scholarly journals Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal keratinocytes

1991 ◽  
Vol 100 (1) ◽  
pp. 153-165 ◽  
Author(s):  
M.D. Kubler ◽  
P.W. Jordan ◽  
C.H. O'Neill ◽  
F.M. Watt
Keyword(s):  
Terminal Differentiation ◽  
Immunoblot Analysis ◽  
Epidermal Keratinocytes ◽  
Basal Cells ◽  
Filamentous Actin ◽  
Human Epidermal Keratinocytes ◽  
Before And After ◽  
Associated Proteins ◽  
Differentiation Marker ◽  
Abundance And Distribution

We have examined the abundance and distribution of actin and several actin-associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for 1 h or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of alpha-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, alpha-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrins that occurs during keratinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrins and the nucleus.

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.

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 Laminin-5 Expression Is Independent of the Injury and the Microenvironment During Reepithelialization of Wounds

1998 ◽  
Vol 46 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Tiina Kainulainen ◽  
Lari Häkkinen ◽  
Sara Hamidi ◽  
Kirsi Larjava ◽  
Matti Kallioinen ◽  
...  
Keyword(s):  
Animal Model ◽  
Basement Membrane ◽  
Leading Edge ◽  
Basement Membrane Zone ◽  
Epidermal Keratinocytes ◽  
Basal Cells ◽  
Human Epidermal Keratinocytes ◽  
Laminin 5 ◽  
Surrounding Matrix ◽  
Cell Layers

We examined the expression of laminin-5 and its integrin receptors during reepithelialization of human wounds. We used suction blisters of skin as a model of keratinocyte migration on a basement membrane matrix and mucosal full-thickness wounds as a model in which keratinocytes migrate in a provisional matrix. An animal model, in which human epidermal keratinocytes were injected into the back of athymic mice, was used to follow the deposition of the basement membrane components. In 4-day-old blisters, about 20–50 cells at the leading edge of the migrating tongue showed cytoplasmic laminin-5 immunostaining. Laminin-5 mRNA was detected in 15–30 cells at the leading edge of the migrating epidermis. α3β1 and α6β4 integrins were found in membrane projections of the migrating basal cells and also in suprabasal cell layers, suggesting their combined role in binding laminin-5. In mucosal wounds, laminin-5 was the only basement membrane zone component that was deposited between the clot and the migrating keratinocytes. In the animal model, linear deposition of laminin-5 and α6β4 integrin was already seen on Day 2, whereas the other basement membrane zone components were not yet organized. The results suggest that, regardless of the injury and the microenvironment, laminin-5 plays an essential role in the interaction between wound keratinocytes and the surrounding matrix.

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 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.

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis

1991 ◽  
Vol 98 (4) ◽  
pp. 577-588 ◽  
Author(s):  
D.D. Vandre ◽  
V.E. Centonze ◽  
J. Peloquin ◽  
R.M. Tombes ◽  
G.G. Borisy
Keyword(s):  
Mitotic Spindle ◽  
Immunoblot Analysis ◽  
Entry And Exit ◽  
Immunofluorescence Analysis ◽  
Temporal And Spatial Distribution ◽  
Microtubule Associated Proteins ◽  
Before And After ◽  
Associated Proteins ◽  
Temporal And Spatial ◽  
Monospecific Antibodies

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 × 10(3) Mr, 350 × 10(3) Mr, a 230–240 X 10(3) Mr doublet, 210 × 10(3) Mr and 120 × 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 × 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 × 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

scholarly journals Identification of Novel Keratinocyte Differentiation Modulating Compounds by High-Throughput Screening

2006 ◽  
Vol 11 (8) ◽  
pp. 977-984 ◽  
Author(s):  
Masaru Honma ◽  
Mark Stubbs ◽  
Ian Collins ◽  
Paul Workman ◽  
Wynne Aherne ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Histone Deacetylase Inhibitors ◽  
Terminal Differentiation ◽  
Mek Inhibitor ◽  
Keratinocyte Differentiation ◽  
Immunofluorescent Staining ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.

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.

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