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.

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.

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.

scholarly journals S100C/A11 is a key mediator of Ca2+-induced growth inhibition of human epidermal keratinocytes

2003 ◽  
Vol 163 (4) ◽  
pp. 825-835 ◽  
Author(s):  
Masakiyo Sakaguchi ◽  
Masahiro Miyazaki ◽  
Mikiro Takaishi ◽  
Yoshihiko Sakaguchi ◽  
Eiichi Makino ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Growth Regulation ◽  
Nuclear Translocation ◽  
Basal Layer ◽  
Human Keratinocytes ◽  
Negative Regulator ◽  
Epidermal Keratinocytes ◽  
Proliferating Cells ◽  
Human Epidermal Keratinocytes

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

Immunocytochemical mapping of the biosynthetic pathways of two major proteins expressed in terminally differentiated epidermal keratinocytes

1990 ◽  
Vol 48 (3) ◽  
pp. 940-941
Author(s):  
M.E. Bisher ◽  
D.R. Roop ◽  
P.M. Steinert ◽  
A.C. Steven
Keyword(s):  
Large Scale ◽  
Cell Envelope ◽  
Picric Acid ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Epidermal Keratinocytes ◽  
Biosynthetic Pathways ◽  
Newborn Mice ◽  
Cornified Cell Envelope ◽  
Lowicryl K4m

In the process of constant renewal of mammalian epidermis, cells divide and move upwards from the basal layer as they embark on a program of terminal differentiation. Cells located higher in the epidermis - the spinous, granular, and cornified layers - represent progressively later steps in this pathway. Commitment to terminal differentiation is accompanied by major changes in gene expression and morphology. Inter alia, large-scale synthesis of several proteins is initiated: these include filaggrin, a protein thought to be responsible for aggregating keratin filaments, and a recently discovered protein called loricrin, which has been implicated as a likely major component of the covalently cross-linked protein lining of the cornified cell envelope. We have used immunoelectron microscopy to investigate the biosynthetic pathways of filaggrin and loricrin.Immediately after sacrifice, skin samples were taken from newborn mice, and diced into blocks of 1-2 mm3. They were then either (i) fixed with 1% glutaraldehyde, 0.2% picric acid, in PBS and embedded in LR White, or (ii) fixed with 1% glutaraldehyde in PBS, and embedded in Lowicryl K4M. Essentially the same conclusions were reached from the experiments with both resins.

CD44 is the major peanut lectin-binding glycoprotein of human epidermal keratinocytes and plays a role in intercellular adhesion

1995 ◽  
Vol 108 (5) ◽  
pp. 1959-1970 ◽  
Author(s):  
D.L. Hudson ◽  
J. Sleeman ◽  
F.M. Watt
Keyword(s):  
Lectin Binding ◽  
Terminal Differentiation ◽  
Variable Region ◽  
Intercellular Adhesion ◽  
Pcr Analysis ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Cd44 Isoforms ◽  
Variant Isoforms

Although binding of peanut agglutinin (PNA) to keratinocytes is often used as a marker of terminal differentiation, the identity of the PNA-binding glycoproteins has been unclear. We now show that an antiserum raised against the glycoproteins recognises isoforms of CD44, the most abundant of which could be labelled with [35S]sulphate, indicating the presence of glycosaminoglycan side chains. RT-PCR analysis showed that keratinocytes expressed at least 5 forms of CD44 containing different numbers of exons from the variable region of the extracellular domain and also expressed the standard ‘haemopoietic’ form of CD44 which lacks the variable exons. Standard and variant isoforms of CD44 were expressed both by proliferating keratinocytes and cells undergoing terminal differentiation, although the level of CD44 mRNAs decreased when keratinocytes were placed in suspension to induce differentiation. The role of CD44 in intercellular adhesion was investigated by plating keratinocytes onto a rat pancreatic carcinoma line transfected with different CD44 isoforms. Keratinocyte adhesion to transfectants expressing variant exons 4–7 was greater than to cells expressing standard CD44 and could be inhibited with hyaluronan or digestion with hyaluronidase. These observations confirm earlier predictions that the PNA-binding glycoproteins of keratinocytes play a role in intercellular adhesion.

scholarly journals Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes.

1991 ◽  
Vol 115 (3) ◽  
pp. 829-841 ◽  
Author(s):  
J C Adams ◽  
F M Watt
Keyword(s):  
Expression Profiles ◽  
Basal Layer ◽  
Cell Types ◽  
Epidermal Keratinocytes ◽  
Integrin Expression ◽  
Adhesive Properties ◽  
Human Epidermal Keratinocytes ◽  
Normal Keratinocytes ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.

scholarly journals Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis.

1984 ◽  
Vol 98 (1) ◽  
pp. 16-21 ◽  
Author(s):  
F M Watt
Keyword(s):  
Calcium Ion ◽  
Molecular Basis ◽  
Culture Medium ◽  
Basal Layer ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Ion Concentration ◽  
Culture Substrate ◽  
Layer I ◽  
Calcium Ion Concentration

How terminally differentiating cells are selectively expelled from the basal layer of epidermis has been a source of interest and speculation for many years. The problem can now be studied in culture, using involucrin synthesis as an early marker of terminal differentiation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the calcium ion concentration of the culture medium, they still begin to synthesize involucrin. Raising the level of calcium ions induces stratification, and cells that are synthesizing involucrin are selectively expelled from the basal layer. I have found that during calcium-induced stratification no new proteins or glycoproteins are synthesized, and the rate of cell division does not change. Movement of involucrin-positive cells out of the basal layer was found to be unaffected by cycloheximide, tunicamycin, or cytosine arabinoside. These results suggest that keratinocytes growing as a monolayer already have the necessary properties to determine their position when stratification is induced. Addition of calcium simply allows formation of desmosomes and other intimate cell contacts required for stratification. The properties of involucrin-positive cells that determine their suprabasal position include a reduced affinity for the culture substrate and preferential adhesion to other cells at the same stage of terminal differentiation. The molecular basis of these adhesive changes is discussed.

Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes

1991 ◽  
Vol 99 (2) ◽  
pp. 397-405
Author(s):  
F.M. Watt ◽  
D.L. Hudson ◽  
A.G. Lamb ◽  
S.R. Bolsover ◽  
R.A. Silver ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Thymidine Incorporation ◽  
Calf Serum ◽  
Terminal Differentiation ◽  
Free Calcium ◽  
Imaging Method ◽  
Standard Medium ◽  
Low Calcium ◽  
Calcium Indicator ◽  
Differentiation Status

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Untangling the Metabolic Reprogramming in Brain Cancer: Discovering Key Molecular Players Using Mass Spectrometry

2019 ◽  
Vol 19 (17) ◽  
pp. 1521-1534 ◽  
Author(s):  
Anatoly Sorokin ◽  
Vsevolod Shurkhay ◽  
Stanislav Pekov ◽  
Evgeny Zhvansky ◽  
Daniil Ivanov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Brain Cancer ◽  
De Novo ◽  
Aerobic Glycolysis ◽  
Lipid Production ◽  
Dietary Fatty Acids ◽  
Metabolic Reprogramming ◽  
Detection Methods ◽  
Malignant Cells ◽  
Proliferating Cells

Cells metabolism alteration is the new hallmark of cancer, as well as an important method for carcinogenesis investigation. It is well known that the malignant cells switch to aerobic glycolysis pathway occurring also in healthy proliferating cells. Recently, it was shown that in malignant cells de novo synthesis of the intracellular fatty acid replaces dietary fatty acids which change the lipid composition of cancer cells noticeably. These alterations in energy metabolism and structural lipid production explain the high proliferation rate of malignant tissues. However, metabolic reprogramming affects not only lipid metabolism but many of the metabolic pathways in the cell. 2-hydroxyglutarate was considered as cancer cell biomarker and its presence is associated with oxidative stress influencing the mitochondria functions. Among the variety of metabolite detection methods, mass spectrometry stands out as the most effective method for simultaneous identification and quantification of the metabolites. As the metabolic reprogramming is tightly connected with epigenetics and signaling modifications, the evaluation of metabolite alterations in cells is a promising approach to investigate the carcinogenesis which is necessary for improving current diagnostic capabilities and therapeutic capabilities. In this paper, we overview recent studies on metabolic alteration and oncometabolites, especially concerning brain cancer and mass spectrometry approaches which are now in use for the investigation of the metabolic pathway.

scholarly journals AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 324
Author(s):  
Matthias Deutsch ◽  
Anne Günther ◽  
Rodrigo Lerchundi ◽  
Christine R. Rose ◽  
Sabine Balfanz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Hippocampal Neurons ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Physiological Role ◽  
High Specificity ◽  
Hcn Channels ◽  
Proliferating Cells ◽  
Neuronal Tissues

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

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