Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension.

Chronic stimulation of the renin-angiotensin system induces an elevation of blood pressure and the development of cardiac hypertrophy via the actions of its effector, angiotensin II. In cardiomyocytes, mitogen-activated protein kinases as well as protein kinase C isoforms have been shown to be important in the transduction of trophic signals. The Ca(2+)/calmodulin-dependent phosphatase calcineurin has also been suggested to play a role in cardiac growth. In the present report, we investigate possible cross-talks between calcineurin, protein kinase C, and mitogen-activated protein kinase pathways in controlling angiotensin II-induced hypertrophy. Angiotensin II-stimulated cardiomyocytes and mice with angiotensin II-dependent renovascular hypertension were treated with the calcineurin inhibitor cyclosporin A. Calcineurin, protein kinase C, and mitogen-activated protein kinase activations were determined. We show that cyclosporin A blocks angiotensin II-induced mitogen-activated protein kinase activation in cultured primary cardiomyocytes and in the heart of hypertensive mice. Cyclosporin A also inhibits specific protein kinase C isoforms. In vivo, cyclosporin A prevents the development of cardiac hypertrophy, and this effect appears to be independent of hemodynamic changes. These data suggest cross-talks between the calcineurin pathway, the protein kinase C, and the mitogen-activated protein kinase signaling cascades in transducing angiotensin II-mediated stimuli in cardiomyocytes and could provide the basis for an integrated model of cardiac hypertrophy.

Lipoproteins and mitogen-activated protein kinase signaling: a role in atherogenesis?

PURPOSE OF REVIEW: Lipoproteins play a critical role in the development of atherosclerosis, which might result partly from their capacity to induce specific intracellular signaling pathways. The goal of this review is to summarize the signaling properties of lipoproteins, in particular, their capacity to induce activation of mitogen-activated protein kinase pathways and the resulting modulation of cellular responses in blood vessel cells. RECENT FINDINGS: Lipoproteins activate the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in all blood vessel cell types. This may require lipoprotein docking to scavenger receptor B1, allowing transfer of cholesterol and sphingosine-1-phosphate to plasma membranes. Subsequent propagation of the signals probably requires the stimulation of G protein-coupled receptors, followed by the transactivation of receptor tyrosine kinases. Lipoprotein-induced extracellular signal-regulated kinase activity favors cell proliferation, whereas lipoprotein-induced p38 mitogen-activated protein kinase activity leads to cell hyperplasia and promotes cell migration. Some signaling pathways and cellular effects induced by lipoproteins have been observed in atherosclerotic plaques and therefore represent potential targets for the development of anti-atherosclerotic drugs. SUMMARY: The main blood vessel cell types have the capacity to activate protein kinase pathways in the presence of lipoproteins. This induces cell proliferation, hyperplasia and migration, known to be dysregulated in atherosclerotic lesions.

Increased expression of p38 mitogen- activated protein kinase is related to the acute renal lesions induced by gentamicin

Mitogen-activated protein kinases (MAPK) may be involved in the pathogenesis of acute renal failure. This study investigated the expression of p-p38 MAPK and nuclear factor kappa B (NF-kappaB) in the renal cortex of rats treated with gentamicin. Twenty rats were injected with gentamicin, 40 mg/kg, im, twice a day for 9 days, 20 with gentamicin + pyrrolidine dithiocarbamate (PDTC, an NF-kappaB inhibitor), 14 with 0.15 M NaCl, im, twice a day for 9 days, and 14 with 0.15 M NaCl , im, twice a day for 9 days and PDTC, 50 mg kg-1 day-1, ip, twice a day for 15 days. The animals were killed 5 and 30 days after the last of the injections and the kidneys were removed for histological, immunohistochemical and Western blot analysis and for nitrate determination. The results of the immunohistochemical study were evaluated by counting the p-p38 MAPK-positive cells per area of renal cortex measuring 0.05 mm². Creatinine was measured by the Jaffé method in blood samples collected 5 and 30 days after the end of the treatments. Gentamicin-treated rats presented a transitory increase in plasma creatinine levels. In addition, animals killed 5 days after the end of gentamicin treatment presented acute tubular necrosis and increased nitrate levels in the renal cortex. Increased expression of p-p38 MAPK and NF-kappaB was also observed in the kidneys from these animals. The animals killed 30 days after gentamicin treatment showed residual areas of interstitial fibrosis in the renal cortex, although the expression of p-p38 MAPK in their kidneys did not differ from control. Treatment with PDTC reduced the functional and structural changes induced by gentamicin as well as the expression of p-p38 MAPK and NF-kappaB. The increased expression of p-p38 MAPK and NF-kappaB observed in these rats suggests that these signaling molecules may be involved in the pathogenesis of tubulointerstitial nephritis induced by gentamicin.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.

Activation of the mitogen-activated protein kinase cascade by pertussis toxin-sensitive and -insensitive pathways in cultured ventricular cardiomyocytes.

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pathways in the activation of the mitogen-activated protein kinase (MAPK) cascade was examined in ventricular cardiomyocytes cultured from neonatal rats. A number of agonists that activate heterotrimeric G-protein-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothelin-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin II. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to control levels within 30-60 min, whereas activation by FCS was more sustained. FPLC on Mono Q showed that carbachol and FCS activated two peaks of MEK and two peaks of MAPK (p42MAPK and p44MAPK). Pretreatment of cells with PTX for 24 h inhibited the activation of MAPK by carbachol, FCS and lysophosphatidic acid, but not that by endothelin-1, phenylephrine or isoprenaline. Involvement of G-proteins in the activation of the cardiac MAPK cascade was demonstrated by the sustained (PTX-insensitive) activation of MAPK (and MEK) after exposure of cells to AlF4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endothelin-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidic acid on PtdIns hydrolysis was small and carbachol was without significant effect even after prolonged exposure. We conclude that PTX-sensitive (i.e. Gi/G(o)-linked) and PTX-insensitive (i.e. Gq/Gs-linked) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.

Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin > BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts

Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1 beta (IL-1 beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by > 70%, > 95% and similar to 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by similar to 60%, similar to 50% and similar to 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38 alpha(-/-) MEF cells provided additional evidence to support the role of p38 alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1 beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38 alpha ERK, JNK and p38 MAPK in mPDL cells.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Roles of p38 mitogen-activated protein kinase pathway in the regulation of SOX9 activity and chondrogenesis

Sox9 is a master transcription factor in chondrocyte differentiation. Several lines of evidence suggest that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in chondrocyte differentiation. In the present study, we examined the roles of p38 in the regulation of SOX9 activity and chondrogenesis. ^ COS7 cells were transfected with a SOX9 expression vector and 4x48-p89, a luciferase construction harboring four tandem copies of a SOX9-dependent 48-bp enhancer in Col2a1. Coexpression of MKK6EE, a constitutively active mutant of MKK6, a MAPKK that specifically activates p38, further increased the activity of the SOX9-dependent 48-bp enhancer about 5-fold, and SOX9 protein levels were not increased under these conditions. This increase in enhancer activity was not observed in a mutant enhancer construct harboring mutations that abolish SOX9 binding. These data strongly suggested that activation of the p38 pathway results in increased activity of SOX9. In addition, the increase of the activity of the SOX9-dependent 48-bp enhancer by MKK6EE was also observed in primary chondrocytes, and this increase was abolished by coexpression of a p38 phosphatase, MKP5, and p38 specific inhibitors. Furthermore, treatment of primary chondrocytes with p38 inhibitors decreased the expression of Col2a1, a downstream target of Sox9, without affecting Sox9 RNA levels, further supporting the hypothesis that p38 plays a role in regulating Sox9 activity in chondrocytes. ^ To further study the role of the p38 MAPK pathway in chondrogenesis, we generated transgenic mice that express MKK6EE in chondrocytes under the control of the Col2a1 promoter/intron regulatory sequences. These mice showed a dwarf phenotype characterized by reduced chondrocyte proliferation and a delay in the formation of primary and secondary ossification centers. Histological analysis using in situ hybridization showed reduced expression of Indian hedgehog, PTH/PTHrP receptor, cyclin D1 and increased expression of p21. In addition, consistent with the notion that Sox9 activity was increased in these mice, transgenic mice that express MKK6EE in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in chondrocyte differentiation and suggests that Sox9 is a downstream target of the p38 MAPK pathway. ^

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de señalización mediadas por proteína quinasas activadas por mitógeno (MAP quinasas) son capaces de integrar y transducir señales ambientales en respuestas celulares. Entre estas señales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son moléculas de patógenos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son moléculas derivadas de las plantas producidas en respuesta a daño celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterización funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que actúa como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulación del patrón estomático, la elongación del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipomórfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a patógenos biótrofos y necrótrofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleción en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de patógenos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulación de la resistencia de las plantas a patógenos. Nuestros datos indican que esta función es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que están implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necrótrofo Plectosphaerella cucumerina y el patrón estomático mediante su interacción genética con la RLK ERECTA (ER), un PRR implicado en la regulación de estos procesos. Por el contrario, la interacción genética entre ER y YDA en la regulación de otros procesos de desarrollo es aditiva en lugar de epistática. Análisis genéticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estomático, es un componente de la ruta de señalización mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el módulo de transducción de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de señalización reguladas por las hormonas de defensa ácido salicílico, ácido jasmónico, ácido abscísico o etileno, y también es independiente de la ruta de metabolitos secundarios derivados del triptófano, que están implicados en inmunidad vegetal. Además, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentración de calcio citoplásmico, la producción de especies reactivas de oxígeno, la fosforilación de MAP quinasas y la expresión de genes de defensa, no están afectadas en el mutante yda11. La expresión constitutiva de la proteína CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En línea con estos resultados, nuestros datos transcriptómicos revelan una sobre-representación en plantas CA-YDA de genes de defensa que codifican, por ejemplo, péptidos antimicrobianos o reguladores de muerte celular, o proteínas implicadas en la biogénesis de la pared celular, lo que sugiere una conexión potencial entre la composición e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Además, análisis de fosfoproteómica indican la fosforilación diferencial de proteínas relacionadas con la pared celular en plantas CA-YDA en comparación con plantas silvestres. El posible papel de la ruta ER-YDA en la regulación de la integridad de la pared celular está apoyado por análisis bioquímicos y glicómicos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composición de la pared celular de estos genotipos en comparación con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a patógenos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.