beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion

Development ◽  
1999 ◽  
Vol 126 (10) ◽  
pp. 2285-2298 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Stem Cells ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Full Length ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Proliferative Potential ◽  
Beta Catenin ◽  
Epidermal Stem Cells ◽  
Armadillo Repeats

We found that cultured human keratinocytes with high proliferative potential, the putative epidermal stem cells, expressed a higher level of noncadherin-associated beta-catenin than populations enriched for keratinocytes of lower proliferative potential. To investigate the physiological significance of this, a series of beta-catenin constructs was introduced into keratinocytes via retroviral infection. Full-length beta-catenin and a mutant containing only nine armadillo repeats had little effect on proliferative potential in culture, the full-length protein being rapidly degraded. However, expression of stabilised, N-terminally truncated beta-catenin increased the proportion of putative stem cells to almost 90% of the proliferative population in vitro without inducing malignant transformation, and relieved the differentiation stimulatory effect of overexpressing the E-cadherin cytoplasmic domain. Conversely, beta-catenin lacking armadillo repeats acted as a dominant negative mutant and stimulated exit from the stem cell compartment in culture. The positive and negative effects of the beta-catenin mutants on proliferative potential were independent of effects on cell-cycle kinetics, overt terminal differentiation or intercellular adhesion, and correlated with stimulation or inhibition of transactivation of a TCF/LEF reporter in basal keratinocytes. We conclude that the elevated level of cytoplasmic beta-catenin in those keratinocytes with characteristics of epidermal stem cells contributes to their high proliferative potential.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

scholarly journals FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1581-1589 ◽  
Author(s):  
Marc Pellegrini ◽  
Sue Bath ◽  
Vanessa S. Marsden ◽  
David C. S. Huang ◽  
Donald Metcalf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Caspase 8 ◽  
Liver Stem Cells ◽  
Hemopoietic Progenitor ◽  
Hemopoietic Progenitor Cells ◽  
Negative Mutant

Abstract The role of caspase-8 and its adaptor Fas-associated death domain (FADD) in lymphocyte apoptosis is well defined, but their functions in other hemopoietic lineages are not clear. We were unable to generate transgenic mice expressing dominant inhibitors of FADD or caspase-8 in hemopoietic cells, possibly because their expression may have precluded production of vital hemopoietic cells. When using a retroviral gene delivery system, fetal liver stem cells expressing a dominant-negative mutant of FADD (FADD-DN) were unable to generate myeloid or lymphoid cells upon transplantation into lethally irradiated mice. However, fetal liver stem cells expressing very low levels of the caspase-8 inhibitor cytokine response modifier A (CrmA) could reconstitute the hemopoietic system. This level of CrmA expression provided some protection against Fas ligand (FasL)–induced apoptosis and promoted accumulation of myeloid cells in the bone marrow, but it did not inhibit mitogen-induced proliferation of B or T lymphocytes. Using an in vitro colony formation assay, we found that fetal liver stem cells expressing FADD-DN, CrmA, or a dominant-negative mutant of caspase-8 could not proliferate in response to cytokine stimulation. These data demonstrate that the enzymatic activity of caspase-8 and its adaptor FADD are required for cytokine-induced proliferation of hemopoietic progenitor cells.

scholarly journals BMPs signal alternately through a SMAD or FRAP–STAT pathway to regulate fate choice in CNS stem cells

2003 ◽  
Vol 161 (5) ◽  
pp. 911-921 ◽  
Author(s):  
Prithi Rajan ◽  
David M. Panchision ◽  
Laura F. Newell ◽  
Ronald D.G. McKay
Keyword(s):  
Stem Cells ◽  
Smooth Muscle ◽  
Cell Fate ◽  
Mammalian Target Of Rapamycin ◽  
Neural Crest Cell ◽  
Cell Types ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Glial Differentiation ◽  
Cytoplasmic Signals

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.

Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 901-909 ◽  
Author(s):  
E. Levine ◽  
C.H. Lee ◽  
C. Kintner ◽  
B.M. Gumbiner
Keyword(s):  
Early Embryo ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Truncated Form ◽  
Adhesive Properties ◽  
Negative Mutant ◽  
E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

scholarly journals Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs

2021 ◽  
Vol 11 ◽  
Author(s):  
Wei Guo ◽  
Xingyuan Ma ◽  
Yunhui Fu ◽  
Chang Liu ◽  
Qiuli Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Cells ◽  
A549 Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Anti Cancer ◽  
Apoptotic Activity ◽  
Reversal Index ◽  
Negative Mutant

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Cadherins regulate aggregation of pancreatic beta-cells in vivo

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2895-2902 ◽  
Author(s):  
U. Dahl ◽  
A. Sjodin ◽  
H. Semb
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Endocrine Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Adhesive Properties ◽  
Pancreatic Islets Of Langerhans ◽  
E Cadherin

It is thought that the cadherin protein family of cell adhesion molecules regulates morphogenetic events in multicellular organisms. In this study we have investigated the importance of beta-cell cadherins for cell-cell interactions mediating the organization of endocrine cells into pancreatic islets of Langerhans. To interfere with endogenous cadherin activity in beta-cells during pancreatic development, we overexpressed a dominant negative mutant of mouse E-cadherin, lacking nearly all extracellular amino acids, in pancreatic beta-cells in transgenic mice. Expression of the truncated E-cadherin receptor displaced both E- and N-cadherin from pancreatic beta-cells. As a result, the initial clustering of beta-cells, which normally begins at 13.5-14.5 days postcoitum, was perturbed. Consequently, the clustering of endocrine cells into islets, which normally begins at 17.5-18 days postcoitum, was abrogated. Instead, transgenic beta-cells were found dispersed in the tissue as individual cells, while alpha-cells selectively aggregated into islet-like clusters devoid of beta-cells. Furthermore, expression of truncated E-cadherin in beta-cells resulted in an accumulation of beta-catenin in the cytoplasm. Thus, we have for the first time shown in vivo that cadherins regulate adhesive properties of beta-cells which are essential for the aggregation of endocrine cells into islets.

Mouse Jagged2 is differentially expressed in hematopoietic progenitors and endothelial cells and promotes the survival and proliferation of hematopoietic progenitors by direct cell-to-cell contact

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 950-957 ◽  
Author(s):  
Schickwann Tsai ◽  
Jutta Fero ◽  
Steve Bartelmez
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Full Length ◽  
Differentially Expressed ◽  
Cell Contact ◽  
Proliferative Potential ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Direct Cell

To study the regulation of the early stages of hematopoiesis, cDNA representational difference analysis was used to isolate genes that were differentially expressed in primitive hematopoietic progenitors. The reasoning was that such genes were more likely to provide functions important to hematopoietic stem cells and progenitors. One of the genes identified through this approach encodes mouse Jagged2(mJagged2). Using quantitative reverse transcription–polymerase chain reaction, it was shown that mJagged2 was differentially expressed in c-kit+ hematopoietic progenitors, including those with the phenotypes of Lin− c-kit+Rhlo Holo and Lin−c-kit+ Rhhi Holo, and that they have been shown to be highly enriched for long-term and short-term repopulating hematopoietic stem cells, respectively. Western blot analyses showed that endothelial cells also expressed high levels of Jagged2, but stromal fibroblasts did not. Using a coculture system we found that exogenous, full-length mJagged2 promoted the survival and proliferation of hematopoietic progenitors, including the high-proliferative potential colony-forming cells. Direct cell-to-cell contact was required for this effect. Taken together, these findings indicate that both c-kit+ hematopoietic progenitors and endothelial cells express Jagged2 and that exogenous, full-length Jagged2 promotes the survival and proliferation of hematopoietic progenitors.

scholarly journals Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Elena Enzo ◽  
Alessia Secone Seconetti ◽  
Mattia Forcato ◽  
Elena Tenedini ◽  
Maria Pia Polito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Skin Diseases ◽  
Clinical Success ◽  
Essential Feature ◽  
Telomerase Activity ◽  
Proliferative Potential ◽  
Epidermal Stem Cells ◽  
Junctional Epidermolysis Bullosa ◽  
Differentiated Cells ◽  
Cell Transcriptome

AbstractAutologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

scholarly journals Antioxidants as an Epidermal Stem Cell Activator

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 958
Author(s):  
Soon-Hyo Kwon ◽  
Kyoung-Chan Park
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Proliferative Potential ◽  
Epidermal Stem Cell ◽  
Epidermal Stem Cells ◽  
Keratinocyte Proliferation ◽  
Cell Niche

Antioxidants may modulate the microenvironment of epidermal stem cells by reducing the production of reactive oxygen species or by regulating the expression of extracellular matrix protein. The extracellular membrane is an important component of the stem cell niche, and microRNAs regulate extracellular membrane-mediated basal keratinocyte proliferation. In this narrative review, we will discuss several antioxidants such as ascorbic acid, plant extracts, peptides and hyaluronic acid, and their effect on the epidermal stem cell niche and the proliferative potential of interfollicular epidermal stem cells in 3D skin equivalent models.

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