Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable.     

Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

In the present study, we demonstrate that erythropoietin (Epo) induces the expression and the release of tissue inhibitors of metalloproteinase-1 (TIMP-1) in a time- and dose-dependent manner in Epo-dependent cell line UT-7 cells and in normal human erythroid progenitor cells from cord blood (CD36+) and required de novo protein synthesis. TIMP-1 was not expressed in the absence of Epo. Inhibition of the mitogen-activated protein kinase pathway by the specific inhibitors PD98059 and U0126 and of phosphatidylinositol 3-kinase by LY294002, strongly inhibited Epo-induced TIMP-1 expression and secretion. In the absence of Epo, both latent and active forms of matrix metalloproteinase-9 (MMP-9) were secreted into media. Upon Epo stimulation, MMP-9 and pro-MMP-9 secretion was inhibited in a dose-dependent manner parallel to TIMP-1 induction. The addition of PD98059, U0126, and LY294002 in the presence of Epo restored MMP-9 production in UT-7 and CD36+ cells. Our findings strongly suggest an inversely coordinated regulation of the TIMP-1 gene and MMP-9 production by Epo via mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.     

促分裂原激活的蛋白激酶(MAPK)信号传导通路的研究进展

MAPK信号传导通路在真核生物细胞的生化和分化、细胞周期调节和细胞凋亡过程中发挥着重要的作用。生物化学研究和分子生物学鉴定表明：在酵母和哺乳动物细胞中MAPK信号传导通路都有一个保守的三组分激活模件,该模件内的激酶引发了一系列的磷酸化级联反应。了解MAPK信号传导通路的组成部分、调控方式和作用机制,有助于对因信号传导通路的调节失控而引起的疾病进行预防和治疗。     

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.     

Organization and regulation of mitogen-activated protein kinase signaling pathways

Mitogen-activated protein kinases (MAPKs) are components of a three kinase regulatory cascade. There are multiple members of each component family of kinases in the MAPK module. Specificity of regulation is achieved by organization of MAPK modules, in part, by use of scaffolding and anchoring proteins. Scaffold proteins bring together specific kinases for selective activation, sequestration and localization of signaling complexes. The recent elucidation of scaffolding mechanisms for MAPK pathways has begun to solve the puzzle of how specificity in signaling can be achieved for each MAPK pathway in different cell types and in response to different stimuli. As new MAPK members are defined, determining their organization in kinase modules will be critical in understanding their select role in cellular regulation.     

Effect of the mitogen-activated kinase inhibitors on monocyte protein-1-stimulated chemotaxis of cells

Monocyte chemoattractant protein (MCP-1) is a major chemoattractant for monocytes and T-lymphocytes although it can cause migration of the HUVECs. We used monocytic cell line THP-1, monocytes of human peripheral blood, and HUVECs to study MCP-1 receptor-mediated cell migration. We showed that THP-1 and the monocytes chemotaxis was decreased in presence of specific inhibitors of p 38 MAP-kinase. Furthermore, it was almost completely diminished by inhibitor of tyrosine kinases. In contrast, MCP-1-stimulated migration of HUVECs was abrogated by specific inhibitor of ERK1/2 MAP-kinases and, to a lesser extent, by blocking tyrosine kinases. These results suggest that intracellular signal pathways activated by MCP-1 in monocytes and HUVECs, are distinct.     

Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.     

ICAM-1-induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways

ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer's disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1beta and IL-1alpha protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1alpha and IL-1beta expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.     

Relations between the mitogen-activated protein kinase and the cAMP-dependent protein kinase pathways: comradeship and hostility

Inter- and intracellular communications and responses to environmental changes are pivotal for the orchestrated and harmonious operation of multi-cellular organisms. These well-tuned functions in living organisms are mediated by the action of signal transduction pathways, which are responsible for receiving a signal, transmitting and amplifying it, and eliciting the appropriate cellular responses. Mammalian cells posses numerous signal transduction pathways that, rather than acting in solitude, interconnect with each other, a phenomenon referred to as cross-talk. This allows cells to regulate the distribution, duration, intensity and specificity of the response. The cAMP/cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascades modulate common processes in the cell and multiple levels of cross-talk between these signalling pathways have been described. The first- and best-characterized interconnections are the PKA-dependent inhibition of the MAPKs ERK1/2 mediated by RAF-1, and PKA-induced activation of ERK1/2 interceded through B-RAF. Recently, novel interactions between components of these pathways and new mechanisms for cross-talk have been elucidated. This review discusses both known and novel interactions between compounds of the cAMP/PKA and MAPKs signalling pathways in mammalian cells.     

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.     

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.