous TNFR2 responsiveness. Moreover, alternative expression of a TNFR2 deletion mutant lacking its cytoplasmic domain rendered the cells unable to activate JNK activity through this receptor subtype. The profile of JNK activation by TNFR1 was more transient than that of TNFR2, with TNFR2-induced JNK activity also being more sensitive to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone. Conversely, only activation of the TNFR1 could stimulate mitogen-activated protein kinase (MAPK) or p38 MAPK activities in a time-dependent manner. The role of TNFR2 activation in enhanced apoptotic cell death was confirmed with agonistic monoclonal antisera in cells expressing high levels of TNFR2. Activation of TNFR2 alone elicited cell death, but full TNF-induced death required stimulation of both receptor types. These findings indicate that efficient activation of TNFR2 by soluble TNFs is achievable with co-stimulation by antisera, and that both receptors differentially modulate extracellular signal-regulated kinases contributing to the cytokine's cytotoxic response.
- cytokine receptors
- protein kinases/phosphatases
- signal transduction
Abbreviations used: ATF-2, activating transcription factor-2; ATA, aurintricarboxylic acid; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FADD/MORT, Fas-associated protein with death domain; FBS, foetal bovine serum; FLICE, Fas-like interleukin 1β-converting enzyme; IAP, inhibitor of apoptosis; LT, lymphotoxin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK, MAPK-activated protein kinase; MEK, MAPK/ERK kinase; MKK, MAPK kinase; PI, propidium iodide; TRADD, TNF-associated protein with a death domain; TNF, tumour necrosis factor-α; TNFR, TNF receptor; TNFR1, type I 55kDa TNFR; TNFR2, type II 75kDa TNFR; TRAF, TNFR-associating factor; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone.
- The Biochemical Society, London ©2001