Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K,PKC and MAPKs

Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin–myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin–myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte re-differentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path.     

TGF-β1 and hypoxia-dependent expression of MKP-1 leads tumor resistance to death receptor-mediated cell death

Sporadic occurrence of transformed tumor cells is under the surveillance of the host immune system and such cells are effectively eliminated by immune-mediated cell death. During tumor progression, the antitumor effects of the tumor microenvironment are suppressed by diverse immunosuppressive mechanisms. In this research, we suggest novel immune evasion strategy of tumor cells through a transforming growth factor (TGF)-β1- and hypoxia-dependent mechanism. Experimental results showed that TGF-β1 and hypoxia induced mitogen-activated protein kinase phosphatase (MKP)-1 expression within 1 h, resulting in attenuation of c-Jun N-terminal kinase (JNK) phosphorylation and subsequent death receptor-mediated cell death. In addition, analysis of microarray data and immunostaining of MKP-1 in hepatocellular carcinoma (HCC) patient samples revealed that expression of MKP-1 is notably higher in tumors than in normal tissues, implying that MKP-1-dependent suppression of immune-mediated cell death takes place only in the tumor. To prove that MKP-1 can act as a mediator of immune escape by tumors, we determined whether chemo-resistance against several anticancer drugs could be overcome by knockdown of MKP-1. Cytotoxic assays showed that chemotherapy with siRNA targeting MKP-1 was significantly more effective than chemotherapy in the presence of MKP-1. Thus, we conclude that TGF-β1 and hypoxia ensure tumor cell survival and growth through expression of MKP-1.     

l-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that l-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that l-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the l-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling.     

Mitogen-activated protein kinase phosphatase-1 (MKP-1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine- N -acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.     

Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Oligomannose-coated liposomes activate ERK via Src kinases and PI3K/Akt in J774A.1 cells

We have previously shown that liposomes coated with a neoglycolipid constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages to induce enhanced expression of co-stimulatory molecules and MHC class II. In this study, we investigated the signaling pathways activated by the Man3-DPPE-coated liposomes (OMLs) in a murine macrophage cell line, J774A.1. In response to OML stimulation, ERK among MAPKs was clearly and transiently phosphorylated in J774 cells. ERK phosphorylation was also induced by treatment of the cells with Man3-DPPE and Man3-BSA, but not by uncoated liposomes. In addition, rapid and transient phosphorylation of Akt and Src family kinases (SFKs) was observed in response to OMLs. OML-induced ERK phosphorylation was inhibited by specific inhibitors of PI3K and SFKs, and OML-induced Akt phosphorylation was inhibited by a inhibitor of SFKs. Therefore, OMLs may activate the PI3K/Akt pathway through phosphorylation of Src family kinases to induce ERK activation.     

Dephosphorylation-induced EZH2 activation mediated RECK downregulation by ERK1/2 signaling

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene, a widely known cancer inhibitor, could effectively suppress cancer metastasis and angiogenesis. Downregulation or loss of RECK expression frequently occurs during cancer progression. However, the mechanism underlying RECK dysregulation has not been fully elucidated. Herein, we reported for the first time that enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, could epigenetically attenuate RECK expression via catalyzing H3K27 trimethylation (H3K27me3) within the RECK promoter. Furthermore, we also proved, for the first time, the involvement of EZH2 in the inhibition of RECK by extracellular signal-related kinases (ERK)-1/2 signaling. Next, we revealed that the modulation of the enzymic activity of EZH2 resulting from posttranslational phosphorylation at the serine-21 site was responsible for the increased enrichment of H3K27me3 at the RECK promoter region by ERK1/2 signaling. Collectively, the results of our study shed more light on the mechanisms responsible for the dysregulation of RECK by the ERK1/2 pathway.