Overexpression of Dominant-Negative Mutant Hepatocyte Nuclear Factor-1  in Pancreatic  -Cells Causes Abnormal Islet Architecture With Decreased Expression of E-Cadherin, Reduced  -cell Proliferation, and Diabetes

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

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

Journal of Cell Science ◽

10.1242/jcs.109.13.3013 ◽

1996 ◽

Vol 109 (13) ◽

pp. 3013-3023 ◽

Cited By ~ 17

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.

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Mutation P291fsinsC in the Transcription Factor Hepatocyte Nuclear Factor-1 is Dominant Negative

Diabetes ◽

10.2337/diab.47.8.1231 ◽

1998 ◽

Vol 47 (8) ◽

pp. 1231-1235 ◽

Cited By ~ 14

Author(s):

K. Yamagata ◽

Q. Yang ◽

K. Yamamoto ◽

H. Iwahashi ◽

J.-i. Miyagawa ◽

...

Keyword(s):

Transcription Factor ◽

Dominant Negative ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Nuclear Factor 1

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Immediate and Delayed Effects of E-Cadherin Inhibition on Gene Regulation and Cell Motility in Human Epidermoid Carcinoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.25.20.9138-9150.2005 ◽

2005 ◽

Vol 25 (20) ◽

pp. 9138-9150 ◽

Cited By ~ 39

Author(s):

Henriette Andersen ◽

Jakob Mejlvang ◽

Shaukat Mahmood ◽

Irina Gromova ◽

Pavel Gromov ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Motility ◽

Family Members ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Short Term ◽

Mesenchymal Transition ◽

Negative Mutant ◽

E Cadherin ◽

Cell Cell

ABSTRACT The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelial-mesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.

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Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β

AJP Renal Physiology ◽

10.1152/ajprenal.00582.2012 ◽

2013 ◽

Vol 305 (1) ◽

pp. F100-F110 ◽

Cited By ~ 6

Author(s):

Yun-Hee Choi ◽

Brian T. McNally ◽

Peter Igarashi

Keyword(s):

Transcription Factor ◽

Epithelial Cells ◽

Nuclear Translocation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Epithelial Tissue ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Lim Domain ◽

Renal Epithelial Cells

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

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