Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates α1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.     

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Electronic supplementary material

The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users.     

Presenilin 2 expression in neuronal cells: induction during differentiation of embryonic carcinoma cells

Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.     

Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-β signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-β and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.     

NOV/CCN3 Induces Adhesion of Muscle Skeletal Cells and Cooperates with FGF2 and IGF-1 to Promote Proliferation and Survival

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin β1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.     

Inclusion of a matrix-attached region in a 7SK pol III vector increases the efficiency of shRNA-mediated gene silencing in embryonic carcinoma cells

RNA interference is a widely used tool for analysis of gene function in mammalian cells. Stable knockdown of specific target genes can be maintained in cell lines and live organisms using vector-based delivery of short hairpins (shRNAs) driven by RNA polymerase III promoters. Here we describe a vector incorporating the human 7SK promoter for shRNA-mediated gene silencing in the P19 embryonic carcinoma stem cell line. Our preliminary experiments with the 7SK shRNA expression vector indicated that its activity could be hindered by random genomic integration. In order to counter this inhibitory mechanism, we inserted a matrix-attached region sequence to generate an episomal vector system. We compared the effects of insertion versus exclusion of the MAR sequence on the shRNA-mediated gene-specific silencing of the beta-tubulin III and Cyclophilin A genes. While the MAR sequence is not strongly correlated with the episomal status of the expression vector, our studies indicate that inclusion of the MAR element significantly enhances the stability of shRNA-mediated gene silencing in the P19 stem cells.     

Induction of a high population of neural stem cells with anterior neuroectoderm characters from epiblast-like P19 embryonic carcinoma cells

Abstract The epiblast, derived from the inner cell mass (ICM), represents the final embryonic founder cell population of mouse embryo and can give rise to all germ layer lineages including the neuroectoderm. The generation of neural stem cells from epiblast-like cells is of great value for studying the mechanism of neural determination during gastrulation stages of embryonic development. Mouse embryonic carcinoma (EC) P19 cells are equivalent to the epiblast of early post-implantation blastocysts. In this study, we establish a feasible induction system that allows rapid and efficient derivation of a high percentage (∼95%) of neural stem cells from P19 EC cell in N2B27 serum-free medium. The induced neural stem cells bear anterior neuroectoderm characters, and can be efficiently caudalized by retinoic acid (RA). These neural stem cells have multilineage potential to differentiate into neurons, astrocytes, and oligodendrocytes. Mechanistic analysis indicates that inhibition of the bone morphogenetic protein (BMP) pathway may be the main reason for N2B27-neural induction, and that fibroblast growth factor (FGF) signaling is also involved in this process. This method will provide an in vitro system to dissect the molecular mechanisms involved in neural induction of early mouse embryos.     

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.     

Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro

A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel–Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.     

Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2′, it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2′, Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.     

Epigenetic activation of the 5-hydroxytryptamine (serotonin) receptor 2C in embryonal carcinoma cells is DNA replication-dependent

The epigenetic states of key regulatory genes must be altered to drive cell fate decisions in differentiating cells. This process must be coupled, at least transiently, to the DNA replication machinery. Only a few genes, however, have been shown to require DNA replication for their activation or repression upon induction of differentiation. We have developed a methodology for examining how gene expression is coupled to cell division during the early stages of differentiation of embryonal carcinoma (EC) cells. Using this approach, we find that the expression of the 5-hydroxytryptamine (serotonin) receptor 2C (Htr2c) is strongly increased in the second division after all-trans retinoic acid addition. We propose that the epigenetic activation of Htr2c in EC cells results from a chromatin remodeling process that requires at least two passages through S phase.     

Resistance to cadmium as a function of Caco-2 cell differentiation: role of reactive oxygen species in cadmium- but not zinc-induced adaptation mechanisms

Cadmium (Cd) is a highly toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium is the first biological barrier crossed by Cd and is also an important target tissue. In the present study, the human intestinal Caco-2 cell line was used to evaluate the impact of a low level of exposure on both undifferentiated and differentiated intestinal cells. As revealed by the LC₅₀ values estimated with the 3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, mature Caco-2 cells were more resistant to Cd. However, following a 24-h exposure to non-cytotoxic levels of Cd (10 μM) or zinc (Zn, 100 μM), threefold increases were obtained in the LC₅₀ values of 7-day-old cells, whereas increased resistance in 21-day-old cells was observed exclusively with Zn. Induction of MT-IIa and HSP70 mRNAs was higher in undifferentiated cells and an increase in cellular glutathione (GSH) content was observed exclusively in these cell cultures. However, the results obtained with cycloheximide used for inhibiting protein synthesis and with l-buthionine sulfoximine (BSO), which inhibits GSH synthesis, revealed that protein synthesis is not a prerequisite to the development of resistance. The presence of 100 mM 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, prevented Cd-induced but not Zn-induced resistance, as well as sensitized cells to Cd toxicity. These results show for the first time differences in constitutive and acquired resistance to Cd as a function of enterocytic differentiation status and suggest the involvement of different mechanisms for Cd- and Zn-induced adaptation in the intestinal cells. Redox signals may trigger Cd-induced adaptation mechanisms but pro-oxidant conditions would eliminate proliferative intestinal cells capability to develop resistance. This would be critical for Cd- but not Zn-induced mechanisms of resistance since Cd but not Zn may cause oxidative stress.     

Ca2 + /calmodulin-dependent protein kinase II mediates apoptosis of P19 cells expressing human tau during neural differentiation with retinoic acid treatment

The involvement of tau phosphorylation in apoptosis resembling Alzheimer's disease (AD) was investigated using a cell model of P19 cells stably expressing human tau441 (tau/P19 cells). Apoptotic cell death was observed specifically in tau/P19 cells during neural differentiation with retinoic acid (RA) treatment. A CaM kinase II inhibitor, KN-93, protected tau/P19 cells from apoptosis, although it stimulated the cell death of wild-type P19 cells (wt/P19 cells). W-7 and calmidazolium, calmodulin antagonists, also specifically inhibited the apoptosis of tau/P19 cells. LiCl, an inhibitor of glycogen synthase 3, a tau kinase, was effective in protecting tau/P19 cells from apoptosis, but the protective effect was less than that of CaM kinase II inhibitor and calmodulin antagonists. Tau in the nuclei of tau/P19 cells was phosphorylated at the sites for CaM kinase II detected by an antibody recognizing a phosphorylated form of tau. These results indicated that CaM kinase II was involved in the apoptosis of tau/P19 cells induced by RA treatment.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.