Abstract
A unique feature of CD40 among the TNF receptor (TNFR) superfamily is its exquisitely contextual effects, as originally demonstrated in normal and malignant B-lymphocytes. We studied renal cell carcinoma (RCC) in comparison to normal (human renal proximal tubule) cells, as a model to better understand the role of CD40 in epithelial cells. CD40 ligation by membrane-presented CD40 ligand (mCD40L), but not soluble CD40 agonist, induced extensive apoptosis in RCC cells; by contrast, normal cells were totally refractory to mCD40L. These findings underline the importance of CD40 ‘signal-quality’ on cell fate and explain the lack of pro-apoptotic effects in RCC cells previously, while confirming the tumour specificity of CD40 in epithelial cells. mCD40L differentially regulated TRAF expression, causing sustained TRAF2/TRAF3 induction in RCC cells, yet downregulation of TRAF2 and no TRAF3 induction in normal cells, observations strikingly reminiscent of TRAF modulation in B-lymphocytes. mCD40L triggered reactive oxygen species (ROS) production, critical in apoptosis, and NADPH oxidase (Nox)-subunit p40phox phosphorylation, with Nox blockade abrogating apoptosis thus implying Nox-dependent initial ROS release. mCD40L mediated downregulation of Thioredoxin-1 (Trx-1), ASK1 phosphorylation, and JNK and p38 activation. Although both JNK/p38 were essential in apoptosis, p38 activation was JNK-dependent, which is the first report of such temporally defined JNK-p38 interplay during an apoptotic programme. CD40-killing entrained Bak/Bax induction, controlled by JNK/p38, and caspase-9-dependent mitochondrial apoptosis, accompanied by pro-inflammatory cytokine secretion, the repertoire of which also depended on CD40 signal quality. Previous reports suggested that, despite the ability of soluble CD40 agonist to reduce RCC tumour size in vivo via immunocyte activation, RCC could be targeted more effectively by combining CD40-mediated immune activation with direct tumour CD40 signalling. Since mCD40L represents a potent tumour cell-specific killing signal, our work not only offers insights into CD40’s biology in normal and malignant epithelial cells, but also provides an avenue for a ‘double-hit’ approach for inflammatory, tumour cell-specific CD40-based therapy.
Highlights
CD40 is a member of the tumour necrosis factor (TNF)receptor (TNFR) superfamily and interaction with itsB cells and antigen-presenting cells, CD40 is expressed by epithelial cells of various origins, including bladder, liver and ovarian carcinoma cells, normal epithelial cells as well as endothelial cells
A unique feature of CD40 among TNF receptor (TNFR) members[28] is its exquisitely contextual influence on cell fate; the consequences of CD40 ligation appear to be different in normal and malignant B-lymphocytes, yet recent evidence suggests that this may apply to epithelial cells
Cross-linked soluble agonist (G28-5 mAb) even in combination with IFN-γ could not trigger death in renal cell carcinoma (RCC) cells. membrane-presented CD40L (mCD40L)-mediated death had apoptotic and A-704 cells were treated with mCD40L for the indicated time periods (1.5, 3 and 6 h) and expression of phosphorylated-mitogen-activated protein kinases (MAPKs) MKK4 (p-MKK4), MKK7 (p-MKK7), JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 μg protein/lane)
Summary
CD40 is a member of the tumour necrosis factor (TNF)receptor (TNFR) superfamily and interaction with itsB cells and antigen-presenting cells, CD40 is expressed by epithelial cells of various origins, including bladder, liver and ovarian carcinoma cells, normal epithelial cells as well as endothelial cells. Official journal of the Cell Death Differentiation Association. After CD40L-mediated activation, CD40 translocates to lipid rafts, where it associates with various TRAFs6 to activate downstream mitogen-activated protein kinases (MAPKs)[7,8]. Recent studies provided evidence that the consequences of CD40 ligation may differ in normal and malignant cells, its effects may be highly context-specific[5]. The ‘quality’ of the CD40 signal may determine whether CD40L–CD40 interactions are pro-apoptotic: extensive receptor cross-linking by membrane-presented CD40L (mCD40L) causes extensive apoptosis, while soluble agonists (e.g. agonistic antibodies) cause little apoptosis[9,10,11,12,13,14]. The mechanisms that define these properties of CD40 have only recently started to become investigated[15]
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