Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin α_IIbβ₃, raising the possibility that it participates in integrin α_IIbβ₃ outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin α_IIbβ₃-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P₂ accumulation. Although agonist-induced integrin α_IIbβ₃ affinity regulation was unaltered in Dok-2^−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin α_IIbβ₃ adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.     

Multi-Lineage Differentiation of Human Umbilical Cord Wharton’s Jelly Mesenchymal Stromal Cells Mediates Changes in the Expression Profile of Stemness Markers

Wharton’s Jelly- derived Mesenchymal stem cells (WJ-MSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal, differentiation and unique immunomodulatory properties. Although many studies have characterized various WJ-MSCs biologically, the expression profiles of the commonly used stemness markers have not yet been addressed. In this study, WJ-MSCs were isolated and characterized for stemness and surface markers expression. Flow cytometry, immunofluorescence and qRT-PCR analysis revealed predominant expression of CD29, CD44, CD73, CD90, CD105 and CD166 in WJ-MSCs, while the hematopoietic and endothelial markers were absent. Differential expression of CD 29, CD90, CD105 and CD166 following adipogenic, osteogenic and chondrogenic induction was observed. Furthermore, our results demonstrated a reduction in CD44 and CD73 expressions in response to the tri-lineage differentiation induction, suggesting that they can be used as reliable stemness markers, since their expression was associated with undifferentiated WJ-MSCs only.     

HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells

BackgroundDuring short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis.ObjectivesWe investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling.ConclusionProlonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.     

Protein Kinase Cα Isoform Regulates the Activation of the MAP Kinase ERK1/2 in Human Glioma Cells: Involvement in Cell Survival and Gene Expression

Protein kinase C is a family of serine/threonine protein kinases involved in many cellular responses, including cell survival and apoptosis. We have recently found that specific inhibition of the PKCα isoform by nucleic acid enzymes induced apoptosis in sensitive cells. Here we show that in PKCα DNA enzyme-treated glioma cells the activation of MAP kinases ERK1/2 is inhibited, whereas their total level was not significantly affected by the treatment. Similar results were obtained when the overall activity of the PKC was inhibited by calphostin, a specific inhibitor for PKC. These results would indicate that the ERK1/2 signaling pathway plays an important role in glioma cell survival and that the PKCα isoform is the main modulator of this pathway. Furthermore, we show that the ERK1/2 signaling pathway is required for the constitutive expression of the basic fibroblast growth factor, a potent mitogen for glioma cell growth.     

Hypoxia-inducible Factor-2α-dependent Hypoxic Induction of Wnt10b Expression in Adipogenic Cells

Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O₂ concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.     

17β-Estradiol Downregulated the Expression of TASK-1 Channels in Mouse Neuroblastoma N2A Cells

TASK channels, an acid-sensitive subgroup of two pore domain K⁺ (K2P) channels family, were widely expressed in a variety of neural tissues, and exhibited potent functions such as the regulation of membrane potential. The steroid hormone estrogen was able to interact with K⁺ channels, including voltage-gated K⁺ (Kv) and large conductance Ca²⁺-activated (BK) K⁺ channels, in different types of cells like cardiac myocytes and neurons. However, it is unclear about the effects of estrogen on TASK channels. In the present study, the expressions of two members of acid-sensitive TASK channels, TASK-1 and TASK-2, were detected in mouse neuroblastoma N2A cells by RT-PCR. Extracellular acidification (pH 6.4) weakly but statistically significantly inhibited the outward background current by 22.9 % at a holding potential of 0 mV, which inactive voltage-gated K⁺ currents, suggesting that there existed the functional TASK channels in the membrane of N2A cells. Although these currents were not altered by the acute application of 100 nM 17β-estradiol, incubation with 10 nM 17β-estradiol for 48 h reduced the mRNA level of TASK-1 channels by 40.4 % without any effect on TASK-2 channels. The proliferation rates of N2A cells were also increased by treatment with 10 nM 17β-estradiol for 48 h. These data implied that N2A cells expressed functional TASK channels and chronic exposure to 17β-estradiol downregulated the expression of TASK-1 channels and improved cell proliferation. The effect of 17β-estradiol on TASK-1 channels might be an alternative mechanism for the neuroprotective action of 17β-estradiol.     

The Effect of TGF-β1 on Expression of Chemokine and Its Receptor in Human Decidual Stromal Cells

为探讨转化生长因子β1(TGF-β1)在蜕膜基质细胞中发挥免疫调节作用的机制,本研究以人妊娠初期的蜕膜基质细胞为研究对象,经0 ng/ml、1 ng/ml、5 ng/ml和10 ng/ml的TGF-β1处理后,运用RT-PCR方法检测趋化因子mRNA的表达,Western-blot检测趋化因子蛋白质的表达.结果表明:在mRNA水平和蛋白水平,高浓度的TGF-β1能够显著的下调蜕膜基质细胞中趋化因子配体CX3CL1、CXCL12和CXCL16的表达,有意义的上调趋化因子受体CXCR4和CXCR6的表达.研究结果提示,TGF-β1对趋化因子配体/受体有显著的调节作用,并通过趋化因子参与母胎界面的免疫调节.