tion and transcription of the genetic code into messenger RNA. This message is then translated into a protein in the ribosomes within the cytosol. The genes for not only COX-2 but also iNOS belong to this group and are upregulated in response to tissue injury, eventually resulting in enhanced product formation, including not only prostaglan-dins and NO but also peroxynitrite and other products of (lipid)peroxidation.

Salicylates can modify the binding of a variety of transcription factors to the promoter region of genes. In many cases, modifications of gene regulation occur via inhibition of kinases that are necessary for activation oftranscriptions factors, allowing for their subsequent binding to the promoter region. Alternatively, there might be a direct interaction with the binding of transcription factors, such as NFkB or nuclear factor of activated T cells (NFAT) to the promoter region, which is kinase independent [149, 157, 158]. In both cases, the result is essentially the same - prevention of gene activation and subsequent product formation.

The understanding of the biological significance ofmodulation ofparticular transcription factors by salicylates is often hampered by the fact that these changes are mainly found in vitro and, in many cases, require extremely high concentrations of salicylates, up to 100 mM (!), to become (statistically) significant. Thus, although from a pharmacological point of view it is interesting to know which genes can be modulated by salicylates, these studies do not necessarily suggest that these changes are biologically significant in vivo. It is also less likely that cell lines in vitro, expressing constitutively active (otherwise inducible) genes after gene transfer or stimulation by tumor promoters that can be directly compared with "normal" somatic nontransfected cells, being the subject of rather transient stimulation by cytokines or related mediators of inflammation, ischemia, or immune re sponses. Thus, transcription factors that are sensitive to salicylates at concentrations above 2 mM are of pharmacological interest but may have limited therapeutic value in vivo. This problem will be discussed in terms of two well known salicylate-sensitive transcription factors: NFkB and C/EBP-p.

Nuclear Factor (NF) NFkB The nuclear factor kB/ RelA family of transcription factors (NFkB/RelA) regulates the expression of numerous genes involved in the control of immune and inflammatory responses, most notably tumor necrosis factor a (TNFa) and interleukin (IL)-1p.NFkB also controls cell survival either as a regulator of the apoptotic program for induction of apoptosis or, more commonly, as its inhibitor. Therefore, NFkB not only controls immediate inflammatory and immune responses [159] but also acts as a central regulator of longer lasting changes, including stress responses [160].

Aspirin and salicylate inhibit NFkB activation via inhibition of IKK-P kinase activity in numerous cells and tissues in vitro, predominantly at millimolar concentrations [161, 162]. Therefore, these actions may not always be detected in intact cells in vivo [151]. The effects of aspirin on NFkB [161] are specific for salicylates and are not seen with indomethacin or other NSAIDs. Direct inhibition of IKK-P by salicylates [154, 163] probably explains the hypoglycemic actions of salicy-

lates [164] and the inhibition of transcriptional activation of "tissue factor"(TF) [165,166]. Another most interestingrecent findingis the inhibition ofinfluenza virus replication in vitro and in vivo by aspirin (not indomethacin) via inhibition of NFkB [167].

NFkB and Apoptosis Sodium salicylate (20mM) produces a strong activation of p38 MAP kinases and cell death by apoptosis, suggesting that this kinase serves as a mediator of induced apoptosis in human fibroblasts and several cell lines including human colon adenocarcinoma cells. This activation of p38-MAPK and the subsequent induction of apoptosis might be important for the antineoplastic effect of the compound [168,169] and is discussed in more detail in Section 2.2.3.

Other Transcription Factors As mentioned above, NFkB is not the only transcription factor that is modified by salicylates. C/EBP-P is another one that can be phosphorylated by several kinases, in particular, ribosomal p90 S6 kinase [170]. C/EBP-P controls transcriptional activation of COX-2, iNOS, and probably other genes that are involved in inflammatory and immune reactions [135, 171] (Sec-tion2.2.1).Theinhibitionofthetranscriptionfactor C/EBP-P by salicylates - in contrast to inhibition of NFkB - is already seen at submillimolar concentrations and, therefore, probably significant also in vivo (Figure 2.19).

Further transcription factors that are potential targets of salicylates are activator protein-1 (AP-1) [153], STAT-6 [172] and NFAT. NFAT shares some homologies with NFkB and becomes activated after dephosphorylation by the phosphatase calcineurin. Salicylates inhibit DNA-binding and activation of this transcription factor without affecting the phos-phorylation status or intracellular localization of NFAT [158]. Interestingly, a new pentafluoropro-poxy derivative of salicylic acid (UR-1505) has recently been shown to block T-cell activation via inhibition of NFAT, eventually resulting in decreased T-cell proliferation and cytokine production [173]. An overview of selected transcription

Intracellular NFkB resides inactive and bound to the inhibitory protein I kB in the cytosol of immunocompe-tent white cells, endothelial cells, and vascular smooth muscle cells as a heterotrimeric complex with IkB. Stimulation of IkB by IKK kinases results in phosphory-lation, cleavage ofthe inhibitor, and translocation ofthe active NFkB heterodimer into the nucleus. IKK kinase activity is stimulated by cytokines, reactive oxygen species, and numerous other stimuli. The activation is mediated by increased activity of an IkB kinase (IKK) complex. The liberated heterodimer p50/p65 activates the genes of IL-1, IL-6, TNFa, ICAM-1, VCAM-1, and others, participating in the regulation of inflammation, immune responses, and cell survival. The net reaction is determined by signaling pathways, distal to NFkB.

Nfkb Translocation
Figure 2.19 Different control of COX-2- and iNOS-promotor activity by salicylates via C/EBPp and NFkB (for further explanation, see the text) (modified after [135, 170, 171]).

factors and affected genes that are modified by salicylates is summarized in Table 2.9.

NFkB and Neuroprotection Grilli et al. [177] showed thataspirinatlowmillimolar (IC50:1.7 mM) concentrations protected rat primary neuronal cultures and hippocampal slices from neurotoxicity elicited by the excitatory amino acid glutamate. Similar albeit less pronounced effects were seen with salicylate but not with indomethacin. The site of action was downstream to the glutamate receptor and involved salicylate-specific inhibition of NFkB activation via blocking phosphorylation of IkB [154, 178]. Another transcription factor, AP-1 remained unaffected [177]. This study was the first to show NFkB-mediated neuroprotective effects of salicylates. This action was not correlated with the anti-inflammatory activity or COX inhibition. More recent studies additionally indicated that glutamate-induced neuronal death involved inhibition of the

Table 2.9 Interactions of salicylates (aspirin and/or salicylate) with activation (act) orinhibition (inh) oftranscription factors via kinase modulation.



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