The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity

LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.     

Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.     

Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK)

Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.     

Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. II. Effects of LFM-A13, a specific Btk inhibitor

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110 gamma or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110 gamma by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.     

Chemosensitizing Anti-Cancer Activity of LFM-A13, a Leflunomide Metabolite Analog Targeting Polo-like Kinases

LFM-A13 (α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide) has recently been identified as an inhibitor of Polo-like kinases (Plk). LFM-A13 does not inhibit other serine/threonine kinases including CDK, CHK, RAF, DAPK, IKK, IRAK, JNK, MAPK, PKC, and SAPK. LFM-A13-treated human cancer cells develop abnormal mitotic spindles and G₂/M-arrest during cell cycle progression. LFM-A13 was not toxic to rodents or dogs at daily dose levels as high as 100 mg/kg. Notably, at a low dose level of 10 mg/kg, which does not result in delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer, LFM-A13 markedly enhanced the anti-cancer activity of the mitotic spindle poison paclitaxel. These results indicate that LFM-A13 may be useful in the treatment of cancer patients.     

Bruton's tyrosine kinase is involved in p65-mediated transactivation and phosphorylation of p65 on serine 536 during NFkappaB activation by lipopolysaccharide

Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor kappaB (NFkappaB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk had no effect on NFkappaB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB.     

Interaction of Bruton's tyrosine kinase and protein kinase Ctheta in platelets. Cross-talk between tyrosine and serine/threonine kinases.

The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCtheta; in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCtheta; upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCtheta; in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCtheta; activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCtheta; in platelets, in which PKCtheta; positively modulates activity of Btk, which in turn feeds back negatively upon PKCtheta;.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation

The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH₂ terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-α, interleukin-6, and interleukin-1β expression are dependent on Tec kinase activity.     

MyD88 adapter-like (Mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction

Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.     

Silencing of Bruton's tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)

Tec family tyrosine kinases, Bruton's tyrosine kinase (Btk), Itk, Bmx, Tec, and Txk, are multi-domain proteins involved in hematopoietic signaling. Here, we demonstrate that human Btk protein can transiently be depleted using double-stranded short RNA interference (siRNA) oligonucleotides. Imaging and Western blotting analysis demonstrate that Btk expression is down regulated in heterologous systems as well as in hematopoietic lineages, following transfection or microinjection of Btk siRNA duplexes. The induction of histamine release, a pro-inflammatory mediator, in RBL-2H3 mast cells was reduced by 20-25% upon Btk down regulation. Similar, results were obtained when the Btk activity was inhibited using the kinase blocker LFM-A13. These results demonstrate a direct role of Btk for the efficient secretion of histamine in allergic responses.     

Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.     

Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.     

Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.

STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.     

Design, synthesis and biological evaluation of novel acrylamide analogues as inhibitors of BCR-ABL kinase

A series of acrylamide analogues were designed and synthesized from Imatinib and Nilotinib as novel BCR-ABL inhibitors by application of the principle of nonclassical electronic isostere. All new compounds were evaluated for their inhibitory effects on the activity of BCR-ABL kinase and the proliferation of K562 leukemia cancer cells in vitro. The acrylamide analogues in which the substituent in C ring was trifluoromethyl group were identified as highly potent BCR-ABL kinase inhibitors. Compound 13f exhibited an IC(50) value as low as 20.6nM in ABL kinase inhibition and an IC(50) value of 32.3nM for antiproliferative activity, about 10.5-fold and 12-fold lower than those of Imatinib respectively. These results suggest that compound 13f is a promising candidate as a novel BCR-ABL kinase inhibitor for further development.     

Potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-Golgi transport revealed by protein kinase inhibitor H89

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of betaCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but betaCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5'-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.