Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.     

Proliferation of Submesothelial Mesenchymal Cells during Early Phase of Serosal Thickening in the Rabbit Bladder Is Accompanied by Transient Keratin 18 Expression

Partial outlet obstruction of the rabbit bladder induces serosal thickening and smooth muscle (SM) hypertrophy. Within thickened serosa, submesothelial (mesenchymal) cells differentiate into SM cells after 30 days of obstruction [S. Buoroet al. Lab. Invest.69, 589–602, 1993]. Here, we show that submesothelial cells transiently express keratin (K) 18 but not K8 soon after obstruction. We investigated a possible relationship between keratin expression and cell proliferation/differentiationin vivoandin vitro.The results of this study indicate that expression of K18 is spatiotemporally related to the pattern of cell proliferation with respect to the localization of an elastic membrane which divides the thickened serosa into an “extrinsic” and an “intrinsic” region. Moreover, K18 is not present in bladder mesenchyma during early development, indicating that its expression in the adult is not attributable to a dedifferentiation process. However, simultaneous K18, K8, and desmoplakin (DP) expression can be induced in normal and thickened serosa upon treatment with bromo-deoxyuridine. Our results indicate that K18 is a marker of proliferating mesenchymal cells in rabbit serosa, whereas the combined expression of K18, K8, and DP might be related to the hypothesized alterations in the stability of gene expression. A model is proposed in which keratin-containing submesothelial cells can act as a “transit” cell phenotype involved in both regenerating mesothelial cells and formation of SM cells.     

Enhanced Expression of αV Integrin Subunit and Osteopontin during Differentiation of HL-60 Cells along the Monocytic Pathway

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for α_V integrin subunit was observed. The α_Vβ₃ integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.     

Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.     

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

Background

Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.

Methodology/Principal Findings

We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the α_2A-adrenoceptor (α_2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14''304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of α_2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation.

Conclusions/Significance

Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.     

Downregulation of HDAC1 Is Involved in the Cardiomyocyte Differentiation from Mesenchymal Stem Cells in a Myocardial Microenvironment

Under myocardial microenvironment, bone marrow-derived mesenchymal stem cells (MSCs) can transdifferentiate into cardiomyocytes (CMs). However, the role of histone deacetylase 1 (HDAC1) in this directed differentiation process remains unclear. The current study is to determine the acetylation regulatory mechanisms that may be involved in the directed CM differentiation from MSCs. MSCs isolated from male Sprague-Dawley (SD) rats were marked with Ad-EGFP and co-cultured with CMs. Flow cytometry was used to sort EGFP-positive (EGFP+) MSCs from the co-culture system. Then, the expression of cardiac troponin T (cTnT) in these MSCs was detected by immunofluorescence assay. In addition, HDAC1 levels at different co-culture times were measured by quantitative real-time polymerase chain reaction (QT-PCR) and Western blotting. At 4 days after co-culture with CMs, the MSCs began to expression detectable levels of cTnT. The expression of HDAC1 in CMs was much lower than that in MSCs. After co-culture with CMs, the expression of HDAC1 in MSCs was significantly decreased in a time dependent manner. In addition, our recent study has also identified that knockdown of the HDAC1 could promote the directed differentiation of MSCs into CMs. The results suggest that HDAC1 has a negative correlation with cardiac cell differentiation from MSCs under a myocardial microenvironment. HDAC1 might play an important role in the directed differentiation of MSCs into CMs in heart.     

Ca2+/Calmodulin-Dependent Kinase Kinase α Is Expressed by Monocytic Cells and Regulates the Activation Profile

Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca²⁺/calmodulin-dependent kinase kinase α (CaMKKα) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKα by mass spectrometry and Western analysis. The function of CaMKKα in monocyte activation was examined using the CaMKKα inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKα, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKα to the nucleus. Finally, to further examine monocyte activation profiles, TNFα and IL-10 secretion were studied. CaMKKα inhibition attenuated PMA-dependent IL-10 production and enhanced TNFα production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKα in the differentiation of monocytic cells.     

抑制MTM1表达促进胚胎干细胞分化

应用随机RNAi文库,筛选了与胚胎干细胞自我更新和分化调控相关基因,发现了多个阳性候选基因,对其中的1个阳性候选基因肌管素1（myotubularin, MTM1）基因进行了深入研究．MTM1是属于蛋白酪氨酸磷酸酶（PTPase）蛋白家族的蛋白,其基因突变导致肌管性肌病．MTM1在胚胎干细胞中的功能到目前为止还不清楚．研究证实,MTM1在小鼠胚胎干细胞系CCE和R1均有表达．应用RNA干扰及集落形成实验证明,MTM1表达抑制后,处于自我更新状况胚胎干细胞集落的比例显著增加,提示MTM1在胚胎干细胞自我更新和分化的调控中起了重要的作用．