Alterations in the spatiotemporal expression pattern and function of N-Cadherin inhibit cellular condensation and chondrogenesis of limb mesenchymal cells in vitro

2002 ◽  
Vol 87 (3) ◽  
pp. 342-359 ◽  
Author(s):  
Anthony M. DeLise ◽  
Rocky S. Tuan
Keyword(s):  
Expression Pattern ◽  
Mesenchymal Cells ◽  
Spatiotemporal Expression ◽  
Cellular Condensation ◽  
And Function

Zyxin Regulates Cell Migration and Differentiation in EMT during Chicken AV Valve Morphogenesis

2013 ◽  
Vol 19 (4) ◽  
pp. 842-854 ◽  
Author(s):  
Na Li ◽  
Richard L. Goodwin ◽  
Jay D. Potts
Keyword(s):  
Cell Migration ◽  
Collagen Gel ◽  
Mesenchymal Cells ◽  
Valve Development ◽  
Focal Adhesion Protein ◽  
Endocardial Cell ◽  
Mesenchymal Transformation ◽  
And Function ◽  
Valve Formation

AbstractDuring heart valve development, epithelial–mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 177-187 ◽  
Author(s):  
S.A. Oberlender ◽  
R.S. Tuan
Keyword(s):  
Pattern Formation ◽  
Cell Aggregation ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Time Period ◽  
Functional Involvement ◽  
Embryonic Limb ◽  
Cellular Condensation

Cell adhesion molecules have been shown to be important mediators of morphogenesis and pattern formation. In this study, we have shown that N-cadherin is expressed in a specific spatiotemporal manner in the developing limb bud during chondrogenesis in vivo and in cultured limb mesenchyme in vitro. The time period of maximal expression of N-cadherin corresponds to the period of active cellular condensation, an event believed to be a necessary prerequisite for chondrogenic differentiation. To directly assess the functional involvement of N-cadherin in cellular condensation, we have examined the effects of perturbing N-cadherin activity on both cell aggregation and chondrogenesis using NCD-2, a rat monoclonal antibody directed against the binding region of N-cadherin. Non-immune rat IgG was used as a control. Our results show that functional N-cadherin is necessary for chondrogenesis to proceed both in vivo and in vitro. Limb mesenchymal cells exhibited characteristic Ca(2+)-dependent cell aggregation in suspension, which was inhibited in the presence of exogenous NCD-2. In micromass cultures of limb mesenchymal cells, NCD-2 inhibited overt chondrogenesis in a dose-dependent manner. Furthermore, NCD-2 inhibition of chondrogenesis in micromass cultures was time-dependent, suggesting that N-cadherin is crucially involved during the latter half of the first 24 hours of culture, a time period most likely corresponding to active cellular condensation. NCD-2 also significantly influenced limb development when injected into embryonic limb buds in vivo. In addition to significant inhibition of chondrogenesis and developmental delays, gross developmental deformities and perturbation of overall pattern formation were also observed. Taken together, these results demonstrate that N-cadherin is functionally required in mediating the cell-cell interactions among mesenchymal cells important for chondrogenesis in micromass culture in vitro and in the intact limb bud in vivo.

Type II PtdInsP kinases: location, regulation and function

2007 ◽  
Vol 74 ◽  
pp. 149-159 ◽  
Author(s):  
Jonathan H. Clarke ◽  
Jonathan P. Richardson ◽  
Katherine A. Hinchliffe ◽  
Robin F. Irvine
Keyword(s):  
Expression Pattern ◽  
Tissue Expression ◽  
Class I ◽  
Type Ii ◽  
Tissue Expression Pattern ◽  
Functional Nature ◽  
Intracellular Vesicles ◽  
And Function

The regulation of the synthesis of PtdIns(4,5)P2 is emerging as being as complex as we might expect from the multi-functional nature of this lipid. In the present chapter we focus on one aspect of inositide metabolism, which is the functions of the Type II PIPkins (Type II PtdInsP kinases). These are primarily PtdIns5P 4-kinases, although in vitro they will also phosphorylate PtdIns3P to PtdIns(3,4)P2. Thus they have three, not necessarily exclusive, functions: to make PtdIns(4,5)P2 by a quantitatively minor route, to remove PtdIns5P and to make PtdIns(3,4)P2 by a route that does not involve a Class I PtdIns 3-kinase. None of these three possible functions has yet been unambiguously proven or ruled out. Of the three isoforms, α and β are widely expressed, the IIα being predominantly cytosolic and the IIβ primarily nuclear. PIPkin IIγ has a much more restricted tissue expression pattern, and appears to be localized primarily to intracellular vesicles. Here we introduce in turn each of the three Type II PIPkins, and discuss what we know about their localization, their regulation and their function.

Human Mesenchymal Cells Control T Regulatory Phenotypes and Functions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2308-2308
Author(s):  
Mauro Di Ianni ◽  
Beatrice Del Papa ◽  
Lorenzo Moretti ◽  
Maria De Ioanni ◽  
Elisabetta Bonifacio ◽  
...  
Keyword(s):  
T Cell ◽  
Suppressor Cells ◽  
In Vitro Study ◽  
Foxp3 Expression ◽  
Mesenchymal Cells ◽  
Regulatory Function ◽  
Cell Populations ◽  
And Function ◽  
Cd127 Expression

Abstract Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Since mesenchymal cells (MSCs) exert an immune regulatory function and suppress T cell proliferation, this in vitro study investigated their role in Treg recruitment and function. hMSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were co-cultured for up to 15 days. Harvested lymphocytes were analysed by flow-cytometry; FoxP3 and CD127 expression were measured by real time PCR; regulatory activity was assessed. Within CD3+ fraction, percentages of CD4+/CD25bright and CD4+/CD25bright/Foxp3+ cells were higher in the presence of hMSCs (42% vs 34% for the CD4+/CD25bright and 20.5 vs 3.5 for the CD4+/CD25bright/Foxp3+). Within CD3+/CD45RA+ and CD3+/CD45RO+ fractions, the T reg starting fraction of the naïve population rose from 0.05% ± 0.01 CD4/CD25 positive cells to 0.2% ± 0.14 and the T reg starting fraction of the memory population rose from 0.3% ± 0.05 CD4/CD25 positive cells to 1.5% ± 0.9. Within CD4+/CD25+, CD4+/CD25+/CD45RA+, CD4+/CD25+/CD45RO+, FoxP3 expression did not change in CD4+/CD25+ cells. It increased 5.38±2.3 fold in CD4+/CD25+/CD45RA+ and 7.98±1.9 fold in CD4+/CD25+/CD45RO+ cells. CD127 expression decreased by -582±29.2 fold in CD4+/CD25+ cells, by -216±17.5 fold in CD4+/CD25+/CD45RA+ and by -71.95±12.6 fold in CD4+/CD25+/CD45RO+ cells. Cytofluorimetric analysis showed CD4+/CD25+/FoxP3+ was up-regulated to 14%±4 and CD127 down-regulated to 4.4%±1.5 vs respectively 0.2%±0.28 and 21.9%±3.5 in controls without MSCs (CD4+/CD25+ alone). FoxP3 up-regulation was more marked in CD4+/CD25bright cells (51%±10 vs 0.1%±0.7 in controls). Sorted Tregs exert potent immunosuppressive activity on CD4+/CD25- cell populations. Inhibitory activity was lost within 15 days when sorted T regs were cultured without hMSC. On day +15 proliferation using CD4+/CD25+ cells as suppressor cells was 1.7±0.8 vs 45.2±12 in controls; proliferation using CD4/CD25+/CD45RA+ as suppressor cells was 5.4±3.1, vs 21.3±11.2 in controls, and proliferation with CD4+/CD25+/RO+ as suppressor cells was 2.3±2 vs 33.5±8.7 in controls. We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was down regulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+ and CD4/CD25+/CD45RO+) were used as fraction for hMSC co-cultures, they maintain FoxP3 expression and CD127 expression is down-regulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. In conclusion, our study demonstrates that MSCs recruit, regulate and maintain T regulatory phenotype and function over time.

CO-Culture with Mesenchymal Cells Modulates TGF-Beta/Smad And Mapk Pathways in T Regulatory Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 676-676 ◽  
Author(s):  
Mauro Di Ianni ◽  
Beatrice Del Papa ◽  
Lorenzo Moretti ◽  
Simonetta Piattoni ◽  
Debora Cecchini ◽  
...  
Keyword(s):  
T Regulatory Cells ◽  
Central Element ◽  
Mesenchymal Cells ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Cells ◽  
Tgf Beta ◽  
Mapk Pathways ◽  
And Function

Abstract We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+ T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+bright constituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+ cells inhibited CD4+/CD25− cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25− control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

scholarly journals The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

2021 ◽  
Vol 15 ◽  
Author(s):  
Yin Chen ◽  
Ruiying Qiang ◽  
Yuan Zhang ◽  
Wei Cao ◽  
Leilei Wu ◽  
...  
Keyword(s):  
Inner Ear ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Elongation Complex ◽  
Cochlear Hair Cells ◽  
Super Elongation Complex ◽  
Proliferation And Differentiation ◽  
And Function

The super elongation complex (SEC) has been reported to play a key role in the proliferation and differentiation of mouse embryonic stem cells. However, the expression pattern and function of the SEC in the inner ear has not been investigated. Here, we studied the inner ear expression pattern of three key SEC components, AFF1, AFF4, and ELL3, and found that these three proteins are all expressed in both cochlear hair cells (HCs)and supporting cells (SCs). We also cultured Lgr5+ inner ear progenitors in vitro for sphere-forming assays and differentiation assays in the presence of the SEC inhibitor flavopiridol. We found that flavopiridol treatment decreased the proliferation ability of Lgr5+ progenitors, while the differentiation ability of Lgr5+ progenitors was not affected. Our results suggest that the SEC might play important roles in regulating inner ear progenitors and thus regulating HC regeneration. Therefore, it will be very meaningful to further investigate the detailed roles of the SEC signaling pathway in the inner ear in vivo in order to develop effective treatments for sensorineural hearing loss.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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