Modulation of autocrine TNF-α-stimulated matrix metalloproteinase 9 (MMP-9) expression by mitogenactivated protein kinases in THP-1 monocytic cells

Matrix metalloproteinase 9 (MMP-9) is implicated in various physiological processes by its ability to degrade the extracellular matrix (ECM) and process multiple regulatory proteins. Normally, MMP-9 expression is tightly controlled in cells. Sustained or enhanced MMP-9 secretion, however, has been demonstrated to contribute to the pathophysiology of numerous diseases, including arthritis and tumor progression, rendering this enzyme a major target for clinical interventions. Here we show that constitutive MMP-9 secretion was abrogated in THP-1 monocytic leukemia cells by addition of neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or TNF receptor type 1 (TNF-R1), as well as by inhibition of TNF-α converting enzyme (TACE). This indicates that MMP-9 production in these cells is maintained by autocrine stimulation, with TNF-α acting via TNF-R1. To investigate the intracellular signaling routes involved in MMP-9 gene transcription, cells were treated with different inhibitors of major mitogen-activated protein kinase (MAPK) pathways. Interruption of the extracellular signal-regulated kinase pathway 1/2 (ERK1/2) using PD98059 significantly downregulated constitutive MMP-9 release. In contrast, blockage of p38 kinase activity by addition of SB203580 or SB202190, as well as inhibition of c-Jun N-terminal kinase (JNK) using L-JNK-I1, clearly augmented MMP-9 expression and secretion by an upregulation of ERK1/2 phosphorylation. Moreover, exogenously added TNF-α augmented MMP-9 synthesis and secretion in THP-1 cells via enhancement of ERK1/2 activity. Taken together, our results indicate that ERK1/2 activity plays a pivotal role in TNF-α-induced MMP-9 production and demonstrate its negative modulation by p38 and JNK activity. These findings suggest ERK1/2 rather than p38 and JNK as a reasonable target to specifically block MMP-9 expression using MAPK inhibitors in therapeutic applications

Classical, novel and atypical isoforms of PKC stimulate ANF- and TRE/AP-1-regulated-promoter activity in ventricular cardiomyocytes

Cultured neonatal rat ventricular myocytes were co-transfected with expression plasmids encoding protein kinase C (PKC) isoforms from each of the PKC subfamilies (classical PKC-α, novel PKC-ε or atypical PKC-ξ) together with an atrial natriuretic factor (ANF) reporter plasmid. Each PKC had been rendered constitutively active by a single Ala→Glu mutation or a small deletion in the inhibitory pseudosubstrate site. cPKC-α, nPKC-ε or aPKC-ξ expression plasmids each stimulated ANF-promoter activity and expression of a reporter gene under the control of a 12-O-tetradecanoylphorbol 13-acetate-response element (TRE). Upregulation of the ANF promoter is characteristic of the hypertrophic response in the heart ventricle and a TRE is present in the ANF promoter. Thus all subfamilies of PKC may have the potential to contribute to hypertrophic response in cardiomyocytes

Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes

AbstractET-1 stimulated MBP kinase activity in cultured cardiomyocytes. Maximal activation (3.5-fold) was at 5 min. EC50 was 0.2 nM. PMA or PE also increased MBP kinase (4- or 2.5-fold, respectively). Pre-treatment with PMA down-regulated the subsequent response to ET-1 or PMA. ET-1- or PMA-stimulated MBP kinase was resolved into 2 major (peaks II and IV) and 2 minor peaks by FPLC on Mono Q. Peaks II and IV were inactivated by either LAR or PP2A. Renatured MBP kinase activities following SDS-PAGE in MBP-containing gels and immunoblot analysis showed that peak II was a p42 MAP kinase and peak IV was a p44 MAP kinase

Doublecortin X (DCX) serine 28 phosphorylation is a regulatory switch, modulating association of DCX with microtubules and actin filaments

Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275–366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1–44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and β-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation

Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an a-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glutubulin) stable MT’s. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy

JNK signaling: Regulation and functions based on complex protein-protein partnerships

The c-Jun N-terminal kinases (JNKs), as members of the mitogenactivated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for -20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. Copyright © 2016, American Society for Microbiology. All Rights Reserved

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

JNK signalling in cancer: In need of new, smarter therapeutic targets

Copyright © 2013 The British Pharmacological Society. This is the accepted version of the following article: Bubici, C. and Papa, S. (2014), JNK signalling in cancer: in need of new, smarter therapeutic targets. British Journal of Pharmacology, 171: 24–37, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/bph.12432/abstract.The JNKs are master protein kinases that regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival and death. It is increasingly apparent that persistent activation of JNKs is involved in cancer development and progression. Therefore, JNKs represent attractive targets for therapeutic intervention with small molecule kinase inhibitors. However, evidence supportive of a tumour suppressor role for the JNK proteins has also been documented. Recent studies showed that the two major JNK proteins, JNK1 and JNK2, have distinct or even opposing functions in different types of cancer. As such, close consideration of which JNK proteins are beneficial targets and, more importantly, what effect small molecule inhibitors of JNKs have on physiological processes, are essential. A number of ATP-competitive and ATP-non-competitive JNK inhibitors have been developed, but have several limitations such as a lack of specificity and cellular toxicity. In this review, we summarize the accumulating evidence supporting a role for the JNK proteins in the pathogenesis of different solid and haematological malignancies, and discuss many challenges and scientific opportunities in the targeting of JNKs in cancer.Kay Kendall Leukemia Fund, Italian Association for Cancer Research and Foundation for Liver Research

Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli