Cellular Mechanical Stress Gradient Regulates Cell Proliferation and Differentiation Patterns

This article has no abstract.     

ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

剪切力对单核细胞趋化蛋白-1的影响

单核细胞趋化蛋白-1（MCP-1）能趋化单核细胞在内皮细胞下聚集,是动脉粥样硬化最早期的病理改变之一.从生物力学的角度对体外培养的人脐静脉内皮细胞（HUVEC）合成和分泌MCP-1的规律作了研究.通过流动小室,HUVEC给予0.4,1.0, 2.0 N/m²的剪应力,运用免疫组化,图象处理及ELISA方法测出不同时间胞浆及灌流液中MCP-1的含量,结果表明HUVEC合成和分泌MCP-1是随剪切力和时间变化而变化的.该工作为进一步理解剪切力诱导动脉粥样硬化的发生提供实验数据.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

PFKFB3 Regulates Oxidative Stress Homeostasis via Its S-Glutathionylation in Cancer

Whereas moderately increased cellular oxidative stress is supportive for cancerous growth of cells, excessive levels of reactive oxygen species (ROS) are detrimental to their growth and survival. We demonstrated that high ROS levels, via increased oxidized glutathione (GSSG), induce isoform-specific S-glutathionylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) at residue Cys206, which is located near the entrance to the 6-phosphofructo-2-kinase catalytic pocket. Upon this ROS-dependent, reversible, covalent modification, a marked decrease in its catalytic ability to synthesize fructose-2,6-bisphosphate (Fru-2,6-P₂), the key glycolysis allosteric activator, was observed. This event was coupled to a decrease in glycolytic flux and an increase in glucose metabolic flux into the pentose phosphate pathway. This shift, in turn, caused an increase in reduced glutathione (GSH) and, ultimately, resulted in ROS detoxification inside HeLa cells. The ability of PFKFB3 to control the Fru-2,6-P₂ levels in an ROS-dependent manner allows the PFKFB3-expressing cancer cells to continue energy metabolism with a reduced risk of excessive oxidative stress and, thereby, to support their cell survival and proliferation. This study provides a new insight into the roles of PFKFB3 as switch that senses and controls redox homeostasis in cancer in addition to its role in cancer glycolysis.     

Rap1A Regulates Osteoblastic Differentiation via the ERK and p38 Mediated Signaling

Rap1A is a member of small G proteins belonging to the Ras family. Recently, an integration of human genome-wide association studies (GWAS) and gene expression profiling study revealed that single-nucleotide polymorphisms (SNPs) within human Rap1A were strongly associated with narrow neck width in women. However, the regulatory role of Rap1A in osteoblasts remains to be elucidated. Here we report that Rap1A is a key regulator in osteoblast differentiation. Rap1A expression and activity were gradually enhanced during the induced differentiation of multipotent mesenchymal progenitor cells (C2C12) and preosteoblastic cells (MC3T3-E1). Knockdown of endogenous Rap1A significantly inhibited the osteogenic marker gene expression and matrix mineralization in cells with osteogenesis. In addition, knockdown of endogenous Rap1A suppressed the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), while overexpression of Rap1A accelerated osteoblast differentiation and enhanced the phosphorylation of ERK and p38. Taken together, our study suggests that Rap1A regulates osteoblast differentiation through modulating the ERK/p38 signaling.     

Ivabradine Prevents Low Shear Stress Induced Endothelial Inflammation and Oxidative Stress via mTOR/eNOS Pathway

Ivabradine not only reduces heart rate but has other cardiac and vascular protective effects including anti-inflammation and anti-oxidation. Since endothelial nitric oxide synthase (eNOS) is a crucial enzyme in maintaining endothelial activity, we aimed to investigate the impact of ivabradine in low shear stress (LSS) induced inflammation and endothelial injury and the role of eNOS played in it. Endothelial cells (ECs) were subjected to LSS at 2dyne/cm², with 1 hour of ivabradine (0.04μM) or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10μM) pre-treatment. The mRNA expression of IL-6, VCAM-1 along with eNOS were measured by QPCR. Reactive oxygen species (ROS) was detected by dihydroethidium (DHE) and DCF, and protein phosphorylation was detected by western blot. It demonstrated that ivabradine decreased LSS induced inflammation and oxidative stress in endothelial cells. Western blot showed reduced rictor and Akt-Ser473 as well as increased eNOS-Thr495 phosphorylation. However, mTORC1 pathway was only increased when LSS applied within 30 minutes. These effects were reversed by ivabradine. It would appear that ivabradine diminish ROS generation by provoking mTORC2/Akt phosphorylation and repressing mTORC1 induced eNOS-Thr495 activation. These results together suggest that LSS induced endothelial inflammation and oxidative stress are suppressed by ivabradine via mTORC2/Akt activation and mTORC1/eNOS reduction.     

Mitochondrial DNA Damage via Augmented Oxidative Stress Regulates Endoplasmic Reticulum Stress and Autophagy: Crosstalk,Links and Signaling

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.     

依达拉奉通过Nrf2信号分子调节氧化应激减轻脑缺血再灌注诱导的神经损伤

本研究旨在探讨依达拉奉(edaravone,ED)在脑缺血再灌注损伤中发挥神经元保护作用与Nrf2信号分子间的关系。体内实验利用脑内脑中动脉闭塞(middle cerebral artery occlusion model,MCAO)建立SD大鼠脑缺血再灌注损伤模型,体外实验采用过氧化氢(H2O2)损伤PC12细胞建立氧化应激模型。通过TTC染色、HE染色、Nissl染色来检测大脑的病理状态。测定活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性,来反映氧化应激水平。此外,通过Hoechst 33342染色和线粒体膜电位(mitochondrial membrane potential,MTP)测定,探究细胞水平的损伤。采用免疫组织化学和蛋白质印记测定Nrf2的表达。构建Nrf2敲除的PC12细胞系,证实Nrf2信号分子抑制氧化应激损伤的作用。结果提示,经依达拉奉给药后,在动物体内水平,TTC染色证实,脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的脑组织梗死体积减小(P<0.001),ROS和MDA水平下降(P<0.01),SOD活性上升(P<0.01);在细胞水平,凋亡细胞减少(P<0.05),MTP上升(P<0.01),ROS和MDA水平下降,SOD活性上升(P<0.01);在分子水平,免疫组化和Western印迹结果均提示,Nrf2蛋白质含量较正常组增加。H2O2诱导Nrf2基因敲除的PC12细胞损伤加重,且依达拉奉的治疗效果明显削弱。综上所述,Nrf2在依达拉奉减轻脑缺血再灌注诱导的氧化应激损伤中发挥关键作用。     

IL-6通过MAPK/ERK信号通路调节BMSCs的软骨定向分化

白细胞介素-6（IL-6）是参与骨髓间充质干细胞（BMSCs）软骨定向分化的重要调节因子. MAPK/ERK信号通路可介导骨关节炎软骨损伤. 然而,IL-6调节BMSCs定向分化为软骨细胞的分子机制尚不清楚. IL-6通过激活MAPK/ERK信号途径,抑制BMSCs的成软骨分化. 本文发现,BMSCs在体外向软骨细胞分化时, Il-6基因表达水平显著下调,同时分泌到培养基中的IL-6蛋白水平亦明显降低. 重组IL-6可抑制BMSCs向软骨细胞分化,软骨分化标志蛋白Runx2和Sox9的诱导表达亦相应下调. IL-6可诱导MAPK/ERK信号通路活化,加入ERK特异性阻断剂后,Runx2和Sox9的诱导表达恢复正常.结果提示,IL-6通过激活MAPK/ERK信号通路抑制BMSCs的软骨细胞分化.炎症因子IL-6对软骨细胞的再生具有不利的影响,该研究为软骨组织工程研究和骨关节炎等软骨疾病的治疗提供有价值的参考.     

Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders, including diabetes, hypertension, and heart disease. This study shows that ultraviolet A (UVA) inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells and its action mechanisms. The mRNA levels of peroxidase proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein α (C/EBPα), but not CCAAT/enhancer-binding protein ((C/EBP) β and δ, were reduced by UVA. Moreover, the mRNA levels of PPAR γ target genes (lipoprotein lipase (LPL), CD36, adipocyte protein (aP2), and liver X receptor α (LXR)) were down-regulated by UVA. Additionally, attempts to elucidate a possible mechanism underlying the UVA-mediated effects revealed that UVA induced migration inhibitory factor (MIF) gene expression, and this was mediated through activation of AP-1 (especially JNK and p42/44 MAPK) and nuclear factor-κB. In addition, reduced adipogenesis by UVA was recovered upon the treatment with anti-MIF antibodies. AMP-activated protein kinase phosphorylation and up-regulation of Kruppel-like factor 2 (KLF2) were induced by UVA. Taken together, these findings suggest that the inhibition of adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells by UVA occurs primarily through the reduced expression of PPAR γ, which is mediated by up-regulation of KLF2 via the activation of MIF-AMP-activated protein kinase signaling.