Molecular cloning of human epidermal transglutaminase cDNA from keratinocytes in culture.

We have isolated a cDNA encoding human epidermal transglutaminase, a key enzyme of terminal differentiation of keratinocytes. A cDNA library from cultured human keratinocytes was screened by a PCR-amplified partial cDNA fragment of the enzyme with oligonucleotide primers based on the homology of the transglutaminase family. The cDNA is 2734 bp coding a protein of 817 amino acids. The several regions including the active site cysteine residue are highly conserved among the transglutaminase family. However, the charged N-terminal domain is unique to the epidermal transglutaminse, suggesting that the region is involved in the function of the enzyme in keratinocytes.     

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.     

Protein-free culture of esophageal cancer cell lines]

We have established 13 esophageal cancer cell lines capable of growing in a protein-free environment. The growth of these cells was not affected by conditioned medium, but the growth of NIH3T3 cells and human fibroblasts was stimulated by conditioned medium. On the other hand, conditioned medium inhibited the growth of human endothelial cells. Amplified int-2 oncogene correlated well with the growth of cells in a protein-free environment but the number of EGF receptors and growth effect of EGF did not relate to such growth. Esophageal cancer cells grow automatically, possibly involving mesenchymal cells via the paracrine system. This results in a poor prognosis in patients.     

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture

Summary In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis, and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes. Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration dependence and stereospecificity for these responses suggest the involvement of theAh receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating basal cells, modulating the responsiveness of these cells to growth factors such as EGF. This paper was presented at the Session-In-Depth on In Vitro Applications in Toxicology at the 34th Annual Meeting of the Tissue Culture Association, Orlando, FL, June 12–16, 1983. Rosemarie Osborne was a Chemical Industry Institute of Toxicology Postdoctoral Fellow.     

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

Epithelial–mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin–vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.     

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-β biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.     

Apoptosis and differentiation of epidermal keratinocytes

We compared some processes characteristic for both apoptosis and terminal differentiation of epidermal keratinocytes. It can be proposed that nonapoptotic programmed cell death takes place during differentiation of keratinocytes. Apoptosis and terminal differentiation of keratinocytes appear to be different events but some similar molecular mechanisms are involved in both processes.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 85–89.Original Russian Text Copyright © 2005 by Terskikh, Vasilev.     

Apoptosis and differentiation of epidermal keratinocytes

We compared some processes characteristic for both apoptosis and terminal differentiation of epidermal keratinocytes. It can be proposed that nonapoptotic programmed cell death takes place during differentiation of keratinocytes. Apoptosis and terminal differentiation of keratinocytes appear to be different processes but some similar molecular mechanisms are involved in these processes.     

Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences

W oolley , R.C. & M orris , J.G. 1990. Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences. Journal of Applied Bacteriology 69 , 718–728.
Several strains of Clostridium acetobutylicum , including strains ATCC 824 and DSM 1731, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted, at a low but measurable frequency, to the parental phenotype, indicating thai their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent-producing and non-solventogenic colonies of Cl. ocetobutylicum .     

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.     

Transforming growth factor-beta up-regulates type VII collagen gene expression in normal and transformed epidermal keratinocytes in culture

Transforming growth factor-betas (TGF-betas) have been shown to enhance the expression of extracellular matrix genes, including several collagens. In this study, the effects of TGF-beta 1 and TGF-beta 2 on the expression of the gene for type VII collagen, the major component of anchoring fibrils, in human epidermal cell cultures were examined. Incubation of human epidermal keratinocytes or oral epidermoid carcinoma KB cells with TGF-beta 1 or TGF-beta 2 markedly (up to 6.3-fold) elevated the alpha 1(VII) collagen mRNA levels. This elevation was accompanied by enhanced synthesis of type VII collagen, as demonstrated by indirect immunofluorescence with a monoclonal antibody. The results indicate that TGF-beta 1 and TGF-beta 2 have similar biological activities with respect to enhanced type VII collagen gene expression.     

Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: Implications for toxicity and medical countermeasures

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 µmol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.     

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.     

Proliferation and differnetiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor,retinoids, and hydrocortisone

Summary Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca²⁺ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors. This work was partly supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds), grant IKW 85–71.     

Co-localization of Rac1 and E-cadherin in human epidermal keratinocytes

The Rac1 small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell cell junctions in epidermal keratinocytes is thought to be dependent on activity of Rac1. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Rac1 expression vectors and analyzed distribution of Rac1 following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP Rac1 proteins distribute to sites of cell cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Rac1 away from sites of cell cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP Rac1 results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Rac1 with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Rac1 neither disrupts cell cell adhesion nor prevents assembly of new cadherin-based adhesion structures.     

Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance

Summary. Keratinocytes are the prevalent cell type of the epidermis, a multilayered cornified epithelium which provides the cellular basis of the outermost barrier between the organism and its environment. By this barrier function the epidermis protects the organism against a variety of environmental hazards such as dehydration and mechanical stress. Under normal conditions, keratinocytes of all layers are interconnected by desmosomes and anchored by hemidesmosomes to a specialised type of extracellular matrix, the basement membrane. When the epidermis is injured, a vitally important response is initiated with the aim to restore the protective function of the epithelium. A fast but provisional sealing is achieved by the deposition of the fibrin clot before within 24 h after wounding keratinocytes from the wound margins begin to migrate into the wound bed, where they start to proliferate and to form the new epithelium. The development of new high-resolution assays for the study of cell migration and motility has potentiated major progress in our understanding of keratinocyte migration in vitro and in situ. The data reviewed here point to a sophisticated cooperation between soluble motogenic growth factors, cell–matrix interactions, and cell-to-cell communications as major parts of the machinery regulating keratinocyte migration.Correspondence and reprints: Institut für Zellbiologie, Universität Bonn, Ulrich-Haberland-Strasse 61a. 53121 Bonn. Federal Republic of Germany.