STAT3, p38 MAP Kinase and NF-{kappa}B Drive Unopposed Monocyte-dependent Fibroblast MMP-1 Secretion in Tuberculosis.

Tissue destruction characterizes infection with Mycobacterium tuberculosis (Mtb). Type I collagen provides the lung's tensile strength, is extremely resistant to degradation, but is cleaved by matrix metalloproteinase (MMP)-1. Fibroblasts potentially secrete quantitatively more MMP-1 than other lung cells. We investigated mechanisms regulating Mtb-induced collagenolytic activity in fibroblasts in vitro and in patients. Lung fibroblasts were stimulated with conditioned media from Mtb-infected monocytes (CoMTb). CoMTb induced sustained increased MMP-1 (74 versus 16 ng/ml) and decreased tissue inhibitor of metalloproteinase (TIMP)-1 (8.6 versus 22.3 ng/ml) protein secretion. CoMTb induced a 2.7-fold increase in MMP-1 promoter activation and a 2.5-fold reduction in TIMP-1 promoter activation at 24 hours (P = 0.01). Consistent with this, TIMP-1 did not co-localize with fibroblasts in patient granulomas. MMP-1 up-regulation and TIMP-1 down-regulation were p38 (but not extracellular signal–regulated kinase or c-Jun N-terminal kinase) mitogen-activated protein kinase–dependent. STAT3 phosphorylation was detected in fibroblasts in vitro and in tuberculous granulomas.STAT3 inhibition reduced fibroblast MMP-1 secretion by 60% (P = 0.046). Deletion of the MMP-1 promoter NF-B–binding site abrogated promoter induction in response to CoMTb. TNF-, IL-1ß, or Oncostatin M inhibition in CoMTb decreased MMP-1 secretion by 65, 63, and 25%, respectively. This cytokine cocktail activated the same signaling pathways in fibroblasts and induced MMP-1 secretion similar to that induced by CoMTb. This study demonstrates in a cellular model and in patients with tuberculosis that in addition to p38 and NF-B, STAT3 has a key role in driving fibroblast-dependent unopposed MMP-1 production that may be key in tissue destruction in patients.

MAP Kinase Phosphatase-1 Protects against Inflammatory Bone Loss

The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-alpha, and IL-1 beta. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1(-/-) mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1(-/-) mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1(-/-) control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.

MAP kinase kinase 1 (MKK1) is essential for transmission of Trypanosoma brucei by Glossina morsitans

MAP kinase kinase 1 (MKK1) is encoded by a single copy gene in Trypanosoma brucei. It has been shown recently that MKK1 is not essential for bloodstream forms [14]. To investigate the requirement for MKK1 in other life-cycle stages we generated null mutants in procyclic forms of a fly-transmissible strain. These grew normally in culture and were able to establish midgut infections in tsetse at normal rates and intensities, but were incapable of colonising the salivary glands. Transformation of null mutants with an ectopic copy of MKK1 enabled parasites to complete the life cycle in tsetse and infect mice. This is the first example of a gene that is indispensable for transmission of T. brucei. It also raises the possibility that activating the MKK1 signalling cascade in vitro might trigger the differentiation and proliferation of life-cycle stages of T. brucei that are currently refractory to culture.

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Does the p38 MAP kinase inhibitor pamapimod have potential for the treatment of rheumatoid arthritis?

Background: Methotrexate alone or in combination with other agents is the standard treatment for moderate-to-severe rheumatoid arthritis. As the biological agents are expensive, they are not usually used until methotrexate has failed to give a good response. Thus, there is scope for the development of cheaper drugs that can be used instead of methotrexate or in addition to methotrexate. Objectives/methods: Pamapimod is a p38α inhibitor being developed for use in the treatment of rheumatoid arthritis. The objective was to evaluate the recent clinical trials of pamapimod in subjects with rheumatoid arthritis. Results: There is no clear cut evidence that pamapimod alone or in the presence of methotrexate is efficacious in subjects with rheumatoid arthritis, but it does cause adverse effects. Conclusion: It is unlikely that pamapimod will be useful in the treatment of rheumatoid arthritis.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Brf1 post-transcriptionally regulates pluripotency and differentiation responses downstream of Erk MAP Kinase

FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.

p38 alpha MAP Kinase Controls IL-17 Synthesis in Vogt-Koyanagi-Harada Syndrome and Experimental Autoimmune Uveitis

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38 alpha mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4(+)IL-17(+) cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4(+) lymphocytes from MSK-1/2-deficient mice, human CD4(+) cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38 alpha MAPK inhibitor and then assayed for IL-17, IFN-gamma, and IL-4 production. RESULTS. The inhibition of p38 alpha MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38 alpha MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4(+) T cells and that inhibition of p38 alpha MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU. (Invest Ophthalmol Vis Sci. 2010;51:3567-3574) DOI: 10.1167/iovs.09-4393

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc.

Dextran sulfate modulates MAP kinase signaling and reduces endothelial injury in a rat aortic clamping model

OBJECTIVE: Mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK1/2, noticeably influence ischemia/reperfusion injury (IRI). The complement inhibitor dextran sulfate (DXS) associates with damaged endothelium denudated of its heparan sulfate proteoglycan (HSPG) layer. Other glycosaminoglycan analogs are known to influence MAPK signaling. Hypothetically therefore, targeted intravascular cytoprotection by DXS may function in part through influencing MAPK activation to reduce IRI-induced damage of the vasculature. METHODS: IRI of the infrarenal aorta of male Wistar rats was induced by 90 minutes clamping followed by 120 minutes reperfusion. DXS (5 mg/mL) or physiologic saline (NaCl controls) was infused locally into the ischemic aortic segment immediately prior to reperfusion. Ninety minutes ischemia-only and heparinase infusion (maximal damage) experiments, as well as native rat aorta, served as controls. Aortas were excised following termination of the experiments for further analysis. RESULTS: DXS significantly inhibited IRI-induced JNK and ERK1/2 activation (P = .043; P =.005) without influencing the p38 pathway (P =.110). Reduced aortic injury, with significant inhibition of apoptosis (P = .032 for DXS vs NaCl), correlated with decreased nuclear factor kappaB translocation within the aortic wall. DXS treatment clearly reduced C1q, C4b/c, C3b/c, and C9 complement deposition, whilst preserving endothelial cell integrity and reducing reperfusion-induced HSPG shedding. Protection was associated with binding of fluorescein labeled DXS to ischemically damaged tissue. CONCLUSIONS: Local application of DXS into ischemic vasculature immediately prior to reperfusion reduces complement deposition and preserves endothelial integrity, partially through modulating activation of MAPKs and may offer a new approach to tackle IRI in vascular surgical procedures. CLINICAL RELEVANCE: The purpose of the present study was to determine the role of dextran sulfate (DXS), a glycosaminoglycan analog and complement inhibitor, in modulating intracellular MAPK signaling pathways, reducing complement activation and ultimately attenuating ischemia/reperfusion injury (IRI) in a rat aortic-clamping model, in part a surrogate model to study the microvasculature. The study shows a role for DXS in ameliorating endothelial injury by reducing IRI-mediated damage and intravascular, local inflammation in the affected aortic segment. DXS may be envisaged as an endothelial protectant in vascular injury, such as occurs during vascular surgical procedures.