EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Biglycan Intensifies ALK5–Smad2/3 Signaling by TGF‐β1 and Downregulates Syndecan‐4 in Cultured Vascular Endothelial Cells

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. A small leucine‐rich dermatan sulfate proteoglycan, biglycan, is one of the predominant types of proteoglycans synthesized by vascular endothelial cells; however, the physiological functions of biglycan are not completely understood. In the present study, bovine aortic endothelial cells in culture were transfected with small interfering RNAs for biglycan, and the expression of other proteoglycans was examined. Transforming growth factor‐β₁ signaling was also investigated, because the interaction of biglycan with cytokines has been reported. Biglycan was found to form a complex with either transforming growth factor‐β₁ or the transforming growth factor‐β₁ type I receptor, ALK5, and to intensify the phosphorylation of Smad2/3, resulting in a lower expression of the transmembrane heparan sulfate proteoglycan, syndecan‐4. This is the first report to clarify the function of biglycan as a regulatory molecule of the ALK5–Smad2/3 TGF‐β₁ signaling pathway that mediates the suppression of syndecan‐4 expression in vascular endothelial cells. J. Cell. Biochem. 118: 1087–1096, 2017. © 2016 Wiley Periodicals, Inc.     

1950 MHz IMT‐2000 field does not activate microglial cells in vitro

Given the widespread use of the cellular phone today, investigation of potential biological effects of radiofrequency (RF) fields has become increasingly important. In particular, much research has been conducted on RF effects on brain function. To examine any biological effects on the central nervous system (CNS) induced by 1950 MHz modulation signals, which are controlled by the International Mobile Telecommunication‐2000 (IMT‐2000) cellular system, we investigated the effect of RF fields on microglial cells in the brain. We assessed functional changes in microglial cells by examining changes in immune reaction‐related molecule expression and cytokine production after exposure to a 1950 MHz Wideband Code Division Multiple Access (W‐CDMA) RF field, at specific absorption rates (SARs) of 0.2, 0.8, and 2.0 W/kg. Primary microglial cell cultures prepared from neonatal rats were subjected to an RF or sham field for 2 h. Assay samples obtained 24 and 72 h after exposure were processed in a blind manner. Results showed that the percentage of cells positive for major histocompatibility complex (MHC) class II, which is the most common marker for activated microglial cells, was similar between cells exposed to W‐CDMA radiation and sham‐exposed controls. No statistically significant differences were observed between any of the RF field exposure groups and the sham‐exposed controls in percentage of MHC class II positive cells. Further, no remarkable differences in the production of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6) were observed between the test groups exposed to W‐CDMA signal and the sham‐exposed negative controls. These findings suggest that exposure to RF fields up to 2 W/kg does not activate microglial cells in vitro. Bioelectromagnetics 31:104–112, 2010. © 2009 Wiley‐Liss, Inc.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Phosphorylation of Phospholipase C‐δ1 Regulates its Enzymatic Activity

Phosphorylation of phospholipase C‐δ₁ (PLC‐δ₁) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC‐δ₁ most potently. It was also demonstrated that PLC‐δ₁ directly bound PKC‐α via its pleckstrin homology (PH) domain. Using deletion mutants of PLC‐δ₁ and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC‐δ₁. In vitro phosphorylation of PLC‐δ₁ by PKC stimulated [³H]PtdIns(4,5)P₂ hydrolyzing activity and [³H]Ins(1,4,5)P₃‐binding of the PLC‐δ₁. On the other hand, endogenous PLC‐δ₁ was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, we determined that Thr209 of PLC‐δ₁ is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC‐δ₁ was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC‐α reduced serine phosphorylation of PLC‐δ₁ detected by an anti‐phosphoserine antibody and PLC‐δ₁‐dependent basal production of inositol phosphates in NIH‐3T3 cells, suggesting PKC‐α activates phosphatase or inactivates another kinase involved in PLC‐δ₁ serine phosphorylation to modulate the PLC‐δ₁ activity in vivo. Taken together, these results suggest that PLC‐δ₁ has multiple phosphorylation sites and phosphorylation status of PLC‐δ₁ regulates its activity positively or negatively depends on the phosphorylation sites. J. Cell. Biochem. 108: 638–650, 2009. © 2009 Wiley‐Liss, Inc.