TNFR family members deliver signals that regulate diverse cellular responses from proliferation and differentiation to growth suppression and apoptosis (Smith et al., 1994
; Cleveland and Ihle, 1995
). Many of these receptors, including Fas, TNFR1, and death receptor 3, share a death domain homology in their cytoplasmic tails through which they transmit apoptotic signals. However, certain receptors such as CD40, TNFR2, and CD30, which lack the death domain, have also been reported to suppress cell growth and survival (Eliopoulos et al., 1996
; Hess and Engelmann, 1996
; Young et al., 1998
). We have shown previously that human hepatocytes and cholangiocytes undergo apoptosis as a result of a cooperative interaction between CD40 and Fas receptors (Afford et al., 1999
). In this model CD40 activation triggers FasL expression by the epithelial cells, leading to autocrine or paracrine activation of Fas and consequent cell death. Despite expressing CD40, Fas, and FasL (our unpublished data), IHECs do not undergo apoptosis in response to CD40 activation but instead proliferate (Figure A). Although we have yet to elucidate the mechanisms responsible for CD40-mediated proliferation of IHEC, results from our present study (Figure B) indicate that NF-κB does play a key role in regulating this process. Indeed, the NF-κB family of transcription factors has been shown to regulate proliferation in several cell types (Hoshi et al., 2000
; Brantley et al., 2001
; Lim et al., 2001
). Endothelial cell proliferation in response to CD40 could be important not only in chronic inflammation but also in angiogenesis and tumor vascularization where 60% of hepatocellular carcinomas express high levels of CD40 (Sugimoto et al., 1999
). Our recent data (Russell, Adams, and Afford, unpublished data) suggest that CD40 ligation on IHECs can modulate other endothelial cell functions, including expression of adhesion molecules and chemokines in a manner similar to that reported with endothelial cells from other tissues (Van Kooten, 2000
). This indicates that CD40 expression by IHECs may have multiple roles beyond regulation of cell fate.
Our present study shows for the first time that after CD40 ligation, differences in activation of the transcription factors NF-κB and AP-1, key components of intracellular apoptotic signaling cascades, occur in primary human hepatocytes compared with endothelial cells, providing a possible mechanism for the apparent cell-specific effects of CD40 ligation. The use of selective inhibitors also demonstrated the contribution of these signaling pathways to cell fate. Confirmation of the activation of NF-κB and AP-1 and the effects of inhibitors on their modulation was also sought and confirmed by studying selected downstream signaling events to CD40.
NF-κB is activated by CD40 and other members of the TNFR family and has a potentially important function in regulating inflammation (Schwabe et al., 2001
) and apoptosis (Foo and Nolan, 1999
). In addition, NF-κB is critical for regulating liver regeneration and development (Iimuro et al., 1998
). In the current study, we report that CD40 activation leads to a transient activation of NF-κB in hepatocytes, but to a sustained up-regulation of NF-κB activity in IHECs that lasts for >24 h. The fact that inhibiting NF-κB with CAPE leads to marked increases in endothelial cell apoptosis at 24 h suggests that this prolonged NF-κB activation is protecting the IHECs from apoptosis.
Many antiapoptotic genes contain NF-κB binding sites (Baeuerle and Henkel, 1994
), including inhibitor of apoptosis protein 2, A20, and TRAFs 1 and 2, and it is likely that CD40 engagement on IHECs leads to the activation of antiapoptotic genes in a NF-κB–dependent manner. NF-κB also binds to promoters of proinflammatory genes such as interleukin-8, monocyte chemoattractant protein-1, intercellular adhesion molecule, and vascular cell adhesion molecule, and the sustained NF-κB up-regulation in IHECs could explain why chemokine and adhesion molecule expression are up-regulated in endothelial cells during CD40 ligation (Van Kooten, 2000
). In agreement with our data, several studies have also shown persistent NF-κB activation after stimulation of the CD40 receptor on B cells (Berberich et al., 1994
; Lee et al., 1995
). It is possible that the sustained NF-κB activity in IHECs could be a consequence of the NF-κB1/RelA dimer being less susceptible to inhibition by IκB proteins. Alternatively, IκBβ can be differentially regulated such that IκBβ produced in response to the initial wave of NF-κB activity is hypophosphorylated and acts as a competitive inhibitor of IκBα but not of NF-κB, leading to persistent NF-κB activity in the nucleus (Phillips and Ghosh, 1997
We observed a transient up-regulation of NF-κB in hepatocytes after CD40 ligation, which peaked at 2 h and was back at baseline by 4 h. Many proapoptotic genes also have NF-κB binding sites on their promoters, for example, FasL (Kasibhatla et al., 1999
), Fas receptor (Gil et al., 1999
; Kuhnel et al., 2000
), and p53 (Wu and Lozano, 1994
), and it is possible that the transient induction of NF-κB may be critical in up-regulating FasL expression (Holtz-Heppelmann et al., 1998
Several studies have demonstrated a relationship between activation of AP-1 and apoptosis (Xia et al., 1995
; Le-Niculescu et al., 1999
; Fan et al., 2001
). The main components of AP-1 are encoded by two families of genes related to the protooncogenes c-jun and c-fos, the products of which form homo- and heterodimers some of which bind promoters of genes involved in apoptosis (i.e., FasL, caspase 3, p53). Induction of AP-1 activity can occur either as a consequence of increased abundance of AP-1 (Angel and Karin, 1991
) or secondary to increased activity. Our results show that the CD40-induced sustained rise in AP-1 activity in hepatocytes is associated with a simultaneous rise in c-Jun and c-Fos protein levels, suggesting that CD40 ligation regulates phosphorylation and expression of the AP-1 family members in these cells. However, it remains to be determined whether the increased protein expression of c-Jun and c-Fos is the result of enhanced synthesis. The sustained rise in AP-1 activity in the absence of sustained NF-κB activity in hepatocytes could allow AP-1 to act unopposed to promote apoptosis. Our previous study has shown increased FasL expression in cultured hepatocytes after CD40 ligation (Afford et al., 1999
), and the present study suggests that this is associated with an increase in AP-1 activity. However, our study also indicates that the AP-1/JNK pathway may not be solely responsible for CD40-induced apoptosis in the epithelial cells because selective ERK and JNK inhibitors failed to completely abrogate CD40-induced apoptosis. It is possible that stage of progression through the cell cycle, or other transcription factors such as signal transducer and activator of transcription 3 (STAT3), may be involved. STAT3 has been implicated in apoptosis (Akira, 2000
; Chapman et al., 2000
), and CD40 ligation in B cells results in tyrosine phosphorylation of Janus tyrosine kinase3, leading to the phosphorylation and subsequent activation of STAT3 (Hanissian and Geha, 1997
). We have previously reported NF-κB and AP-1 activation in response to CD40 ligation in primary human cholangiocytes (Afford et al., 2001
), and further experiments have confirmed that both hepatocytes and cholangiocytes show similar signaling responses to CD40 activation (our unpublished data for cholangiocytes). These data suggest that in both liver epithelial cell types a common mechanism exists for regulation of cell survival, which is distinct from the response of hepatic endothelial cells.
Recent studies provide possible explanations for the different cellular fates of epithelial and endothelial cells in response to CD40 ligation. We have reported that differences in response to CD40 death signal transduction in carcinoma cells may be due to the differential signals emanating from two distinct CD40 cytoplasmic tail domains: the membrane proximal domain and the TRAF-interacting PXQXT motif (Eliopoulos et al., 2000
). Tsukamoto et al. (1999)
also demonstrated differential NF-κB regulation by these two CD40 domains. Another recent study reports the existence of CD40 isoforms generated through posttranscriptional and posttranslational alternative splicing (Tone et al., 2001
), providing another potential mechanism for differential signaling and hence cell fate in response to CD40 activation.
We conclude that CD40 activation in primary human hepatic epithelial and endothelial cells is associated with differential activation of the NF-κB and AP-1 transcription factors and disruption of these signaling pathways can alter cell fate in primary human hepatocytes or IHECs in vitro. These events could explain why hepatocytes are lost through apoptosis in inflammatory liver disease, whereas endothelial cells are not, even though they all show increased expression of CD40 (Afford et al., 1999
). Such differential responses of hepatocytes and endothelial cells make teleological sense in the context of inflammation where the endothelium needs to actively recruit effector cells by expressing adhesion molecules and chemokines, whereas infected epithelial cells need to be destroyed to control either viral or bacterial pathogens. Therapeutic strategies targeted at modulation of CD40-mediated NF-κB/AP-1–dependent mechanisms are of potential importance. This study illustrates that such approaches will need to take into account the wide range of functional consequences that can occur in primary cells after CD40 ligation.