Differentiation Of Normal Human Keratinocytes Research Articles

AP-1 Activity during Normal Human Keratinocyte Differentiation: Evidence for a Cytosolic Modulator of AP-1/DNA Binding

Increased levels of c-fos and c-jun expression have been observed in differentiating epithelial cells. However, no data are available on activator protein 1 (AP-1) activity during keratinocyte differentiation. In this work we investigated c-fos and c-jun gene expression and AP-1-(12- O-tetradecanoylphorbol-13-acetate)-responsive enhancer element (TRE) binding activity during keratinocyte differentiation utilizing both authentic and in culture-reconstituted human epidermis. We demonstrate that: (i) in reconstituted epidermis, non-differentiated and differentiated keratinocytes express equivalent levels of c-Jun, while in reconstituted epidermis permanently grafted onto athymic mice, as well as in authentic epidermis, c-Jun is predominantly expressed in the granular layer of the tissue. Equivalent levels of c-fos expression have been found in all the layers of both reconstituted and authentic epidermis. (ii) Nuclear extracts from cultures enriched in differentiated keratinocytes display an 80-90% reduction of AP-1 activity when compared to extracts from cultures enriched in nondifferentiated cells. (iii) Cytosolic extracts obtained from cultures enriched in differentiated cells reduce, in a concentration-dependent manner, the AP-1 activity present in nuclear extracts of both mammalian and Drosophila cells. (iv) The specific TRE binding activity of a recombinant c-Jun protein is significantly reduced by cytosolic extracts of differentiated keratinocytes, while the specific DNA binding of the purified recombinant human homeoprotein HOX4B is not. (v) The dephosphorylation, by alkaline phosphatase, of cytosolic extracts increases the inhibitory activity already present or makes evident a latent activity.

LDL Receptors in Keratinocytes

The presence of sufficient amounts of cholesterol in the epidermis is necessary for proper functioning of plasma membranes in the viable epidermal cell layers and also for the barrier quality of lipid intercellular bilayers of the stratum corneum. Cholesterol can be generated by local epidermal synthesis, or imported from the circulation as low-density lipoprotein (LDL), which is internalized by the cells by receptor-mediated endocytosis. Because of the complex structure of the skin, a model consisting of cultured human keratinocytes has been used to study in detail the regulation of epidermal sterologenesis in relation to keratinocyte differentiation. Experimental modulation of the differentiation of normal human keratinocytes has been achieved by varying extracellular calcium concentration or by comparison of a number of human squamous carcinoma cell lines and normal keratinocytes. These studies have clearly demonstrated a reciprocal correlation between the ability of cells to differentiate and LDL receptor activity. Regulation of LDL receptor expression has been found to occur at the DNA, mRNA, and protein levels, depending on the cell line studied. In normal but not malignant keratinocytes, the induction of keratinocyte differentiation was associated not only with a decrease of functional LDL receptors but also with changes in their cellular distribution. This conclusion is drawn from the observations that only in normal human keratinocytes, cultured at physiologic calcium concentrations, high levels of intracellular, cytoskeleton-associated receptors were found. Differentiation-related modulations of the LDL-receptor expression and of the cellular LDL-receptor distribution found in cultured keratinocytes were in agreement with observations made in the epidermis in situ.

Transforming growth factor-β1 acts cooperatively with sodium n-butyrate to induce differentiation of normal human keratinocytes

Growth factors with established biological activity toward cultured normal human epidermal keratinocytes (NHEKs) (e.g., transforming growth factor-β, TGF-β; retinoic acid, RA) initiate programmed changes in cellular maturation which differ with regard to the specific differentiation pathway (normal or abnormal) analyzed. Sodium butyrate (NaB) initiates one form of epidermal differentiation leading to enhanced cornified envelope (CE) formation which involves abrogation of the normally inhibitory effect of RA on NHEK maturation. NaB also induces TGF-β mRNA in the maturing suprabasal compartment, suggesting that TGF-β may play a role in NaB-initiated NHEK differentiation. Treatment with TGF-β1 alone, however, only marginally increased (by twofold) the number of detergent-resistant CEs compared to control NHEKs and did not alter the prevalence of fully mature enucleated CEs. TGF-β1 was quite effective in inducing significant levels of CE expression when used simultaneously with suboptimal concentrations of NaB. The cooperative action of suboptimal NaB and TGF-β1 generated numbers of CEs which, in fact, exceeded the incidence of mature CEs formed in response to optimal levels of NaB alone. Neutralizing antibodies to TGF-β, moreover, effectively reduced the incidence of CE formation in cultures treated with optimal NaB concentrations, further implicating endogenous TGF-β activity in the NaB-initiated NHEK differentiation model. It is suggested, therefore, that within the NaB-induced pathway of NHEK differentiation, TGF-β can positively modulate expression of the differentiated phenotype but alone is insufficient for generation of mature CEs.

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH) 2D 3, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D 3], and that 1,25(OH) 2D 3 endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH) 2D 3 is produced from 25(OH)D 3 by skin keratinocytes, 25(OH)[ 3H]D 3 was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH) 2D 3 was 1,25(OH) 2D 3. Most of the 1,25(OH) 2D 3 endogenously produced from 25(OH)D 3 remained within the cells. To examine the time course of 1,25(OH) 2D 3 production, the amount of 1,25(OH)[ 3H]D 3 was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D 3. These data indicate that keratinocytes rapidly convert 25(OH)D 3 to 1,25(OH) 2D 3 and that 1,25(OH) 2D 3 is not released into the medium. To determine whether endogenously produced 1,25(OH) 2D 3 is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH) 2D 3 and 25(OH)D 3 on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10 −8 M and 10 −7 M 1,25(OH) 2D 3 and to 80.5% and 23.9% by 10 −8 M and 10 −7 M 25(OH)D 3, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10 −7 M 1,25(OH) 2D 3, and to 11.8% by 2.5·10 −7 M 25(OH)D 3. These results clearly indicate that 25(OH)D 3 is as effective on human keratinocytes as 1,25(OH) 2D 3 in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH) 2D 3. The possibility that the effect of 25(OH)D 3 is mediated through binding to the 1,25(OH) 2D 3 receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D 3 for the 1,25(OH) 2D 3 receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH) 2D 3. Thus, these results suggest that 1,25(OH) 2D 3 endogenously produced in keratinocytes from 25(OH)D 3 is involved in the regulation of their growth and differentiation in vitro.

Uncoupling of the calcium-sensing mechanism and differentiation in squamous carcinoma cell lines

Increases in the intracellular calcium (Ca i) levels, induced either by extracellular calcium or by calcium ionophores, stimulate the terminal differentiation of normal human keratinocytes in culture (NHK). Despite extensive differences in phenotypic expression, squamous carcinoma cell lines (SCC lines) display only partial terminal differentiation even in the presence of normal extracellular calcium. Therefore, in this study, we evaluated whether the inability of SCC lines to differentiate normally is due to a defect in achieving adequate levels of Ca i. Membrane-bound transglutaminase activity and involucrin levels of the various SCC lines were lower than those of NHK and correlated with their low extent of cornified envelope formation. Ionomycin, a calcium ionophore, acutely increased cornified envelope formation of NHK 60- to 70-fold, but only initiated a 1- to 5-fold increase in SCC lines. Yet resting Ca 1 levels in and the Ca 1 response to various agents of SCC lines were similar or higher than those of NHK. Extracellular calcium evoked a rapid, transient and a slower, sustained increase of Ca i. Extracellular ATP increased Ca i by a rapid release from intracellular sources. Ionomycin, on the other hand, increased Ca i from both intracellular compartments and extracellular sources. Thus, these studies indicate that the abnormalities in differentiation among SCC lines do not appear to involve their calcium-sensing mechanism. An uncoupling of the Ca i changes to the synthesis of the precursor molecules required for differentiation may be responsible for the defect in differentiation displayed by these SCC lines.

Growth inhibition of human keratinocytes by antisense c-myc oligomer is not coupled to induction of differentiation

To study the relationship between cell growth and differentiation in human keratinocytes, we examined the effect of the antisense oligomer of c-myc mRNA. This oligomer is stable in culture medium. A 24 h incubation of cells with 5 μM antisense c-myc oligomer resulted in a 48.2% decrease in c-myc protein and inhibited cell growth by 80.7% compared to the sense c-myc oligomer. In contrast, antisense c-myc oligomer had no effect on differentiation when the population of involucrin-positive cells and cornified envelope formation were used as differentiation markers. These results show that antisense c-myc oligomer inhibits cell growth but does not induce differentiation in normal human keratinocytes. Therefore, cell growth and differentiation are not necessarily coupled in these cells.

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor‐β

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-β 1

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-β 1

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.

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.