Differentiation Of Normal Human Keratinocytes Research Articles

Keratinocyte Growth Inhibition by High-Dose Epidermal Growth Factor Is Mediated by Transforming Growth Factor β Autoinduction: A Negative Feedback Mechanism for Keratinocyte Growth

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

The η isoform of protein kinase C inhibits UV-induced activation of caspase-3 in normal human keratinocytes

Protein kinase C (PKC) fulfills a central role in the decision of cell fate in keratinocytes. Both PKCδ and PKCη induce growth inhibition and differentiation of normal human keratinocytes (NHK). Here we show that PKCδ and PKCη play opposite roles in UVB-induced apoptosis in NHK. PKCδ enhanced UVB-induced caspase-3 activity, while overexpression of PKCη reduced it. In keeping with these observations, the dominant negative mutant of PKCδ significantly inhibited the activation of caspase-3, whereas dominant negative PKCη increased it in a dose (MOI)-dependent manner. Unlike PKCδ, cleavage and translocation to mitochondria of PKCη were not observed, resulting in no detection of cytochorome c release. Furthermore, UV-induced activation of p38 MAP kinase, which suppressed the caspase-3 activity in NHK, was blocked by dominant negative PKCη. These findings suggest that PKCη negatively regulates UV-induced apoptosis through its localization, resistance to cleavage, and the p38 MAPK pathway.

Differential Expression of a Novel Gene in Response to hsp27 and Cell Differentiation in Human Keratinocytes

The 27 kDa heat shock protein (hsp27) is expressed in keratinocytes in a differentiation-related pattern. Keratinocyte differentiation involves a coordinated program of expression and interaction of specific differentiation-related genes and proteins. To investigate the functional role of hsp27 in these processes we used a differential display approach to identify genes that might be regulated by the expression of hsp27 in human keratinocytes. mRNA was extracted from the human squamous carcinoma cell line A431 and a subclone stably transfected with human hsp27. Reverse transcriptase differential display polymerase chain reaction was performed using one base anchored oligo-dT and arbitrary primers. Differentially expressed genes were confirmed by northern blot analysis and further characterized by sequencing. Their expression in human skin and other tissues was investigated by northern blot and in situ hybridization. Out of five fragments detected with the initial reverse transcriptase differential display polymerase chain reaction screen one could be confirmed by northern blot to be downregulated in hsp27-overexpressing A431. This mRNA (G24) is not only downregulated by overexpression of hsp27 in A431 but also during differentiation in normal human keratinocytes in culture and in situ, situations where hsp27 is known to be induced. According to sequence analysis G24 represents a novel gene that does not code for a protein and thus might belong to the growing family of noncoding RNAs. These results not only demonstrate for the first time that overexpression of hsp27 by gene transfer is associated with regulation of gene expression but also reveal a novel differentiation-associated gene in human keratinocytes.

Modulation of Keratinocyte Gene Expression and Differentiation by PPAR-Selective Ligands and Tetradecylthioacetic Acid

Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression was linked to differentiation, and accordingly, expression of PPARalpha and PPARgamma was in essence confined to suprabasal cells. Differentiation was not accompanied by significant changes in the expression of the coactivators CREB-binding protein, p300, steroid receptor coactivator 1, or the corepressors nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order PPARdelta >> PPARalpha > PPARgamma. All selective PPAR ligands marginally induced transglutaminase-1 expression with the PPARdelta-selective ligand L165041 being the most potent. The PPARalpha- and PPARgamma-selective ligands Wy14643 and BRL49653 had negligible effect on involucrin expression, whereas a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease in proliferation and a robust upregulation of the expression of involucrin and transglutaminase. Our results indicate that tetradecylthioacetic acid may affect keratinocyte gene expression and differentiation via PPAR-dependent and PPAR-independent pathways, and that the latter play an important role.

Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D

Lipids that are synthesized de novo in the epidermis, including fatty acids, oxysterols, 1,25-dihydroxyvitamin D3, and farnesol, can regulate the differentiation of normal human keratinocytes (NHK). Cholesterol sulfate (CS), an epidermal lipid that is produced in the upper nucleated layers of the epidermis coincident with terminal differentiation, has been shown to play a role in the regulation of the late stages of keratinocyte differentiation, including formation of the cornified envelope. In the present study, we determined i) whether CS regulates involucrin (INV), an early keratinocyte differentiation marker, and ii) the mechanism by which CS regulates differentiation. mRNA and protein levels of INV, a precursor protein of the cornified envelope, increased 2- to 3-fold in NHK incubated in the presence of CS. In contrast, cholesterol had no effect on INV protein or mRNA levels. Transcriptional regulation was assessed in NHK transfected with INV promoter-luciferase constructs. CS increased luciferase reporter activity approximately 2- to 3-fold in NHK transfected with a 3.7-kb INV promoter construct. Deletional analysis revealed a CS-responsive region of the INV promoter located between bp −2452 and −1880. A 5-base pair (bp) mutation of the AP-1 site (bp −2117 to −2111) within this responsive region abolished CS responsiveness, suggesting a role for the AP-1 complex in the regulation of INV transcription by CS. Electrophoretic mobility shift analysis demonstrated increased binding of nuclear extracts isolated from CS-treated NHK to AP-1 DNA as compared with vehicle-treated controls. Incubation of the nuclear extract with the appropriate antibodies showed that the AP-1 DNA-binding complex contained Fra-1, Fra-2, and Jun D. Western blots demonstrated that CS treatment increased the levels of Fra-1, Fra-2, and Jun D, and Northern analyses revealed that CS increased mRNA levels for these same AP-1 factors. These data indicate that CS, an endogenous lipid synthesized by keratinocytes, regulates the early stages of keratinocyte differentiation, and may do so through its ability to modulate levels of AP-1 proteins. —Hanley, K., L. Wood, D. C. Ng, S. S. He, P. Lau, A. Moser, P. M. Elias, D. D. Bikle, M. L. Williams, and K. R. Feingold. Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D.

β-Endorphin Stimulates Cytokeratin 16 Expression and Downregulates μ-Opiate Receptor Expression in Human Epidermis

It has been reported that opioid peptides modulate the differentiation of normal human keratinocytes and that mu-opiate receptors are expressed in human epidermis. The regulation of keratinocyte differentiation is particularly important in psoriasis, and one of the markers for hyperproliferative and differentiating skin diseases is cytokeratin 16. The finding that the endogenous mu-opiate receptor ligand beta-endorphin is increased in serum of patients with psoriasis indicates that the mu-opiate system may play an important role in the pathophysiology of the skin. In this study, we addressed the question whether there is a link between mu-opiate receptor regulation and cytokeratin 16 expression in normal and psoriatic skin. Firstly, we demonstrate that beta-endorphin concentrations between 16 and 1000 nM significantly downregulate mu-opiate receptor expression in epidermis of cultured human skin after 48 h. Secondly, we show that beta-endorphin regulates cytokeratin 16 expression in the epidermis of skin organ cultures exposed to 41-125 nM beta-endorphin for 48 h, leading to elevated cytokeratin 16 production. As expected, the expression of cytokeratin 16 was detected primarily in the suprabasal layer. The same pattern was observed in psoriatic lesional skin, i.e., mu-opiate receptor expression was significantly downregulated and cytokeratin 16 expression upregulated. These results suggest that the mu-opiate receptor system and its ligand beta-endorphin are involved in the pathogenesis of psoriasis, especially in terms of differentiation.

Effects of Diisopropylamine Dichloroacetate on Proliferation and Differentiation of Normal Human Keratinocytes in vitro

We found that diisopropylamine dichloroacetate (DADA), known as a vasodilator, enhanced growth of keratinocytes in 4 days culture at 1–30 μg/ml, and such promoting effects of cell proliferation were reconfirmed by measuring DNA synthesis using [³H]thymidine incorporation. On the other hand, this substance enhanced synthesis of keratin K1, a potent marker of differentiation in keratinocytes, at 1–100 μg/ml in low calcium (0.1 mM) or high calcium medium (1.25 mM). Moreover, the formation of cornified envelope, another potent marker of differentiation in keratinocytes, was also promoted by DADA at a concentration of 0.1–10 mM which includes valid concentration of DADA for the enhancement of keratin K1 formation (1–100 μg/ml: 0.05–0.5 mM DADA). These results indicate that DADA has a double function, enhancement of both proliferation and differentiation of cells, which could be linked to the turnover of skin epidermis. Furthermore, in order to analyze the effect of DADA on keratinocytes, we examined the effects of each component of this substance, diisopropylamine (DIA) and dichloroacetate (DCA), on keratinocytes. As the result of these investigations, evidence was found that DCA was effective on enhancement of cell growth, but DIA was ineffective. Moreover, we found that DCA was effective on keratinocyte differentiation by evaluating the enhancement of a differentiation marker, formation of cornified envelopes, within 10 mM, while DIA was not effective. Therefore, we concluded that only DCA was an active component of the DADA molecule for the proliferation and the differentiation of keratinocytes in vitro.

Adenovirus-mediated gene transfer to keratinocytes--a review.

The introduction and expression of a foreign gene provide a powerful tool for investigating functions and regulation of a gene of interest; however, keratinocytes have a major drawback in that foreign genes are hardly transfected by conventional methods and stable transformants are most difficult to establish in normal keratinocytes with a limited short life span. To overcome these problems, we used an adenovirus vector, Ax, developed by Saito et al, which yields desired recombinant viruses at an efficiency about 100-fold that of conventional methods, and by which genes are expressed at a high level under the control of a composite CAG promoter. We established Ax vectors carrying various isoforms of protein kinase C (PKC). Using these vectors, we found that the eta and delta isoforms of PKC, but not the alpha and zeta isoforms, mediate terminal differentiation in normal human keratinocytes. These Ax-vectors are also applicable to organ culture of mouse embryos. Advantages and disadvantages of adenovirus vectors and their use for keratinocyte biology are reviewed.

Differentiation of normal human keratinocytes influences hexavalent chromium uptake and distribution and the ability of cells to withstand Cr(VI) cytotoxicity.

The degree of differentiation of normal human keratinocytes determines the biology of the cells to a large extent. We have previously documented that keratinocytes from different donors differ significantly in their ability to withstand hexavalent chromium [Cr(VI)]-induced cytotoxicity. Several factors may contribute to this differing donor sensitivity to Cr(VI). The aims of this study were to investigate to what extent keratinocyte differentiation might influence Cr(VI) uptake and the ability of cells to withstand Cr(VI)-induced cytotoxicity. Keratinocytes from different donors were cultured under identical conditions and exposed to Cr(VI) (as potassium dichromate) at different points during their maturation process. The degree of differentiation of the cells was assessed using a quantitative assay for involucrin and related to the Cr(VI) cytotoxicity experienced by the cells. Chromium content was measured in whole cell, cytosolic and particulate fractions. While proliferative keratinocytes exposed to Cr(VI) showed a high degree of cytotoxicity to dichromate exposure, the more differentiated cells showed significantly less cytotoxicity but a higher uptake of the metal ion into the cells. The relative percentage of cytosolic chromium was high in the proliferative cells and decreased as the cells matured, suggesting that differentiated cultures were binding most of the chromium to the particulate fraction. Total chromium also increased during differentiation. The use of the channel-blocking agent 4, 4'-diisothiocyanate-2-2'-stilbenedisulphonic acid confirmed the spatial differences of chromium accumulation in the phenotypically different cultures, in that it prevented Cr(VI) entry into the proliferative cells and attenuated dichromate cytotoxicity in these cultures, but had no effect on the Cr(VI) uptake in differentiated cells, nor did it reduce its cytotoxicity. These data support the hypothesis that the upper differentiated layers of the epidermis are able to offer considerable physical protection to the lower proliferative layers from chemical pro-oxidants.

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.

Induction of Differentiation in Normal Human Keratinocytes by Adenovirus-Mediated Introduction of the η and δ Isoforms of Protein Kinase C

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-dihydroxyvitamin D3 in normal human keratinocytes.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10(-6) M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10(-9) M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading.

All‐trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25‐Dihydroxyvitamin D3 in normal human keratinocytes

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10−6 M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10−9 M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading. J. Cell. Physiol. 174:1–8, 1998. © 1998 Wiley-Liss, Inc.

Enkephalins modulate differentiation of normal human keratinocytes in vitro.

Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine-enkephalin, leucine-enkephalin and the synthetic DADL) inhibited cell differentiation dose-dependently, while beta-endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine-enkephalin and leucine-enkephalin, but not that of DADL. Furthermore, methionine-enkephalin had a slight inhibitory effect on the proliferation of keratinocytes. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.

Ultraviolet radiation B induces differentiation and protein kinase C in normal human epidermal keratinocytes.

Mid-wave ultraviolet radiation (UVB, 280-320 nm) is highly efficient at inducing erythema, pyrimidine dimers in DNA, oncogene expression and initiation of cutaneous tumors. These UVB-induced responses of epidermal cells have been correlated with the direct effects of UVB on DNA. However, UVB has also been shown to have biologic effects at the cellular level that appear to mimic some of the membrane-associated effects produced by phorbol ester tumor promoters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA). For example, we have previously shown that both UVB irradiation and TPA treatment are followed by release of arachidonic acid and a rapid, dose-dependent inhibition of epidermal growth factor (EGF) binding. TPA generates cellular responses through activation of a phospholipid-dependent, calcium-sensitive protein kinase, protein kinase C (PKC). The primary goal of the studies described here was to compare the cellular effects of TPA with those of UVB with special regard to PKC and keratinocyte growth control, using normal human epidermal keratinocytes. The results obtained showed that both TPA and UVB radiation induced differentiation in normal human keratinocytes. UVB radiation, however, increased both cytosolic and membrane-associated levels of PKC, in contrast to TPA, which increased PKC primarily in the membrane fraction. PKC is probably not the initial chromophore or target molecule of UVB, but because activation of PKC has been shown to be essential for keratinocyte differentiation, differentiation induced by UVB may be caused by activation of PKC by UVB-induced release of diacylglycerol or arachidonic acid.

Effect of a chemically-synthesized acylglucosylceramide, epidermoside, on normal human keratinocyte differentiation

Epidemosides ( N-( O-linoleoyl)-ω-hydroxy fatty acyl sphingosyl glucose) are found exclusively in the epidermis, not in dermis, and are thought to play important role in forming the mammalian epidermal permeability barrier. A species of epidermoside isolated from guinea pig epidermis and named lipokeratinogenoside has been shown to enhance fetal rat keratinocyte differentiation. In the present investigation, we studied the effects of a chemically synthesized equivalent of human epidermoside on the viability and differentiation of cultured human keratinocytes (HK Cells). The chemically-synthesized epidermoside was not toxic to cultured HK Cells at concentrations of 0.01 to 10 μg/ml. When 10 μg/ml of the chemically-synthesized epidermoside was added to keratinocyte growth medium containing 1.2 mM Ca 2 +, HK Cells showed a 5.6-fold increase of keratin content compared to the vehicle treated control at 144 h of cultivation, and they also displayed morphological changes suggestive of differentiation. A similar increase of cellular keratin content was observed in HK cells treated with tetradecanoyl phorbol-13 myristyl-12 acetate (TPA), an agent known to enhance the differentiation of keratinocytes. Lipokeratinogenoside also increased the keratin content of cultured HK cells. These results suggest that epidermosides have an ability to enhance keratinocyte differentiation. Epidermoside could thus be a key molecule, not only as a constituent of the epidermal permeability barrier, but also as a regulator of keratinocyte differentiation.

Squamous Carcinoma Cell Lines Fail to Respond to 1,25-Dihydroxyvitamin D Despite Normal Levels of the Vitamin D Receptor

Squamous carcinoma cells (SCC) fail to differentiate under conditions that are favorable for the growth and differentiation of normal human keratinocytes. Human keratinocytes differentiate from a highly proliferative basal cell to a terminally differentiated cornified cell in culture in the presence of physiological levels of extracellular calcium. 1,25-Dihydroxyvitamin D (1,25[OH]2D3) potentiates this process. Previous studies have shown that the differentiation process in keratinocytes is associated with increased expression of the genes for involucrin and transglutaminase, the products of which participate in cornified envelope formation. The mRNA for involucrin and transglutaminase was not detected in the SCC lines studied (viz. SCC4, 12B2, 12F2, A431, and HACAT) when they were grown in serum free medium. Addition of at least 2% fetal bovine serum for 48 h triggered the expression of these genes, which could then be maintained in the absence of serum. Serum was not required for induction of these genes in keratinocytes. In these cells, 1,25(OH)2D3 stimulated the expression of involucrin and transglutaminase in a concentration-dependent manner, while the SCC lines failed to respond to 1,25(OH)2D3 regardless of whether these cells had been pre-exposed to serum. An important factor that mediates 1,25(OH)2D3-stimulated gene expression is the vitamin D receptor, but vitamin D receptor mRNA levels in all the SCC lines examined were comparable to those in keratinocytes. Furthermore, the vitamin D receptor protein levels in SCC lines as assessed by ligand-binding analysis were comparable to those of keratinocytes. Thus, the mediators of 1,25(OH)2D3 action on gene expression other than the vitamin D receptor may be missing or defective in SCC lines, whereas the mediators of as yet undefined agents in serum may be better expressed in SCC lines than in keratinocytes. Our results indicate that, although SCC lines are capable of expressing the genes for the proteins involved in differentiation, the control of the expression of these genes by 1,25(OH)2D3 is abnormal in SCC despite the presence of a functional vitamin D receptor in concentrations equivalent to those in keratinocytes.

Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor.

Extracellular calcium concentrations (Cao) > 0.1 mM are required for the differentiation of normal human keratinocytes in culture. Increments in Cao result in acute and sustained increases in the intracellular calcium level (Cai), postulated to involve both a release of calcium from intracellular stores and a subsequent increase in calcium influx through nonspecific cation channels. The sustained rise in Cai appears to be necessary for keratinocyte differentiation. To understand the mechanism by which keratinocytes respond to Cao, we measured the acute effects of Cao on Cai and calcium influx in keratinocytes at various stages of differentiation. We then demonstrated the existence of the calcium receptor (CaR) in keratinocytes and determined the effect of calcium-induced differentiation on its mRNA levels. Finally, we examined the role of Cai in regulating both the initial rise in Cai after the switch to higher Cao and the activity of the nonspecific cation channel through which calcium influx occurs. Our data indicate that the acute Cai response to Cao is lost as the cells differentiate and increase their basal Cai. These data correlated with the decrease in CaR mRNA levels in cells grown in low calcium. However, calcium influx as measured by 45Ca uptake increased with differentiation in 1.2mM calcium, consistent with the increase in CaR mRNA in these cells as well as the calcium-induced opening of the nonspecific cation channels. We conclude that the keratinocyte contains a CaR that regulates both the initial release of Cai from intracellular stores and the subsequent increase in calcium flux through nonspecific calcium channels. A rising level of Cai may turn off the release of calcium from intracellular stores while potentiating the influx through the nonspecific cation channels. Differentiation of keratinocytes appears to increase the CaR, which may facilitate the maintenance of the high Cai required for differentiation.

1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces the differentiation of normal human keratinocytes, in part by increasing their basal intracellular calcium levels (Cai) over a period of hours. Agonists such as ATP acting through membrane receptors cause an immediate but transient increase in Cai accompanied by an increase in inositol trisphosphate (IP3). Treatment of keratinocytes for 24 h with 1 nM 1,25(OH)2D3 resulted in a two- to four-fold potentiation of the Cai response of these cells to ATP. This potentiation was inhibitable with cycloheximide, unaccompanied by a change in total intracellular calcium pools, but associated with an increase in basal IP3 levels and ATP-stimulated IP3 production. Treatment with 1,25(OH)2D3 raised the protein and mRNA levels of phospholipase C isoenzymes, particularly phospholipase C-beta 1 in a dose-dependent manner. These studies indicate that 1,25(OH)2D3 modulates the keratinocyte signal transduction pathway by induction of phospholipase isoenzymes, a previously undescribed action for this hormone.

Effects of 1,25 Dihydroxyvitamin D3 and Calcium on Growth and Differentiation and on c-fos and p53 Gene Expression in Normal Human Keratinocytes

Calcium enhances keratinocyte differentiation, and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is both antiproliferative and prodifferentiative in many cell types, including normal human keratinocytes. In the present study, we examined the combined effects of calcium and 1,25(OH)2D3 on parameters of growth and differentiation and on c-fos and p53 gene expression in normal human keratinocytes. Exposure of normal human keratinocytes to 1,25(OH)2D3 markedly reduced [3H] thymidine incorporation and cell number at low and high medium Ca++ concentrations. Simultaneously, cells in the G0/G1 phase of the cell cycle increased significantly and those in the S phase fell precipitously. 1,25(OH)2D3 and calcium also induced keratinocyte differentiation independently, as assessed by immunocytochemistry and by induction of involucrin mRNA. Both Ca++ and 1,25(OH)2D3 were shown, by nuclear run-on assays, to increase involucrin gene transcription. A rapid, transient elevation in c-fos protooncogene expression preceded these effects when epidermal growth factor was present alone. When 1,25(OH)2D3 was added to quiescent keratinocytes, there was a marked augmentation of c-fos mRNA accumulation at low and high medium Ca++ concentrations. Varying medium Ca++ concentrations had no effect on c-fos mRNA levels. Increasing medium Ca++ concentrations from 0.15 to 2.0 mM produced marked elevations of p53 mRNA accumulation and of the rate of p53 gene transcription, whereas 1,25(OH)2D3 had no effect. These results, therefore, suggest that 1,25(OH)2D3 and calcium act in concert to modulate the expression of two important cell-cycle-associated genes, which may be important components in the initial programming of growth and differentiation of normal human keratinocytes.