scholarly journals Regulation of cell surface beta 1 integrin levels during keratinocyte terminal differentiation.

1995 ◽  
Vol 128 (6) ◽  
pp. 1209-1219 ◽  
Author(s):  
N A Hotchin ◽  
A Gandarillas ◽  
F M Watt
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
De Novo ◽  
Terminal Differentiation ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Confocal Immunofluorescence Microscopy ◽  
Affinity Modulation ◽  
Confocal Immunofluorescence ◽  
Beta 1 Integrin

Integrins of the beta 1 family play a central role in controlling adhesion and terminal differentiation within the epidermis. When human epidermal keratinocytes undergo terminal differentiation, intracellular transport of newly synthesized integrins is inhibited, and mature receptors are lost from the cell surface. We have examined the mechanisms underlying these processes, using an experimental model in which keratinocytes are placed in suspension to induce terminal differentiation. The block in intracellular transport was keratinocyte- and integrin-specific since it was not observed when fibroblasts were placed in suspension and did not affect E-cadherin synthesis in suspended keratinocytes. Newly synthesized beta 1 integrins associated with an endoplasmic reticulum resident protein, calnexin; the association was prolonged when keratinocytes were placed in suspension, suggesting a role for calnexin in the inhibition of transport. After 24 h, the level of beta 1 integrin mRNA declines in suspended keratinocytes, reflecting inhibition of gene transcription, but in fibroblasts, the level remained constant. Transport of integrins could be blocked in both adherent keratinocytes and fibroblasts by inhibiting total protein synthesis, raising the possibility that transport is coupled to de novo integrin synthesis. The fate of receptors on the surface of keratinocytes was followed by confocal immunofluorescence microscopy, immunoelectron microscopy, and biochemical analysis: with the onset of terminal differentiation, endocytosed receptors were transported to the lysosomes. These experiments reveal novel mechanisms by which integrin levels can be controlled. Together with our earlier evidence for transcriptional regulation and affinity modulation of integrins, they highlight the complexity of the mechanisms which ensure that the onset of terminal differentiation is linked to detachment of keratinocytes from the underlying basement membrane.

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions

1993 ◽  
Vol 106 (1) ◽  
pp. 175-182 ◽  
Author(s):  
F.M. Watt ◽  
M.D. Kubler ◽  
N.A. Hotchin ◽  
L.J. Nicholson ◽  
J.C. Adams
Keyword(s):  
Extracellular Matrix ◽  
Actin Cytoskeleton ◽  
Intracellular Transport ◽  
Terminal Differentiation ◽  
The State ◽  
Cell Surface Expression ◽  
Epidermal Keratinocytes ◽  
Integrin Subunit ◽  
Human Epidermal Keratinocytes ◽  
Beta 1 Integrin

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Decreased expression of fibronectin and the alpha 5 beta 1 integrin during terminal differentiation of human keratinocytes

1991 ◽  
Vol 98 (2) ◽  
pp. 225-232 ◽  
Author(s):  
L.J. Nicholson ◽  
F.M. Watt
Keyword(s):  
Messenger Rna ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Fibronectin Receptor ◽  
Beta 1 Integrin ◽  
Differentiation Marker ◽  
Unit Gravity Sedimentation

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

Production of scatter factor by ndk, a strain of epithelial cells, and inhibition of scatter factor activity by suramin

1991 ◽  
Vol 98 (3) ◽  
pp. 385-394
Author(s):  
J.C. Adams ◽  
R.A. Furlong ◽  
F.M. Watt
Keyword(s):  
Cell Surface ◽  
Single Cells ◽  
Terminal Differentiation ◽  
Epidermal Keratinocytes ◽  
Factor Activity ◽  
Cell Type ◽  
Activity Assay ◽  
Scatter Factor ◽  
Human Epidermal Keratinocytes ◽  
3T3 Fibroblasts

ndk are a strain of human epidermal keratinocytes that do not undergo terminal differentiation and which grow as single cells rather than compact colonies. We show that ndk are motile and secrete an epithelial scatter factor that has the same biochemical and immunological properties as the scatter factor previously purified from ras-transformed 3T3 fibroblasts. We have found that suramin, a polyanionic detergent, will reverse the activity of scatter factor from either cell type in the standard MDCK activity assay. When added to ndk cultures, suramin causes the cells to grow in coherent patches. This morphological change is accompanied by alterations in the distribution of actin and integrins, but not by stratification or terminal differentiation. The effect is reversed upon removal of suramin. We propose that the motile phenotype of ndk is due, at least in part, to autocrine production of scatter factor and that suramin may be useful for further studies of scatter factor binding to the cell surface.

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 Terminal differentiation of human epidermal keratinocytes involves mitochondria- and caspase-dependent cell death pathway

2003 ◽  
Vol 10 (7) ◽  
pp. 850-852 ◽  
Author(s):  
C Allombert-Blaise ◽  
S Tamiji ◽  
L Mortier ◽  
H Fauvel ◽  
M Tual ◽  
...  
Keyword(s):  
Cell Death ◽  
Terminal Differentiation ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Cell Death Pathway ◽  
Dependent Cell

scholarly journals Biosynthesis, Processing, and Intracellular Transport of GM2Activator Protein in Human Epidermal Keratinocytes

1997 ◽  
Vol 272 (8) ◽  
pp. 5199-5207 ◽  
Author(s):  
Gereon J. Glombitza ◽  
Elisabeth Becker ◽  
Hans Wilhelm Kaiser ◽  
Konrad Sandhoff
Keyword(s):  
Intracellular Transport ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes

scholarly journals Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation

1994 ◽  
Vol 124 (4) ◽  
pp. 589-600 ◽  
Author(s):  
KJ Hodivala ◽  
FM Watt
Keyword(s):  
De Novo ◽  
Lectin Binding ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Ion Concentration ◽  
Epidermal Keratinocytes ◽  
Integrin Expression ◽  
Standard Medium ◽  
Proliferating Cells ◽  
Low Calcium

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.

scholarly journals β1 Integrins Regulate Keratinocyte Adhesion and Differentiation by Distinct Mechanisms

2000 ◽  
Vol 11 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Laurence Levy ◽  
Simon Broad ◽  
Dagmar Diekmann ◽  
Richard D. Evans ◽  
Fiona M. Watt
Keyword(s):  
Point Mutations ◽  
Terminal Differentiation ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Proximal Part ◽  
Epidermal Keratinocytes ◽  
Β1 Integrin ◽  
Primary Human Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Β1 Integrins

In keratinocytes, the β1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick β1 integrin subunits. We examined the ability of adhesion-blocking chick β1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick β1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by β1 integrins in normal keratinocytes was “do not differentiate” (transduced by ligand-occupied receptors) as opposed to “do differentiate” (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the β1 subunit. We conclude that distinct signaling pathways are involved in β1 integrin–mediated adhesion and differentiation control in keratinocytes.

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