Type II arginine methyltransferase PRMT5 regulates gene expression of inhibitors of differentiation/DNA binding Id2 and Id4 during glial cell differentiation

PRMT5 is a type II protein arginine methyltranferase that catalyzes monomethylation and symmetric dimethylation of arginine residues. PRMT5 is functionally involved in a variety of biological processes including embryo development and circadian clock regulation. However, the role of PRMT5 in oligodendrocyte differentiation and central nervous system myelination is unknown. Here we show that PRMT5 expression gradually increases throughout postnatal brain development, coinciding with the period of active myelination. PRMT5 expression was observed in neurons, astrocytes, and oligodendrocytes. siRNA-mediated depletion of PRMT5 in mouse primary oligodendrocyte progenitor cells abrogated oligodendrocyte differentiation. In addition, the PRMT5-depleted oligodendrocyte progenitor and C6 glioma cells expressed high levels of the inhibitors of differentiation/DNA binding, Id2 and Id4, known repressors of glial cell differentiation. We observed that CpG-rich islands within the Id2 and Id4 genes were bound by PRMT5 and were hypomethylated in PRMT5-deficient cells, suggesting that PRMT5 plays a role in gene silencing during glial cell differentiation. Our findings define a role of PRMT5 in glial cell differentiation and link PRMT5 to epigenetic changes during oligodendrocyte differentiation.     

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.     

Survival and differentiation of mammary epithelial cells in mammary gland development require nuclear retention of Id2 due to RANK signaling

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.     

Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.     

Constitutive and inducible expression of the epithelial antigen MUC1 (CD227) in human T cells

The activated type 1 insulin-like growth factor (IGF-IR) increases the expression of Id1 proteins in mouse embryo fibroblasts (MEF). Up-regulation depends on a functional receptor and on multiple pathways originating from different domains of the receptor. In MEF, Id1 protein expression is also up-regulated by serum and certain oncogenes. Signaling through Stat3 plays an important, but not exclusive, role in the up-regulation of Id1 protein levels. In all instances, the increase in Id1 protein expression is paralleled by a corresponding increase in Id1 promoter activity, as measured with a reporter gene.     

Prophylactic effects of sulforaphane on depression-like behavior and dendritic changes in mice after inflammation

Inflammation plays a role in the pathophysiology of depression. Sulforaphane (SFN), an isothiocyanate compound derived from broccoli, is a potent activator of the NF-E2-related factor-2 (Nrf2), which plays a role in inflammation. In this study, we examined whether the prevention effects of SFN in lipopolysaccharide (LPS) induced depression-like behavior in mice. Pretreatment with SFN significantly blocked an increase in the serum tumor necrosis factor-α (TNF-α) level and an increase in microglial activation of brain regions after a single administration of LPS (0.5 mg/kg). Furthermore, SFN significantly potentiated increased serum levels of IL-10 after LPS administration. In the tail-suspension test and forced swimming test, SFN significantly attenuated an increase of the immobility time after LPS administration. In addition, SFN significantly recovered to control levels for LPS-induced alterations in the proteins such as brain-derived neurotrophic factor, postsynaptic density protein 95 and AMPA receptor 1 (GluA1) and dendritic spine density in the brain regions. Finally, dietary intake of 0.1% glucoraphanin (a glucosinolate precursor of SFN) food during the juvenile and adolescence could prevent the onset of LPS-induced depression-like behaviors and dendritic spine changes in the brain regions at adulthood. In conclusion, these findings suggest that dietary intake of SFN-rich broccoli sprout has prophylactic effects on inflammation-related depressive symptoms. Therefore, supplementation of SFN-rich broccoli sprout could be prophylactic vegetable to prevent or minimize the relapse by inflammation in the remission state of depressed patients.     

MAPK-activated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease

MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon gamma-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2-/-) mice demonstrated significant inhibition in release of tumor necrosis factor alpha, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1alpha on stimulation with LPS + interferon gamma or amyloid-beta peptide (1-42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon gamma-stimulated or amyloid-beta peptide (1-42)-stimulated MK2-/- microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with beta-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor alpha, macrophage inflammatory protein 1alpha, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented.     

Crucial role of inhibitor of DNA binding/differentiation in the vascular endothelial growth factor-induced activation and angiogenic processes of human endothelial cells

Angiogenesis plays a pivotal role in the aggressive proliferation of synovial cells in rheumatoid arthritis. We have previously reported the overexpression of inhibitor of DNA binding/differentiation (Id) in the endothelial cells within the synovial tissues of rheumatoid arthritis. In this study, we investigated the role of Id in inflammation and angiogenesis in an in vitro model using HUVECs. Vascular endothelial growth factor (VEGF) and TGFbeta induced the expression of Id1 and Id3 in HUVECs. Forced expression of Id induced proliferative activity in HUVECs accompanied by down-regulation of p16INK4a. Overexpression of Id enhanced expression of ICAM-1 and E-selectin, and induced angiogenic processes such as transmigration, matrix metalloproteinase-2 and -9 expression, and tube formation. In contrast, knockdown of Id1 and Id3 with RNA interference abolished proliferation, activation, and angiogenic processes of HUVECs induced by VEGF. These results indicated that Id plays a crucial role in VEGF-induced signals of endothelial cells by causing activation and potentiation of angiogenic processes. Based on these findings, it was proposed that inhibition of expression and/or function of Id1 and Id3 may potentially be of therapeutic value for conditions associated with pathological angiogenesis.     

Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines

Purpose

It is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases, although the mechanisms through which this occurs remain unclear. Among mitogen-activated protein kinase (MAPK) targets, mitogen- and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression. In this study, the roles of MSK1 activation in neuroinflammation were investigated.

Methods

The bacterial lipopolysaccharide (LPS)-induced brain injury model was performed on Sprague-Dawley rats. The dynamic expression changes and the cellular location of p-MSK1 in the brain cortex were detected by Western blot and immunofluorescence staining. The synthesis of inflammatory cytokines in astrocytes was detected by enzyme-linked immunosorbent assay (ELISA).

Results

Phosphorylated MSK1 (p-MSK1 Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1 in astrocytes was also observed in inflamed cerebral cortex. At 1 day after LPS stimulation, iNOS, TNFα expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1 (Thr-581) may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS in primary astrocytes stimulated the synthesis of inflammatory cytokines, through MAPKs signaling pathways. In cultured primary astrocytes, both knock-down of total MSK1 by small interfering RNAs (siRNA) or specific mutation of Thr-581 resulted in higher production of certain cytokines, such as TNFα and IL-6.

Conclusions

Collectively, these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of inflammation.