Differential Induction of Nuclear Factor-κB and Activator Protein-1 Activity after CD40 Ligation Is Associated with Primary Human Hepatocyte Apoptosis or Intrahepatic Endothelial Cell Proliferation

Abstract

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-κB activity in hepatocytes ,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-κB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH2-terminal kinase 1/2) and expression of inhibitor κBα were entirely consistent, and thus confirmed the profiles of NF-κB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-κB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH2-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by ∼50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-κB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-κB activation and a lack of AP-1 activation in IHECs result in proliferation.

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

“…Thus, JNK and AP-1 are essential for CD40-mediated death in UCC cells, which accords with the critical role for AP-1 reported in CD40-induced hepatocyte death. 10 The CD40 apoptotic pathway was not influenced by p38 and is not associated with ERK activity, in agreement with the well-established role for ERKs in cell proliferation and carcinogenesis, and in inhibition of apoptosis induced by death ligands such as TRAIL. We then examined whether TRAF3 functions as a link between CD40 engagement and induction of the JNK/AP-1 pathway.…”

“…10,14 CD40 ligation in malignant cells can also induce expression of antiapoptotic proteins via PI3-K and ERK activation, 35 thereby explaining why protein synthesis blocking is necessary for apoptosis induction by soluble agonists. However, inactivation of the MEK/ERK and p38 pathways did not alter the apoptotic response to mCD40L in our study.…”

“…[8][9][10] Conversely, CD40-mediated apoptosis has been documented in biliary, hepatocyte and carcinoma cells ectopically expressing CD40, where death occurs by autocrine/paracrine induction of death ligands such as TNFa, FasL and TNFrelated apoptosis-inducing ligand (TRAIL). [10][11][12][13][14] However, apoptosis induced by soluble CD40 agonists depends on concurrent blocking of protein synthesis by cycloheximide (CHX). By contrast, membrane-presented agonists efficiently induce apoptosis in carcinoma cells without pharmacological intervention, while sparing homologous normal epithelial cells.…”

“…Activation of AP-1 and subsequent induction of cell death is well documented, 26 and a direct link between CD40-mediated induction of AP-1 and apoptosis has been suggested. 10,14 Furthermore, de novo expression of CD40 can induce JNK activity. 17 A role for TRAF3 is implied from findings that TRAF3 specifically blocks TRAF2-mediated activation of the noncanonical NF-kB pathway following CD40 ligation 27 and TRAF3 overexpression inhibits NF-kB activator 1 (Act1) protein and thus interferes with proliferation, 28 while a dominant-negative TRAF3 mutant impairs CD40-mediated growth inhibition in malignant urothelial cells.…”

A CD40 Kozak sequence polymorphism and susceptibility to antibody-mediated autoimmune conditions: the role of CD40 tissue-specific expression

“…60 Therefore, it is possible that CD40 overexpression on thyrocytes may drive anti-apoptotic pathways which would be expected to drive the autoimmune response towards GD. These two potential mechanisms are not mutually exclusive, as an inflamed thyrocyte may function as a more efficient facultative APC.…”