Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

Effects of dexamethasone,heat shock,and serum responses on the inhibition of hsc70 synthesis by antisense RNA in NIH 3T3 cells

A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5′-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA. © 1995 Wiley-Liss, Inc.     

Up-regulation of Axin2 by dexamethasone promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells

Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/β-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/β-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear β-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear β-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPARγ-positive and aP2-positive cells and decreased the mRNA expression of PPARγ2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling β-catenin expression.     

A member of the Y-box protein family interacts with an upstream element in the alpha1(I) collagen gene.

Transforming growth factor beta (TGF-beta) stimulates protein complex formation on a TGF-beta response element (TAE) found in the distal portion (-1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE-protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein-TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-beta treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-beta treatment. Therefore, the increased binding activity seen in TGF-beta-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter-reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-beta response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-beta addition to fibroblasts, suggesting a role for this protein in TGF-beta signaling.     

A member of the Y-box protein family interacts with an upstream element in the α1(I) collagen gene

Transforming growth factor beta (TGF-β) stimulates protein complex formation on a TGF-β response element (TAE) found in the distal portion (−1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE–protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein–TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-β treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-β treatment. Therefore, the increased binding activity seen in TGF-β-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter–reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signaling.     

Nonspecific and specific interactions of Y-box-binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of protein synthesis in animal cells

Y-box-binding protein 1 (YB-1) is an animal multifunctional DNA/RNA-binding protein that is involved in reproduction, storing, and expression of genetic information. YB-1 accompanies mRNA throughout its life, from synthesis to degradation, and has a high specific and nonspecific affinity for RNA. In the nucleus YB-1 regulates mRNA processing. In the cytoplasm YB-1 is responsible for global and selective regulation of protein synthesis, as well as the mRNA life. This review focuses on the role of YB-1 in regulating translation. The possible mechanisms of the positive and negative effects of YB-1 on this process are considered. The recent original data are described, supporting the role of YB-1 as a major structural component of mRNP. Data about specific interactions of YB-1 with RNA are summarized for the first time.     

Expression of YB-1 enhances production of murine leukemia virus vectors by stabilizing genomic viral RNA

Murine leukemia virus (MLV)-based retroviral vectors is widely used for gene transfer and basic research, and production of high-titer retroviral vectors is very important. Here we report that expression of the Y-box binding protein 1 (YB-1) enhanced the production of infectious MLV vectors. YB-1 specifically increased the stability of viral genomic RNA in virus-producing cells, and thus increasing viral RNA levels in both producer cells and virion particles. The viral element responsive to YB-1 was mapped to the repeat sequence (R region) in MLV genomic RNA. These results identified YB-1 as a MLV mRNA stabilizer, which can be used for improving production of MLV vectors.     

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.