Differentiation Of Normal Keratinocytes Research Articles

Calcium regulation of growth and differentiation of normal human keratinocytes: Modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.

Regulation of growth and differentiation of normal and psoriatic epidermal keratinocytes

Regulation of growth and differentiation of normal and psoriatic epidermal keratinocytes

Altered levels of endogenous retrovirus‐like sequence (VL30) RNA during mouse epidermal cell carcinogenesis

Gene expression during mouse keratinocyte carcinogenesis was examined in a clonal cell model. Tumor cells from three separate initiated cell lineages were compared with their nontumorigenic precursors and with the progenitor cell strain prior to treatment with 7,12-dimethylbenz[a]anthracene (DMBA). The steady-state levels of VL30 RNA in normal and papilloma cells were regulated by extracellular Ca2+ (which controls proliferation and differentiation in normal epidermal keratinocytes) and culture density. In contrast, steady-state levels of VL30 RNA were not regulated by these factors in the squamous cell carcinoma or the anaplastic carcinoma cells. VL30 expression was Ca2+ dependent in the initiated cell precursors within each tumor cell lineage, suggesting that the loss of response to extracellular Ca2+ was associated with the malignant conversion stage of carcinogenesis. No differences between normal and tumor cells were found in the cellular RNA levels of five additional proto-oncogenes. The mouse epidermal cell model should provide a means for direct assessment of a potential functional role of VL30 sequences in cancer development.

Increased Cholesterol Sulfate and Cholesterol Sulfotransferase Activity in Relation to the Multi-step Process of Differentiation in Human Epidermal Keratinocytes

In this study the synthesis of cholesterol sulfate is examined in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK) in culture. During the exponential growth phase, NHEK cells exhibit a relatively high colony-forming efficiency and appear undifferentiated on the basis of their morphology and expression of biochemical characteristics. At confluence, the cells undergo terminal differentiation that is characterized by the commitment to terminal cell division (reduction in colony-forming ability) and expression of the differentiated phenotype. An accumulation of cholesterol sulfate accompanies this program of differentiation. This accumulation of cholesterol sulfate parallels the increase in transglutaminase type I activity and the competence to form cross-linked envelopes, whereas it precedes the "spontaneous" formation of cross-linked envelopes. Increased cholesterol sulfotransferase activity appears to account for the increase in cholesterol sulfate. The cholesterol sulfate accumulation, as well as the increase in cholesterol sulfotransferase and transglutaminase activity, are inhibited by retinoids. However, the presence of retinoids does not prevent NHEK cells from undergoing terminal cell division at confluence. Two NHEK cell lines expressing SV40-large T antigen also undergo terminal differentiation at confluence and start to accumulate cholesterol sulfate. Two other, differentiation-defective cell lines do not exhibit an increase in cholesterol sulfate at confluence. These results show that epidermal keratinocytes in culture, like cells in the epidermis, accumulate cholesterol sulfate when undergoing squamous differentiation. This program appears to consist of a retinoid-insensitive step (commitment to terminal cell division) and a retinoid-sensitive step (expression of the squamous differentiated phenotype).

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin.

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum‐free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.

Turnover of Inositol Phospholipids in Cultured Murine Keratinocytes: Possible Involvement of Inositol Triphosphate in Cellular Differentiation

The relationship between the turnover of inositol phospholipids (PtdIns) and the growth and differentiation of normal murine keratinocytes in culture was studied. Addition of myo-[U-14C]inositol to freshly plated cells resulted in a linear incorporation of radiolabel into the inositol phospholipids of the proliferating basal cells in culture during the initial 36 h, after which time the rate of radiolabel incorporation into the cells declined. The decrease in the incorporation of the radiolabel into the PtdIns, particularly the more highly phosphorylated PtdIns-4P and PtdIns4,5P2, correlated with the marked hydrolysis of these polyphosphoinositides and the rapid hydrolytic release of the inositol phosphates (InsP2 and InsP3). The transient accumulation of the InsPs correlated with the onset of differentiation of these cells. To ascertain whether the above observations of keratinocytes that were undergoing normal proliferative and differentiating phases in culture are consistent with the more synchronized populations of proliferative and differentiating cells, we investigated the turnover of PtdIns in a Ca2+-regulated system of homogenous populations of proliferating mouse keratinocytes in 0.09 mM Ca2+, and a differentiating population in 1.8 mM Ca2+. Our data from system revealed rapid hydrolysis of the PtdIns in the prelabeled low-Ca2+ proliferating cells immediately after a switch from the low to normal extracellular Ca2+ medium. Associated with this hydrolysis was the rapid and transient accumulation of the InsPs (maximum of 60 sec). The hydrolysis of the PtdIns and the accumulation of the InsP3 were not observed when the prelabeled proliferating cells were switched from a low to a low extracellular Ca2+ medium. These results suggest that the rapid hydrolysis of the PtdIns, particularly PtdIns4,5P2, which was accompanied by the hydrolytic release of InsP3, could be the initiating signal to program proliferating keratinocytes into differentiation.

Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.

Azelaic acid vs. placebo: effects on normal human keratinocytes and melanocytes. Electron microscopic evaluation after long-term application in vivo

The effects of topically applied 20% azelaic acid (AA) on normal human epidermis were investigated vs. placebo in a double blind study by electron microscopy in 15 volunteers. After 3 months of local application twice daily, the pattern of epidermal keratinization was found altered in skin treated with AA. In particular, the number and thickness of tonofilament bundles and the number of keratohyaline granules seemed decreased; the remaining granules were smaller, occasionally showing irregular electron densities. The perinuclear endoplasmic reticulum and the cytoplasmic cisternae were enlarged and swollen mitochondria were regularly observed in most malpighian keratinocytes. Thorough quantitative evaluation of the number and distribution of melanocytes by a MOP-videoplan computer system showed no differences between verum and placebo sites, although, the mean number of melanocytes had increased in both, as compared to the untreated controls taken before onset of therapy. No significant qualitative changes of the normal melanocytes were found. These findings indicate that azelaic acid may influence the differentiation of normal human keratinocytes by reducing the synthesis of keratin precursors and may, therefore, act as a mild antikeratinizing agent, whereas, the pigmentary system in normal human epidermis does not show any specific change after 3 months of treatment with AA.

Phospholipase C mimics the differential effects of phorbol-12-myristate-13-acetate on the colony formation and cornification of cultured normal and transformed human keratinocytes

Phorbol ester tumour promoters like phorbol-12-myristate-13-acetate (PMA) and serum-derived factors inhibit growth and induce terminal differentiation in normal human and mouse keratinocytes but have a much reduced effect on their transformed counterparts. These observations may be relevant to potency of PMA and wounding as tumour promoters in mouse epidermis. Since some serum factors produced during wounding are thought to exert their effects through the production of diacylglycerol (DAG-the proposed physiological ligand for the phorbol ester receptor) from phospholipids by the activation of phospholipase C (PC) we have compared the effects of PC with PMA in cultures of normal and transformed human keratinocytes. The addition of PC from Clostridium perfringens (0.1-3.0 units/ml) to the culture medium of normal human keratinocytes produced similar morphological changes to PMA and also mimicked the effects of the phorbol ester on cloning efficiency and cornified envelope formation. Most importantly PC, like PMA, had a very weak effect on the human squamous cell carcinoma lines SCC-12B and SCC-15. All the effects of PC were abolished by boiling the enzyme. These results are discussed in relation to the proposed role of serum factors in tumour promotion by deep skin wounding and their mechanistic relationship to phorbol esters.

A sensitive method to quantify the terminal differentiation of cultured epidermal cells

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Yl, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [ 35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.

Immunocytochemical detection of involucrin in precancerous and cancerous non-pigmented conjunctival tumors

Involucrin is a precursor of the cross-linked envelope protein of the human stratum corneum. Its appearance in the upper layers of the epidermis reflects normal differentiation of keratinocytes. This study uses an immunoperoxidase technique for localization of involucrin in paraffin sections of normal conjunctiva, conjunctival dysplasia, carcinoma in situ, and invasive carcinoma. Clinicoimmunocytochemical correlations are presented. The results demonstrate that the distribution patterns of involucrin differ in precancerous and cancerous conjunctival lesions: normal limbal conjunctiva shows involucrin only in the three superficial cell layers; the fornix conjunctivae contains no involucrin. All 23 conjunctival dysplasias show an involvement also of deeper layers of the epithelium, sparing the basal layers. Three carcinomas in situ and one invasive squamous cell carcinoma, however, demonstrate an involvement of all layers of the epithelium. The involucrin staining pattern helps in histologic differential diagnosis of epithelial lesions of the conjunctiva.

Calcium-Regulated Differentiation of Normal Human Epidermal Keratinocytes in Chemically Defined Clonal Culture and Serum-Free Serial Culture

An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK). Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures, for frozen storage, and for serial culture. The need for feeder layers, conditioned medium, serum, and specialized culture surfaces has been eliminated entirely. With an optimal level of calcium ion (0.3 mM), colony-forming efficiency is about 30 percent and cellular multiplication rate is 0.96 doublings per day in the defined medium. A high-calcium concentration (1.0 mM) induces stratification and terminal differentiation, which can be quantified by counting cornified envelopes that are resistant to boiling in sodium dodecyl sulfate plus dithiothreitol. Under optimal conditions with wBPE present, cellular senescence occurs after about 40 population doublings. Scanning electron microscopy (SEM) has verified the occurrence of stratification during differentiation in the defined medium with high calcium. High-voltage electron microscopy (HVEM) after detergent extraction of human epidermal keratinocyte (HK) colonies grown in the defined medium with low and high calcium has revealed specific changes in the intermediate filament network and keratohyalin granules corresponding to changes in cellular differentiation. Indirect immunofluorescence studies have verified that the intermediate filament network observed with HVEM is composed of keratin proteins.