dlk modulates mitogen-activated protein kinase signaling to allow or prevent differentiation

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals.     

Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42

Cyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 [1]) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2 h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10 μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42.     

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.     

Ouabain-sensitive and ouabain-resistant isoforms of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in cerebellar granule cells control MAP kinase activity

Three-hour incubation of rat cerebellar granule cells with 0.1 μM ouabain increases intracellular levels of Ca²⁺ ions and reactive oxygen species (ROS) resulting in pronounced activation of Mitogen-Activated Protein Kinase (MAPK). Higher concentrations of ouabain induce further increases in MAPK activity. The activating effect of ouabain is attenuated by the NMDA-receptor antagonists MK-801 and D-AP5. The data obtained suggest that similar to NMDA receptors ouabain-sensitive and ouabain-resistant isoforms of Na⁺,K⁺-ATPase are actively involved in intracellular signaling cascades controlling proliferative activity of neuronal cells.     

Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.     

MAP kinase signaling in diverse effects of ethanol

Chronic ethanol abuse is associated with liver injury, neurotoxicity, hypertension, cardiomyopathy, modulation of immune responses and increased risk for cancer, whereas moderate alcohol consumption exerts protective effect on coronary heart disease. However, the signal transduction mechanisms underlying these processes are not well understood. Emerging evidences highlight a central role for mitogen activated protein kinase (MAPK) family in several of these effects of ethanol. MAPK signaling cascade plays an essential role in the initiation of cellular processes such as proliferation, differentiation, development, apoptosis, stress and inflammatory responses. Modulation of MAPK signaling pathway by ethanol is distinctive, depending on the cell type; acute or chronic; normal or transformed cell phenotype and on the type of agonist stimulating the MAPK. Acute exposure to ethanol results in modest activation of p42/44 MAPK in hepatocytes, astrocytes, and vascular smooth muscle cells. Acute ethanol exposure also results in potentiation or prolonged activation of p42/44MAPK in an agonist selective manner. Acute ethanol treatment also inhibits serum stimulated p42/44 MAPK activation and DNA synthesis in vascular smooth muscle cells. Chronic ethanol treatment causes decreased activation of p42/44 MAPK and inhibition of growth factor stimulated p42/44 MAPK activation and these effects of ethanol are correlated to suppression of DNA synthesis, impaired synaptic plasticity and neurotoxicity. In contrast, chronic ethanol treatment causes potentiation of endotoxin stimulated p42/44 MAPK and p38 MAPK signaling in Kupffer cells leading to increased synthesis of tumor necrosis factor. Acute exposure to ethanol activates pro-apoptotic JNK pathway and anti-apoptotic p42/44 MAPK pathway. Apoptosis caused by chronic ethanol treatment may be due to ethanol potentiation of TNF induced activation of p38 MAPK. Ethanol induced activation of MAPK signaling is also involved in collagen expression in stellate cells. Ethanol did not potentiate serum stimulated or Gi-protein dependent activation of p42/44 MAPK in normal hepatocytes but did so in embryonic liver cells and transformed hepatocytes leading to enhanced DNA synthesis. Ethanol has a 'triangular effect' on MAPK that involve direct effects of ethanol, its metabolically derived mediators and oxidative stress. Acetaldehyde, phosphatidylethanol, fatty acid ethyl ester and oxidative stress, mediate some of the effects seen after ethanol alone whereas ethanol modulation of agonist stimulated MAPK signaling appears to be mediated by phosphatidylethanol. Nuclear MAPKs are also affected by ethanol. Ethanol modulation of nuclear p42/44 MAPK occurs by both nuclear translocation of p42/44 MAPK and its activation in the nucleus. Of interest is the observation that ethanol caused selective acetylation of Lys 9 of histone 3 in the hepatocyte nucleus. It is plausible that ethanol modulation of cross talk between phosphorylation and acetylations of histone may regulate chromatin remodeling. Taken together, these recent developments place MAPK in a pivotal position in relation to cellular actions of ethanol. Furthermore, they offer promising insights into the specificity of ethanol effects and pharmacological modulation of MAPK signaling. Such molecular signaling approaches have the potential to provide mechanism-based therapy for the management of deleterious effects of ethanol or for exploiting its beneficial effects.     

MAP kinase mediates silica-induced fibrotic nodule formation and collagen accumulation in fibroblasts

It is well known that silica generates fibrosis around them in animals and human. However, the pathogenesis and mechanism of silica-induced fibrosis are still poorly understood. Here, we established a new strategy through which the effects of silica on fibrotic nodule formation, key extracellular matrix accumulation, and the mechanism involved were explored. To achieve this, human dermal fibroblasts were directly exposed to silica gel for various durations. Fibrotic nodule formation was evaluated by their microscopic appearance, type-1 procollagen, and fibronection expression in cell lysate and MMP-1 and-3 in conditioned media were analyzed by Western blotting. The results show an easily formation of nodule-like structures around silica gel in an in vitro-cultured system. The findings further revealed that silica gel stimulates collagen and fibronectin expression, while down-regulates matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) released in conditioned medium. To explore the mechanism involved, P38 and ERK1/2 Mitogen-Activated Protein Kinase (MAPK) signaling pathways were evaluated. Result showed that silica inhibits P38 and extracellular signal-regulated kinases (ERK1/2) MAP kinase phosphorylation. The addition of ERK1/2 inhibitor increases silica-stimulated type-1 collagen expression, reduces MMP-1 release and further enhances silica-induced nodule formation in dermal fibroblasts. These findings indicate that the inhibition of ERK1/2 MAPK signaling pathway may contribute to silica-caused fibrosis. In summary, our findings suggest that silica can directly cause fibrotic phenotype when fibroblasts contact with silica particles independent of any inflammation and other factors may exist in an in vivo condition.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Mineral trioxide aggregate promotes odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp stem cells

Mitogen-activated protein kinase (MAPK) pathways are involved in stem cell differentiation. However, the odontoblastic differentiation-inducing effects by mineral trioxide aggregate (MTA) via MAPK pathways have not been clarified in human dental pulp stem cells (DPSCs). In this study we investigated the effects of MTA on cell viability and production of differentiation markers, and the involvement of MAPK signaling pathways in cultured human DPSCs. Cells were cultured with MTA, and the viability and differentiation productions of the cells were determined using the MTT assay and real-time PCR analysis, respectively. MAPK activation was measured by western blotting. MTA at concentrations of 20 and 10 mg/ml was toxic for human DPSCs. MTA significantly increased the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), type I collagen (COLI), osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs and induced the phosphorylation of p42 and p44 (p42/44), p38 and c-Jun N-terminal kinases 1 and 2 (JNK1/2) MAPK. Furthermore, the inhibitor of p42/44 MAPK attenuated the MTA-induced odontoblastic differentiation. These data indicated that MTA-induced odontoblastic differentiation of human DPSCs was via MAPK pathways, which may play a key role in the repair responses of dentin-pulp-like complexes.     

Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway

Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the NSD1 gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13 contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated NSD1 expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.     

Regulation of Plasmodium falciparum glideosome associated protein 45 (PfGAP45) phosphorylation

The actomyosin motor complex of the glideosome provides the force needed by apicomplexan parasites such as Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) to invade their host cells and for gliding motility of their motile forms. Glideosome Associated Protein 45 (PfGAP45) is an essential component of the glideosome complex as it facilitates anchoring and effective functioning of the motor. Dissection of events that regulate PfGAP45 may provide insights into how the motor and the glideosome operate. We found that PfGAP45 is phosphorylated in response to Phospholipase C (PLC) and calcium signaling. It is phosphorylated by P. falciparum kinases Protein Kinase B (PfPKB) and Calcium Dependent Protein Kinase 1 (PfCDPK1), which are calcium dependent enzymes, at S89, S103 and S149. The Phospholipase C pathway influenced the phosphorylation of S103 and S149. The phosphorylation of PfGAP45 at these sites is differentially regulated during parasite development. The localization of PfGAP45 and its association may be independent of the phosphorylation of these sites. PfGAP45 regulation in response to calcium fits in well with the previously described role of calcium in host cell invasion by malaria parasite.     

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.