Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes

The establishment of cadherin-dependent cell–cell contacts in human epidermal keratinocytes are known to be regulated by the Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell–cell adhesion are not well understood. In this study we utilized green fluorescent protein (GFP)-tagged Rac1 expression vectors to examine the subcellular distribution of Rac1 and its effects on E-cadherin–mediated cell–cell adhesion. Microinjection of keratinocytes with constitutively active Rac1 resulted in cell spreading and disruption of cell–cell contacts. The ability of Rac1 to disrupt cell–cell adhesion was dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell–cell contacts in small preconfluent colonies was achieved through the selective recruitment of E-cadherin–catenin complexes to the perimeter of multiple large intracellular vesicles, which were bounded by GFP-tagged L61Rac1. Similar vesicles were observed in noninjected keratinocytes when cell–cell adhesion was disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes was dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrated that reorganization of the actin cytoskeleton was important for vesicle formation. Characterization of these Rac1-induced vesicles revealed that they were endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibited uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin was a clathrin-independent mechanism. This was supported by the observation that caveolin, but not clathrin, localized around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibited formation of cadherin vesicles. Our data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin.

Download Full-text

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.

Download Full-text

The Role of the Calcium Sensing Receptor in Regulating Intracellular Calcium Handling in Human Epidermal Keratinocytes

Journal of Investigative Dermatology ◽

10.1038/sj.jid.5700633 ◽

2007 ◽

Vol 127 (5) ◽

pp. 1074-1083 ◽

Cited By ~ 58

Author(s):

Chia-Ling Tu ◽

Wenhan Chang ◽

Daniel D. Bikle

Keyword(s):

Intracellular Calcium ◽

Calcium Handling ◽

Epidermal Keratinocytes ◽

Calcium Sensing Receptor ◽

Human Epidermal Keratinocytes ◽

Calcium Sensing

Download Full-text

Calcium-Sensing Receptor Activation Increases Cell-Cell Adhesion and ß-Cell Function

Cellular Physiology and Biochemistry ◽

10.1159/000341439 ◽

2012 ◽

Vol 30 (3) ◽

pp. 575-586 ◽

Cited By ~ 21

Author(s):

Claire E. Hills ◽

Mustafa Y.G. Younis ◽

Jeanette Bennett ◽

Eleftherios Siamantouras ◽

Kuo-Kang Liu ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Function ◽

Receptor Activation ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Cell Cell

Download Full-text

Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

Molecular Biology of the Cell ◽

10.1091/mbc.12.7.1983 ◽

2001 ◽

Vol 12 (7) ◽

pp. 1983-1993 ◽

Cited By ~ 14

Author(s):

Sun-Ho Kee ◽

Peter M. Steinert

Keyword(s):

Cell Adhesion ◽

Cell Lines ◽

Epidermal Keratinocytes ◽

Cell Periphery ◽

Microtubule Disruption ◽

Low Calcium ◽

High Calcium ◽

Long Term Treatment ◽

E Cadherin ◽

Cell Cell

The association of the cytoskeleton with the cadherin–catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin–catenin–actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.

Download Full-text

Inhibition of N-glycosylation by tunicamycin attenuates cell–cell adhesion via impaired desmosome formation in normal human epidermal keratinocytes

Bioscience Reports ◽

10.1042/bsr20171641 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 3

Author(s):

Seon-Pil Jin ◽

Jin Ho Chung

Keyword(s):

Cell Adhesion ◽

Adhesive Strength ◽

Epidermal Keratinocytes ◽

Primary Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Tissue Integrity ◽

Cell Adhesive ◽

Protein Functions ◽

The Stability ◽

Cell Cell

N-Glycosylation affects protein functions such as location, stability, and susceptibility to proteases. Desmosomes in keratinocytes are essential to maintain epidermal tissue integrity to protect against environmental insults. However, it is not yet known whether N-glycosylation affects desmosomal functions in primary keratinocytes. Tunicamycin is an inhibitor of N-glycosylation that has been a useful tool in glycobiology. Therefore, we investigated the effect of inhibiting N-glycosylation by tunicamycin treatment on desmosomes in primary keratinocytes. In our experiments, cell–cell adhesive strength was reduced in tunicamycin-treated primary keratinocytes. TEM showed that desmosome formation was impaired by tunicamycin. Desmogleins (Dsgs) 1 and 3, which constitute the core structure of desmosomes, were well transported to the cell–cell borders, but the amount decreased and showed an aberrant distribution at the cell borders in tunicamycin-treated keratinocytes. The stability of both desmoglein proteins was also reduced, and they were degraded through both proteasomal and lysosomal pathways, although inhibiting degradation did not restore the cell–cell adhesion. Finally, tunicamycin induced desmosomal instability, enhancing their disassembly. In conclusion, these results indicate that N-glycosylation is critical to the desmosome complex to maintain cell–cell adhesive strength in primary keratinocytes.

Download Full-text