Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.     

Initial hyperproliferation and incomplete terminal differentiation of cultured human keratinocytes from lesional and uninvolved psoriatic skin

Human epidermal keratinocytes (KCs) were isolated from lesional and from uninvolved skin of 8 patients with chronic plaque-like psoriasis and from the normal skin of 8 healthy volunteers. Primary KC cultures, grown on 3T3 cell feeder layers, were examined over a period of 4 weeks and their plating efficiency, colony growth area, DNA synthesis and ultrastructural cell differentiation were evaluated. Psoriatic KCs formed colonies one day earlier than non-psoriatic controls and proliferated faster during the first 2 weeks, as assessed by the mean colony growth area and 3H-thymidine incorporation. After 4 weeks, however, no significant differences were observed between the in vitro proliferation parameters of normal and psoriatic KCs. At the ultrastructural level, cultures of lesional psoriatic KCs consisted of more cell layers with adherent transitional cells and incomplete formation of cornified envelopes, even after 4 weeks, while KCs from uninvolved psoriatic skin were characterized by a transient delay of in vitro maturation. These results indicate that the characteristic hyperproliferation of psoriatic KCs may only be maintained over a short period of primary culture, whereas defective terminal differentiation of lesional psoriatic KCs was maintained throughout the culture period.     

Camptothecin induces differentiation, tissue transglutaminase and apoptosis in cultured keratinocytes

Abstract: Cultured normal human adult keratinocytes were exposed to (S)-(+)- camptothecin over the concentration range 10^-5 to 10^-10 M. The dose-dependent inhibition of growth was recorded using cell counting. The induction of terminal differentiation was demonstrated by the relative increase in squamous and cornified cells, and the concomitant decrease in small, proliferative cells, with an overall decrease in total cell numbers on going from 10^-10 to 10^-6 M concentration of the drug. The induction of apoptosis was studied by assay of two types of transglutaminase, "tissue" and "keratinocyte", and by assay of histonelinked mono- and oligonucleosomes. Induction of apoptosis was accompanied with increase in "tissue" transglutaminase and in the amount of nucleosomes, the latter being indicative of endonuclease activity. This activity was sharply increased at a camptothecin concentration of 10^-5 M, and may have been faciliated by "tissue" transglutaminase at lower concentrations. The data suggest that camptothecin restricts keratinocyte growth by several mechanisms including apoptosis and emphasize its possible use in topical therapy for psoriasis.     

Recombinant gamma interferon induces HLA-DR expression on cultured human keratinocytes

In normal human epidermis, expression of HLA-DR antigen is restricted to Langerhans cells (LC) and acrosyringial epithelium. However, in diseases such as lichen planus and graft-vs.-host, HLA-DR antigen appears to be expressed by keratinocytes, although the exact source of the HLA-DR is unclear. Two possibilities are that (1) the HLA-DR is shed by neighboring immunocompetent cells, or (2) that the keratinocytes are synthesizing the antigen themselves. Recently, gamma interferon has been shown to induce HLA-DR biosynthesis and expression on human malignant melanoma cells lines and on normal vascular endothelium. We report here that pure recombinant human gamma interferon (100 units/ml) induces HLA-DR expression on 60-70% of cultured human adult keratinocytes depleted of LC within 2-4 days of culture as determined by fluorescence-activated cell sorter (FACS) analysis using monoclonal antibodies. No residual LC or lymphocytes could be detected in these cultures. This is the first demonstration of HLA-DR expression by cultured human keratinocytes. This expression may be of functional significance in antigen presentation and cell-mediated cytotoxicity involving the epidermis.     

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes

Background Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B₁ receptor (B₁R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. Objectives To determine the consequence of kinin B₁R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. Methods Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. Results The major proteins phosphorylated, after B₁R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170‐ and 125‐kDa proteins were identified as EGFR and p125^FAK, respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12‐myristate 13‐acetate, indicating the participation of protein kinase C. B₁R stimulation did not increase [Ca²⁺]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr⁸⁴⁵, Tyr⁹⁹² and Tyr¹⁰⁶⁸ of EGFR. B₁R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B₁R stimulation, produced an additional increase in filaggrin expression. Conclusions The kinin B₁R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.     

Long-wave ultraviolet light induces phospholipase activation in cultured human epidermal keratinocytes

Long wave ultraviolet radiation (UVA) has been shown to play an important role in the overall response of skin to solar radiation, including sunburn, tanning, premature aging, and non-melanoma skin cancer. UVA induction of inflammation in human skin is thought to be mediated by membrane lipid derived products. In order to investigate the mechanism of this response we examined the effect of UVA on phospholipid metabolism of human epidermal keratinocytes in culture. Keratinocytes were grown in serum free low calcium medium. The cells were prelabeled with [3H] arachidonic acid or [3H] choline and irradiated with UVA (Honle 2002-Hg vapor lamp). Identification and quantitation of specific membrane phospholipid-derived components was achieved using high-performance liquid chromatography, paper chromatography, and radioimmunoassay. UVA resulted in a linear dose dependent release of [3H] arachidonic acid into medium between 1 and 20 joule/cm2. This response was inhibited in an oxygen-reduced environment. The radiolabel released was predominantly free arachidonate and cyclooxygenase metabolites. Cyclooxygenase metabolites prostaglandin E2 and prostacyclin derivative, 6-keto-prostaglandin F1a, were stimulated following UVA irradiation, but the lipoxygenase metabolite, leukotriene B was not detected. Maximal release was measured immediately after irradiation and changed little over 24 h post-irradiation. UVA stimulated an increase of [3H] choline metabolites glycerophosphorylcholine and phosphorylcholine in media extracts suggesting UVA activation of phospholipase C and phospholipase A2 or diacylglyceride lipase.     

Growth and differentiation of human keratinocytes cultured on eye lens capsules

Summary The efficiency of the outgrowth of human epidermal and hair-follicle-sheath keratinocytes was studied using three different growth substrates: plastic, type-I collagen and bovine eye lens capsules (the Epicult system). It was shown that the eye lens capsule is the best substrate, since a higher percentage of cultures showed outgrowth, and the outgrowth of epidermal keratinocytes was much more rapid. This effect is related to the faster migration (not proliferation) of cells grown on lens capsules as compared to the two other substrates. The view that lens capsules can replace the basement membrane present in vivo was supported by the finding that two basement-membrane components, i.e., laminin and fibronectin, are present on lens capsules. It was shown that, in cultures grown on lens capsules, bullous-pemphigoid antigen is restricted to the basal layer, indicating that the differentiation of these cells is comparable to that of keratinocytes grown on irradiated, non-viable pig dermis.     

Ganglioside-induced terminal differentiation of human keratinocytes: early biochemical events in signal transduction

Previous studies have indicated that GQ_1b, a tetrasialoganglioside containing two disialosyl residues, may be an important regulator of cellular differentiation in murine keratinocytes. In the present study, we examined the effect of gangliosides on the differentiation of human keratinocytes. Current evidence indicates that GQ_1b induces cornified envelope formation and enhancement of transglutaminase (TGase) activity, which are characteristic parameters of terminal differentiation in human cultured keratinocytes, while the other gangliosides, GT_1b and GM₁, are much less effective. The mass contents of inositol 1,4,5-trisphosphate (1,4,5-IP₃) and the intracellular calcium concentration ([Ca⁺]_i) were also measured in keratinocytes exposed to gangliosides. A rapid increase in 1,4,5-IP₃ occurred at 30 s following stimulation, but no significant difference at the maximum level was observed among the three gangliosides in contrast to the finding in murine keratinocytes. In addition, [Ca⁺] increases occurred concurrently with the 1,4,5-IP₃ generation by the three gangliosides. On the other hand, [Ca⁺] transients were unaffected by chelating extracellular Ca⁺ with EGTA. It is thus considered that the mobilization by 1,4,5-IP₃ from internal stores plays a crucial role. These [Ca⁺]_i profiles were also indistinguishable between the gangliosides. Taken together, in human keratinocytes, gangliosides differentially affect some other as yet unidentified site(s) in the post-calcium transmission pathway(s) which leads to TGase activation.     

Calcipotriol (MC 903), a novel vitamin D3 analogue stimulates terminal differentiation and inhibits proliferation of cultured human keratinocytes

Summary The hormonally active form of vitamin D₃, 1,25-dihydroxy vitamin D₃ [1,25-(OH)₂-D₃; calcitriol], regulates the differentiation and proliferation of epidermal keratinocytes in vitro. MC 903 (calcipotriol) is a novel vitamin D₃ analogue which is at least 100 times less potent than 1,25-(OH)₂-D₃ in its effects on calcium homeostasis. The present study compared the effects of MC 903 and 1,25-(OH)₂-D₃ on terminal differentiation and proliferation of cultured normal human keratinocytes. Keratinocytes were grown in McCoy's 5A medium supplemented with penicillin (50 IU/ml), streptomycin (50 g/ml), l-serine (4×10^–4 M), and 10% human type AB serum. MC 903, 1,25-(OH)₂-D₃ or 1-OH-D₃ (10^–12 M–10^–8 M) was added with each feeding when cultures became preconfluent. After incubation for 24 h with D₃ vitamins, cultures were extracted for transglutaminase, and the enzyme activity was indexed against DNA content. The activity of transglutaminase, the enzyme reponsible for cross-linking the proteins of the cornified envelope, was maximally stimulated by 388% with MC 903 (10^–8 M), by 328% with 1,25-(OH)₂-D₃ (10^–8 M), and by 27% with 1-OH-D₃ (10^–8 M) compared with vehicle. After incubation for 2 weeks the number of keratinocytes with cornified envelopes had increased by 288% with MC 903 (10^–8 M), by 360% with 1,25-(OH)₂-D₃ (10^–8 M), and by 149% with 1-OH-D₃ (10^–8 M) compared with vehicle. Simultaneously the incorporation of (³H)thymidine into DNA was decreased by 64% with MC 903 (10^–8 M), by 71% with 1,25-(OH)²-D₃ (10^–8 M), and by 10% with 1-OH-D₃ (10^–8 M). There was a corresponding decrease in cell number. These results demonstrate that both MC 903 and 1,25-(OH)₂-D₃ are potent modulators of keratinocytes differentiation and proliferation in vitro. Because MC 903 is much less active than 1,25-(OH)₂-D₃ in causing hypercalcemia, this compound is a candidate for the treatment of skin diseases characterized by aberrant epidermal differentiation and proliferation.Parts of this study were presented at the ESDR meeting in Munich, May, 1988     

E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.     

UVB induces a biphasic response of HIF-1alpha in cultured human keratinocytes

Abstract:  Hypoxia in the skin is important in chronic degenerative dermo-epidermal changes, inflammation, photoageing and carcinogenesis. In these processes, vascular endothelial growth factor (VEGF) plays a crucial role and is known to be affected by ultraviolet radiation (UVR). Hypoxia-inducible factor-1 (HIF-1) closely regulates the expression of VEGF in several experimental settings. We set out to study the impact of acute UVB irradiation on the level of HIF-1 as a major regulator of hypoxia-induced genes. Effects of UVB exposure on HIF-1α expression were investigated in HaCaT cells after a single irradiation by Western blots. Downstream target gene expression was measured by quantitative real-time polymerace chair reaction (PCR). UVB treatment resulted in an initial decrease of the HIF-1α protein level followed by a subsequent prolonged increase. If cells were exposed to additional UVB irradiation, another decrease in HIF-1α was provoked, similar to the original effect. The observed changes followed a strict timeline and were dose-dependent. The role of the PI3K/AKT pathway was examined. No change in the total level of AKT after UVB treatment was seen; however, its phosphorylation level was found to be markedly higher. In accordance with these observations, wortmannin, an inhibitor of PI3-kinase effectively blocked the UVB-induced increase in HIF-1α. In agreement with previous findings, UVB irradiation increased VEGF and haem oxygenase-1 mRNA levels determined by quantitative real-time PCR. It is concluded that changes in HIF-1α expression underlie the alterations in expression of VEGF upon UVB irradiation. Our findings indicate the involvement of PI3K in UVB-mediated HIF-1α upregulation.     

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2×10⁻¹⁰ mol/l) or by pharmacological doses of biotin (10⁻⁵ mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.     

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2×10^?10 mol/l) or by pharmacological doses of biotin (10^?5 mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.     

Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium

Normal human proliferative keratinocytes can be serially cultured in serum-free medium. The medium and culture conditions that have been developed support the growth of these cells in the absence of a feeder layer and in the absence of contaminating fibroblasts. Using this system, the nutritional and hormonal factors that affect the growth and differentiation of these cells have been studied in the absence of undefined supplements. The data suggest that the control of proliferation and differentiation in keratinocytes may take place by unique mechanisms. The defined serum-free conditions that have been developed provide an excellent system for the study of nutritional disorders that affect human epidermal cells. In addition, the culture system can be used to propagate large numbers of cells for use in autografts for cases of severe burns.     

神经酰胺诱导培养角质形成细胞分化的实验研究

目的：观察神经酰胺对无血清培养角质形成细胞分化的影响。方法：选择两种神经酰胺（C2和C6）,分别添加于无血清培养的角质形成细胞液中,并在添加后0、3、6、12、24h分别回收总RNA,用逆转录PCR（RT－PCR）方法,检测角质形成细胞分化的特异性标记：TGase1、IVL、KRT、、KRT10等mRNA表达情况。结果：神经酰胺添加后,C2－神经酰胺对TGase1 mRNA、IVL mRNA表达在12小时均明显上升,在24小时分别升高达阴性对照的5.7倍和5.2倍;但C2－神经酰胺对KRT1 mRNA和KRT10 mRNA表达升高不明显。C6－神经酰胺对TGase1 mRNA、IVL mRNA表达大致与C2－神经酰胺相似,只是24小时表达比C2－神经酰胺降低,分别为阴性对照的3.3倍和3.5倍;但C6－神经酰胺对KRT1 mRNA和KRT10 mRNA表达显著高于C2－神经酰胺,24小时分别高达5.4倍和10倍。结论：神经酰胺能明显升高无血清培养的角质形成细胞对分化特异性标记蛋白mRNA的表达,诱导角质形成细胞的分化。     

The growth and differentiation of cultured newborn rat keratinocytes

Keratinocytes were cultured from adult and newborn rat epidermis using the 3T3 feeder cell technique. By modifying culture conditions a long-lived line of newborn rat keratinocytes was developed which showed a plating efficiency of 40% and a doubling time of 16 h. The cells produced stratified colonies with tonofilaments, desmosomes, cell envelopes, and keratohyaline granules. When the cells were grown on a collagen gel they formed a thick stratum corneum and many keratohyaline granules. The fibrous proteins synthesized by the newborn rat cultured keratinocytes were different than those of newborn rat epidermis but similar to those of adult rat cultured keratinocytes. A histidine-rich basic protein was identified by immunologic techniques but it appeared to be more heterogeneous than that of newborn rat epidermis. A cell envelope precursor protein was identified by dansyl cadaverine incorporation studies and was identical to a major envelope precursor of newborn rat epidermis. The growth characteristics, colony morphology, and biochemical markers did not change for up to 40 passages and there was no evidence of malignant transformation. Because of their case of growth and long-term survival these cells are useful for studying a variety of problems related to keratinization.