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Despite a dogma that apoptosis does not induce inflammation, Fas ligand (FasL), a well‐known death factor, possesses pro‐inflammatory activity. For example, FasL induces nuclear factor κB (NF‐κB) activity and interleukin 8 (IL‐8) production by engagement of Fas in human cells. Here, we found that a dominant negative mutant of c‐Jun, a component of the activator protein‐1 (AP‐1) transcription factor, inhibits FasL‐induced AP‐1 activity and IL‐8 production in HEK293 cells. Selective inhibition of AP‐1 did not affect NF‐κB activation and vice versa, indicating that their activations were not sequential events. The FasL‐induced AP‐1 activation could be inhibited by deleting or introducing the lymphoproliferation (lpr)‐type point mutation into the Fas death domain (DD), knocking down the Fas‐associated DD protein (FADD), abrogating caspase‐8 expression with small interfering RNAs, or using inhibitors for pan‐caspase and caspase‐8 but not caspase‐1 or caspase‐3. Furthermore, wildtype, but not a catalytically inactive mutant, of caspase‐8 reconstituted the FasL‐induced AP‐1 activation in caspase‐8‐deficient cells. Fas ligand induced the phosphorylation of two of the three major mitogen‐activated protein kinases (MAPKs): extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) but not p38 MAPK. Unexpectedly, an inhibitor for JNK but not for MAPK/ERK kinase inhibited the FasL‐induced AP‐1 activation and IL‐8 production. These results demonstrate that FasL‐induced AP‐1 activation is required for optimal IL‐8 production, and this process is mediated by FADD, caspase‐8, and JNK.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1β and IL-18 maturation is well known, ASC also induces NF-κB activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-κB-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-κB activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation.

Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator (tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAi(Met) promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudotype viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.