Andrew Paul

Stress-activated Protein Kinases: Activation, Regulation and Function

The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes. Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists. Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation. More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1). These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-alpha (TNF-alpha), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion. Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses. In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress.

Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-kappaB signaling pathway.

Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IκBα and IκBβ and the related protein NF-κB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-κB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-κB was still observed. Cysteine peptidase inhibitors prevented IκBα, IκBβ, and NF-κB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IκBα in vitro. LPS-mediated IκBα and IκBβ degradation was not affected by these inhibitors, confirming that the site of degradation of IκBα, IκBβ, and NF-κB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-κB and consequently inhibit IL-12 production.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF‐κB (kidney), expression of iNOS and COX‐2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF‐κB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF‐κB to DNA and the degradation of IκBα, IKBβ, and IκBε. Selective inhibition of iNOS activity with L‐NIL reduced the circulatory failure and liver injury, while selective inhibition of COX‐2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF‐κB and thus prevention of the expression of NFκB‐dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX‐2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.—McDonald, M. C., Mota‐Filipe, H., Paul, A., Cuzzocrea, S., Abdelrahman, M., Harwood, S., Plevin, R., Chatterjee, P. K., Yaqoob, M. M., Thiemermann, C. Calpain inhibitor I reduces the activation of nuclear factor‐κB and organ injury/dysfunction in hemorrhagic shock. FASEB J. 15, 171–186 (2001)

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.

Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

1 In RAW 264.7 macrophages, lipopolysaccharide (LPS) and γ‐interferon (IFNγ) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2 LPS‐induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3 LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFNγ did not potentiate LPS‐stimulated PKC activity. 4 The protein kinase C inhibitor, Ro‐318220, abolished both LPS‐ and IFNγ‐stimulated protein kinase C activity and the induction of NOS activity. 5 LPS‐ and IFNγ‐induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS‐stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6 Nicotinamide, an inhibitor of poly‐ADP ribosylation, abolished LPS‐ and IFNγ‐induced NOS activity. 7 Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP‐ribosylation factor, ARF, reduced LPS‐ and IFNγ‐induced NOS activity by approximately 80%. 8 These results suggest the involvement of protein kinase C, tyrosine kinase and ploy‐ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFNγ.

The effects of cardamonin on lipopolysaccharide‐induced inflammatory protein production and MAP kinase and NFκB signalling pathways in monocytes/macrophages

In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)‐induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action.

Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells

In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon γ (IFNγ) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time‐ and concentration‐dependent manner. Incubation with the direct activator of protein kinase C (PKC), phorbol 12‐myristate 13‐acetate (PMA) alone, did not result in detectable iNOS expression in either cell type. Chronic PMA pretreatment resulted in significant down‐regulation of α, β and ε isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20–30% reduction in LPS‐induced iNOS expression. In contrast, IFNγ alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively. Pre‐incubation with PKC inhibitors (calphostin C and H‐7) showed similar effects upon stimulated induction of iNOS. In RASM cells chronic PMA pretreatment resulted in down‐regulation of α and ε PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin. Co‐incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin. Differential sensitivity to PKC inhibitors (calphostin C and H‐7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction. In addition the PKC inhibitor compound Ro‐31–8220 abolished stimulated induction in both cell types in response to all treatments. These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.

Inhibitory kappa B Kinases as targets for pharmacological regulation.

The inhibitory kappa B kinases (IKKs) are well recognized as key regulators of the nuclear factor kappa B (NF‐κB) cascade and as such represent a point of convergence for many extracellular agents that activate this pathway. The IKKs generally serve to transduce pro‐inflammatory and growth stimulating signals that contribute to major cellular processes but also play a key role in the pathogenesis of a number of human diseases. Therefore, the catalytic IKKs represent attractive targets for intervention with small molecule kinase inhibitors. IKK isoforms are assembled as variable multi‐subunit IKK complexes that regulate not only NF‐κB dimers, but also protein substrates out‐with this cascade. Consequently, close consideration of how these individual complexes transduce extracellular signals and more importantly what impact small molecule inhibitors of the IKKs have on functional outcomes are demanded. A number of adenosine triphosphate (ATP)‐competitive IKKβ‐selective inhibitors have been developed but have demonstrated a lack of activity against IKKα. A number of these chemicals have also exhibited detrimental outcomes such as cellular toxicity and immuno‐suppression. The impact of small molecule inhibitors of IKK catalytic activity will therefore be reappraised, examining the advantages and potential disadvantages to this type of intervention strategy in the treatment of diseases such as arthritis, intestinal inflammation and cancer. Furthermore, we will outline some emerging strategies, particularly the disruption of protein–protein interactions within the IKK complex, as an alternative route towards the development of novel pharmacological agents. Whether these alternatives may negate the limitations of ATP‐competitive molecules and potentially avoid the issues of toxicity will be discussed.

Differential modulation of TLR3- and TLR4-mediated dendritic cell maturation and function by progesterone.

The role of progesterone in modulating dendritic cell (DC) function following stimulation of different TLRs is relatively unknown. We compared the ability of progesterone to modulate murine bone marrow-derived DC cytokine production (IL-6 and IL-12) and costimulatory molecule expression (CD40, CD80, and CD86) induced by either TLR3 or TLR4 ligation and determined whether activity was via the progesterone receptor (PR) or glucocorticoid receptor (GR) by comparative studies with the PR-specific agonist norgestrel and the GR agonist dexamethasone. Progesterone was found to downregulate, albeit with different sensitivities, both TLR3- and TLR4-induced IL-6 production entirely via the GR, but IL-12p40 production via either the GR or PR. Of particular significance was that progesterone was able to significantly inhibit TLR3- but not TLR4-induced CD40 expression in bone marrow-derived DCs. Stimulation of the PR (with progesterone and norgestrel) by pretreatment of DCs was found to sustain IFN regulatory factor-3 phosphorylation following TLR3 ligation, but not TLR4 ligation. Overall, these studies demonstrate that progesterone can differentially regulate the signaling pathways employed by TLR3 and TLR4 agonists to affect costimulatory molecule expression and cytokine production.

Effects of L-canavanine, an inhibitor of inducible nitric oxide synthase, on endotoxin mediated shock in rats

The effects of L-canavanine, an inhibitor of nitric oxide synthase, on endotoxin-induced shock was investigated in the pentobarbitone anesthetized rat. Endotoxin infusion (2.5 mg kg-1 h-1 over 6 h) produced progressive and marked hypotension and hypoglycemia. Electron microscopy showed marked changes in the kidney, comprising severe endothelial cell disruption and the accumulation of platelets in the blood vessels. In the lung, there was marked accumulation of polymorphonuclear leukocytes in small blood vessels and endothelial disruption. Treatment with L-canavanine (10 mg kg-1 by bolus injection each hour starting 70 min after endotoxin or saline infusion) significantly reduced endotoxin-induced hypotension, without any effect on the hypoglycemia. This treatment markedly reduced the endotoxin-induced electron microscopical changes in the kidneys and lungs. Although L-canavanine, like L-NAME, inhibited both cerebellar constitutive and splenic inducible nitric oxide synthase in vitro, in contrast to L-NAME it did not modify either arterial blood pressure or carotid artery blood flow in control rats. The data are consistent with L-canavanine being a selective inhibitor of inducible nitric oxide synthase, at least in vivo, and suggest that inhibitors of this enzyme may be beneficial in endotoxin-induced shock.