Cultured Mouse Keratinocytes Research Articles

The Proximal Promoter of the Mouse Loricrin Gene Contains a Functional AP-1 Element and Directs Keratinocyte-specific but Not Differentiation-specific Expression

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.

Evidence that Diazoxide Promotes Calcium Influx in Mouse Keratinocyte Cultures by Membrane Hyperpolarization

We have used fura-2/AM to investigate the effect of diazoxide on the cytosolic calcium concentration (Cai) in primary mouse keratinocyte cultures. Treatment of keratinocytes with 100 microM diazoxide induced a transient peak in Cai, followed by a sustained elevation. Depletion of medium calcium by addition of EGTA abolished the diazoxide-induced Cai response, indicating that the agent promoted calcium influx without release of calcium from intracellular stores. The diazoxide-induced rise in Cai was inhibited both by addition of 60 mM KCl to the assay buffer and by preincubation with glibenclamide, a specific K+ channel blocker. In addition, studies with the membrane potential-sensitive fluorescent probe bis-oxonol demonstrated that diazoxide hyperpolarized keratinocyte membranes. These findings suggest that keratinocytes possess K+ channels, and that the previously reported proliferation effects of K+ channel openers such as diazoxide on keratinocytes may result from hyperpolarization-induced elevation of Cai.

Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium.

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca2+ concentrations (Cao) from 0.05 mM to > or = 0.1 mM. The addition of Sr2+ (> or = 2.5 mM) to medium containing 0.05 mM Ca2+ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Cao to 0.12 mM. Sr2+ in 0.05 mM Ca2+ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr2+ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca2+ in the medium, the Sr2+ effects are reduced, and Sr2+ is ineffective when both Ca2+ and serum are deleted from the medium. Sr2+ treatment increases the ratio of fluorescence intensity of the intracellular Ca2+ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca2+ and/or Sr2+. At doses sufficient to induce differentiation, Sr2+ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of 85Sr2+ by primary keratinocytes are similar to those of 45Ca2+. At low concentrations, the initial uptake of both 45Ca2+ and 85Sr2+ reaches a plateau within 1 hr; at higher concentrations, the uptake of both 45Ca2+ and 85Sr2+ increases continuously for 12 hr. In keratinocytes pre-equilibrated with 45Ca2+ in 0.05 mM Ca2+ medium, Sr2+ causes an increase of 45Ca2+ uptake, which is dependent on the presence of serum. These results suggest that Sr2+ utilizes the same signalling pathway as Ca2+ to induce keratinocyte terminal differentiation and that Ca2+ may be required to exert these effects.

The long terminal repeat of VL30 retrotransposons contains sequences that determine retinoic acid-induced transcription in cultured keratinocytes.

The characterization of retinoic acid (RA)-regulated gene transcription in keratinocytes has important implications as to the function of retinoids in epidermal homeostasis and to the central role retinoids play in the pharmaco-therapy of a variety of skin disorders. We show that cultured mouse keratinocytes (Balb/MK) responded to RA with induced expression of VL30 retrotransposons. The induction was rapid, was present at nanomolar concentrations of RA, was independent of new protein synthesis, and occurred both in proliferating and differentiated keratinocytes. The long terminal repeat of a VL30 retrotransposon, expressed in mouse epidermis in vivo, was found to contain two RA-responsive elements (RREs) that independently conferred RA responsiveness on a heterologous promoter in both cultured Balb/MK cells and normal human keratinocytes. Functional characterization and in vitro binding analysis showed that the sequence requirement for binding of retinoid X receptor alpha (RXR alpha) and retinoic acid receptor (RAR, either alpha, beta, or gamma) heterodimers, correlated with the sequence requirement for RA-induced transcription in keratinocytes. The VL30 RREs differed functionally from the RA response element present in the RAR-beta 2 promoter, in that the VL30 RREs were non-responsive in fibroblasts cultured from human skin. The non-responsiveness correlated with a reduced complex formation between a VL30 RRE and endogenously expressed nuclear factors present in skin fibroblasts. The data suggest that a direct repeat of two half-sites, spaced by two base pairs, with the consensus sequence T(A/G)AACTTTT(T/C)ACC(T/C), bound RAR-RXR heterodimers and mediated, at constitutive receptor levels, a primary RA response on gene transcription specifically in keratinocytes.

Identification of control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression.

To define DNA regulatory elements that mediate the response of the keratin 1 (K1) gene to Ca(2+)-induced differentiation, regions spanning the 5'- and 3'-flanking sequences, coding regions, and introns from the human K1 gene were cloned into vectors containing the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into cultured mouse keratinocytes. A 4.3-kilobase (kb) region located 3' to the K1 gene stimulated CAT activity in response to increasing Ca2+ concentrations from 0.05 mM (basal cells) to 1.2 mM (differentiated cells). The 4.3-kb fragment was also active in human epidermal cells but inactive in NIH 3T3 cells and primary mouse fibroblasts. Deletion analysis localized the activity to the terminal 1682 base pairs (bp) of the flanking sequence which retained Ca2+ sensitivity in epidermal cells but was not active in mesenchymal cells. Removal of a 207-base pair element created an enhancer which was active in both epidermal and mesenchymal cells but was still Ca(2+)-inducible. Further deletions identified two elements which functioned synergistically to give maximal Ca(2+)-sensitive activity. Stably transfected epidermal cell lines expressed CAT under the direction of these elements when grafted onto nude mice to reconstitute an intact epidermis. Previously reported keratin regulatory motifs were not contained in the 1682-bp fragment, but an AP-1 site was identified in one of the synergistic subunits.

DNA-mediated gene transfection into primary cultures of adult mouse keratinocytes.

An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32 degrees C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 micrograms plasmid DNA and 10 micrograms pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.

Altered expression of a mouse epidermal cytoskeletal protein is a sensitive marker for proliferation induced by tumor promoters.

Treatment of mouse skin with tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) or 8-methoxypsoralen and ultraviolet light (PUVA) caused a marked decrease in the expression of p48, an acidic epidermal protein. This protein was specifically identified in mouse epidermis and confluent PAM 212 mouse keratinocytes in culture. Extraction of p48 required strong denaturing conditions (greater than 8 M urea) as well as a reducing agent, indicating that this protein was associated with the cytoskeleton. Positive immunoblot staining of p48 with antikeratin monoclonal antibodies following two-dimensional polyacrylamide gel electrophoresis suggested that this protein may be related to the acidic keratins. The sensitivity of this protein to topically applied mitogens and tumor promoters implies that p48 expression is important in normal epidermal cell growth and differentiation.

Analysis of phospholipid metabolism in murine keratinocytes transformed by the v-ras oncogene: relationship of phosphatidylinositol turnover and cytokine stimulation to the transformed phenotype.

Introduction of a v-rasHa oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGF alpha and hyperproliferate in culture medium with 0.05 mM Ca2+. Whereas normal keratinocytes respond to elevated medium Ca2+ by cessation of proliferation and induction of terminal differentiation, v-rasHa keratinocytes are not induced to differentiate by Ca2+. We now demonstrate that v-rasHa keratinocytes have elevated basal levels of phosphatidylinositol, inositol phosphates and diacylglycerols in 0.05 mM Ca2+ medium. Basal turnover of phosphatidylcholine is not altered by the rasHa oncogene. The generation of inositol phosphates is even further stimulated in v-rasHa cells by an increase in extracellular Ca2+ or by exposure to aluminum fluoride. Thus, the v-rasHa gene product does not stimulate the inositol phospholipid pathway maximally and additional phosphatidylinositol is available for turnover in response to inducers of phospholipase C activity. TGF alpha and medium conditioned by v-rasHa keratinocytes, both of which stimulate proliferation of normal cells in 0.05 mM Ca2+, transiently increased phosphatidylinositol turnover in normal keratinocytes but did not inhibit Ca(2+)-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGF alpha caused hyperproliferation, resistance to Ca(2+)-induced differentiation and morphological changes identical to those of v-rasHa keratinocytes. These results provide a link between the biological consequences of v-rasHa gene expression and biochemical changes which are known to alter the keratinocyte phenotype.

Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2.

Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.

Ganglioside GQ1b-induced terminal differentiation in cultured mouse keratinocytes. Phosphoinositide turnover forms the onset signal

Investigations were undertaken to see whether mouse keratinocyte differentiation was elicited by gangliosides. Among the gangliosides tested only GQ1b, a tetrasialoganglioside containing two disialosyl residues, induced keratinocyte differentiation, as indicated by the formation of cornified envelopes, enhancement of transglutaminase activity and suppression of DNA synthesis. Upon stimulation with GQ1b the mass content of Ins(1,4,5)P3 and the intracellular Ca2+ levels were markedly enhanced in a time- and dose-dependent manner, whereas no significant changes were observed with other gangliosides, thereby indicating activation of phospholipase C for the onset of keratinocyte differentiation. Furthermore, only GQ1b promoted the translocation of protein kinase C (PKC) from cytosol to membrane. Inhibition of PKC with H-7 or down-regulation of the enzyme by prolonged pre-treatment with phorbol 12,13-dibutyrate greatly suppressed transglutaminase activity and formation of cornified envelopes induced by GQ1b. These results demonstrate that the tetrasialoganglioside GQ1b generates the initial differentiation signal in mouse keratinocytes through phosphoinositide turnover, and also suggest that PKC activation may act at certain, as yet unidentified, stages of differentiation processes.

Permeability characteristics of cultured mouse keratinocytes compared to hairless mouse skin

Abstract The feasibility of using cultured mouse keratinocytes as an in vitro model for hairless mouse skin for permeability studies was explored in this study. Basal keratinocytes from neonatal mice were cultured onto microporous membranes using an air-liquid interface which has been reported to induce formation of stratum corneum. The permeability characteristics of the cultured keratinocytes were compared to hairless mouse skin using a series of n-alkanols. Because of the difficulty in inducing the keratinocytes to form a complete stratum corneum, the use of cultured keratinocytes as a model for studying solute transport is problematic.

A comparison of peptidase activities and peptide metabolism in cultured mouse keratinocytes and neonatal mouse epidermis.

One of the barriers to transdermal delivery of peptides is the metabolic activity of the epidermis. To define this metabolic activity, aminopeptidase activity and Leu-enkephalin metabolism were measured in the epidermis obtained from neonatal mouse skin and in cultured mouse keratinocytes. Aminopeptidase activity was measured fluorometrically using leucine, tyrosine, lysine, and aspartic acid derivatives of beta-naphthylamine as substrates. Similarities in substrate kinetic values (Km and Vmax) and substrate specificity of the enzyme(s) in homogenates prepared from neonatal mouse skin epidermis and cultured mouse keratinocytes strongly suggest that the keratinocytes in culture express the same aminopeptidase(s) with the same relative activity as in neonatal skin. The Km and Vmax values for aminopeptidase(s) with different substrates in epidermis homogenates are as follows: leucine beta-naphthylamide (11 microM and 38 nmol.min-1.mg-1), tyrosine beta-naphthylamide (21 microM and 18 nmol.min-1.mg-1), and lysine beta-naphthylamide (11 microM and 35 nmol.min-1.mg-1). Aspartic acid beta-naphthylamide and glutamic acid beta-naphthylamide were not hydrolyzed by these homogenates at pH 7.4 (37 degrees C). Leu-enkephalin hydrolysis by the homogenates from cultured mouse keratinocytes and neonatal mouse epidermis gave similar Km (32 and 24 microM). Vmax (9.77 and 7.55 nmol.min-1.mg-1) and Ki (223 and 194 microM) values. In addition, the cellular homogenates gave similar metabolite profiles for Leu-enkephalin.

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 Does Not Stimulate DNA Synthesis of Cultured Human Keratinocytes Growth-Arrested in Growth-Factor-Depleted Medium

Interleukin-1, a multifunctional cytokine, plays a central role in inflammatory processes. Several reports have appeared demonstrating that IL-1 stimulates growth of keratinocytes under certain experimental conditions, and we have shown previously that it can act as a strong stimulator of DNA synthesis in murine keratinocytes that have been growth-arrested by removal of growth factors (GF) from the medium for several days. Using the same assay system, we report here that, in contrast to cultured mouse keratinocytes, growth-arrested adult and newborn human keratinocytes do not respond to IL-1 with an increase in DNA synthesis. The experiments were performed with primary and secondary cells and cells propagated with and without feeder layer prior to the assay. A growth response to IL-1 in the absence of exogenous GF was only observed in non-growth-arrested human keratinocytes, i.e., when the cytokine was added for 24 to 48 h immediately after removal of other GF from the culture. Because keratinocytes continue to grow during this time anyway, although at a reduced rate, the additional growth observed with IL-1 could be explained as an enhancement of the growth-promoting effect by factors not previously completely removed from the culture. We would like to conclude from our results that, contrary to the findings in cultured mouse keratinocytes, IL-1 has no direct mitogenic effect on cultured human keratinocytes, but can still act as a growth promoter under certain conditions, apparently in concert with other GF.

Sphingolipid Metabolism in Organotypic Mouse Keratinocyte Cultures

Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine. These results indicate that considerable diversity of ceramide structures occurs among mammalian species and that cultured keratinocytes may only partially reproduce the in vivo complement of ceramides. Using labeled serine in keratinocyte cultures, we have also demonstrated the de novo synthesis of ceramides and the transfer of label from glucosylceramides to ceramides during terminal differentiation of lifted cultures. The covalently bound corneocyte lipid envelope, which has recently been characterized in pig and human epidermis, was also present in mouse epidermis and was reproduced by the lifted cultures. Very-long-chain omega-hydroxyceramides were the dominant bound lipid and labeling studies in culture indicated that they were derived from ceramides synthesized in the viable epidermis.

A novel epidermal cell differentiation inhibitor (EDIN): Purification and characterization from Staphylococcusaureus

A factor inhibiting the calcium-induced terminal differentiation of cultured mouse keratinocytes was purified to homogeneity from the extracellular products of S. aureus E-1 and designated 'epidermal cell differentiation inhibitor' (EDIN). EDIN activity was sensitive to trypsin and heat-labile, suggesting that EDIN is a protein. EDIN gave a single band with a molecular weight of 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was found to be a single chain polypeptide, having an isoelectric point higher than 9. The N-terminal amino acid sequence of EDIN was determined as A-D-V-K-N-F-T-D-L. EDIN inhibited the differentiation of not only mouse but also human keratinocytes in culture.

Ca 2+-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca 2+ (low Ca 2+) incorporated [1− l4C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca 2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1− 14C]AA and reincubated in label-free medium containing 1.2 mM Ca 2+ (normal Ca 2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca 2+ stimulation of phosphoinositide turnover [1]. Cell shift to normal Ca 2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1− 14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca 2+ are discussed.

Tyrosine Phosphorylation Is an Early and Specific Event Involved in Primary Keratinocyte Differentiation

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.

Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.

The v‐ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes

Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v-rasHa gene by viral transduction. Previous studies indicated that v-rasHa-transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with greater than 0.1 mM Ca2+, a culture condition in which normal cells mature into squames. The current studies demonstrate that v-rasHa keratinocytes do not express transcripts or protein for epidermal early differentiation markers keratins 1 and 10 when cells are challenged with 0.12 mM Ca2+, which is a signal for expression of these genes in normal cells. Both transcript and protein for the late differentiation marker loricrin are also diminished in v-ras keratinocytes, but filaggrin, also a late differentiation-related gene product, is expressed in nearly normal amounts but at a different Ca2+ optimum. Modification of intracellular Ca2+ with ionomycin failed to restore the expression of any suprabasal keratinocyte markers. In contrast to the effects on normal products of keratinocyte differentiation, the introduction of the v-rasHa gene facilitated the expression of keratins 8 (K8) and 18 (K18). These keratins are characteristic of embryonic cells and cells of simple adult epithelia but not stratified squamous epithelia such as skin. Like normal differentiation markers, the expression of K8 and K18 was dependent both on the v-ras oncogene and the Ca2+ concentration of the culture medium, with greater than 0.1 mM Ca2+ being optimal. At the optimal Ca2+ level, the majority of v-ras keratinocytes expressed K8 and K18 after 96 h, and many cells had reduced amounts of the normal keratinocyte cytokeratin K14. These studies indicate that the v-ras gene causes substantial reprogramming of epidermal physiology, producing an unusual phenotype devoid of early suprabasal markers but at least partially permissive for late marker expression. Furthermore, the Ca2(+)-dependent expression of K8 and K18 suggests that a normal signalling pathway used in keratinocyte differentiation is diverted to an abnormal endpoint.