维胺酯对HaCaT细胞增殖和分化的影响

目的 探讨维胺酯对体外培养角质形成细胞(HaCaT细胞)增殖和分化的影响.方法 将浓度为2,5,10,15,20,25,30 μg/mL的维胺酯作用于培养的HaCaT细胞,采用MTT法检测维胺酯对HaCaT细胞体外增殖的影响,流式细胞仪测定细胞周期及凋亡率变化,逆转录-聚合酶链反应(RT-PCR)半定量检测分化标记物角蛋白10及内披蛋白mRNA的表达水平.结果 2 μg/mL维胺酯处理的HaCaT细胞,48 h时表现出对细胞增殖的抑制作用,随着时间延长和药物剂量加大,抗增殖作用愈明显;当药物质量浓度达到30 μg/mL时,48 h和72 h时的抑制率分别为57.67%和82.00%.与对照组相比,经维胺酯作用48 h后,细胞G₁期比例显著增加,S期与G₂期比例则显著下降,并可抑制G₁/G₂期转换,但对细胞凋亡无影响.细胞内披蛋白mRNA表达水平随维胺酯处理浓度增高而上升,药物浓度达30μg/mL时,表达水平由对照组的40.80%增高至156.12%;而角蛋白10 mRNA表达水平则下降,由96.46%降至14.60%.结论 维胺酯具有抑制角质形成细胞增殖及诱导其分化的作用.     

Protein kinase C isozymes regulate proliferation and high cell density-mediated differentiation in HaCaT keratinocytes

Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density-induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKC alpha, beta, gamma, delta, epsilon, eta, theta, zeta), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the lat (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocyte-specific transglutaminase-1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down-regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density-induced differentiation of HaCaT cells.     

Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.     

Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation

Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes μ-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo , to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D₃ enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.     

靶向角蛋白17基因的小干涉RNA对角质形成细胞增生与凋亡的影响

目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。     

Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro

Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases.     

Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers—K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.     

Similarly potent action of 1,25-dihydroxyvitamin D3 and its analogues,tacalcitol, calcipotriol,and maxacalcitol on normal human keratinocyte proliferation and differentiation

BACKGROUND: The active vitamin D3 regulates proliferation and differentiation of epidermal keratinocytes. Recently topical vitamin D3, tacalcitol, calcipotriol, and maxacalcitol are widely used for psoriasis. OBJECTIVE: To examine the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cultured normal keratinocytes (NHK) and compared its effect with those of various vitamin D3 analogues. METHODS: Cell proliferation of NHK cells was analyzed by MTS, BrdU and 3H-thymidine incorporation. The expression of involucrin, transglutaminase 1, keratin 5 and keratin 1 was investigated by western blot and PCR amplification and quantitative assay. Furthermore, we performed cornified cell envelope (CE) formation assay. RESULTS: 1,25(OH)2D3, tacalcitol, calcipotriol, and maxacalcitol decreased NHK cell proliferation in a concentration-dependent manner and the maximal effect was observed at 10(-7) M. There was no significant difference in the anti-proliferative effect among the active vitamin D3 analogues. The expression of involucrin and transglutaminase 1 were induced by 1,25(OH)2D3 and its analogues in mRNA and protein levels. CE formation was also induced by 1,25(OH)2D3 and its analogues. There was no significant difference in the potency among these chemicals. Keratin 5 and 1 expression was not altered by these active vitamin D3 analogues. CONCLUSIONS: The present study demonstrated that active vitamin D3 analogues, tacalcitol, calcipotriol, and maxacalcitol, suppress keratinocyte proliferation and induce differentiation with similar potency.     

Detecting expression of keratins 8/18 in human HaCaT keratinocytes

Differences in treatment solution affect the efficiency of keratin extraction in cultured human squamous cell carcinomas, malignant melanomas, and melanocytes. Using an aqueous solution that is excellent for cultured cells, we focused this study on the expression of keratin subunits in the spontaneously immortalized human keratinocyte cell line HaCaT. We extracted several keratin (K) subunits, namely K4, K7, K8, K15, K17, and K18, and ATP synthase alpha-chain, in addition to those previously reported by Boukamp et al. (J Cell Biol 1988;106:761-771) in human HaCaT keratinocytes. In particular, K8 and K18 subunits, which are related to tumorigenesis, may be very important subunits within the specificities of immortalized HaCaT cells. Vimentin, which is frequently co-expressed in cultured epithelial cell lines, was not expressed.     

Cyclic AMP differentially regulates cell proliferation of normal human keratinocytes through ERK activation depending on the expression pattern of B-Raf

Intracellular cyclic AMP (cAMP) increased by extracellular stimuli induces various biological effects, such as cell proliferation, differentiation, and migration. Previous reports regarding the effect of cAMP on keratinocyte proliferation are contradictory and indicate that the effect apparently depends on cellular density. Recent studies have revealed that cAMP signaling regulates cell proliferation by modulating mitogen-activated protein kinase (MAPK) activity. The precise mechanism by which cAMP affects keratinocyte proliferation and/or the crosstalk between the cAMP and MAPK signaling pathways, however, remain to be determined. Using normal human keratinocytes (NHK), we investigated the effect of cAMP on keratinocyte proliferation and its molecular mechanism in terms of cellular density. In confluent NHK, cyclic AMP decreased extracellular regulated kinase (ERK) phosphorylation and cell proliferation in a Ras-independent and Rap1-dependent manner. The decreased cell proliferation by cAMP was blocked by the MEK-1 inhibitor, PD98059. In contrast, in subconfluent NHK, cAMP increased ERK phosphorylation and cell proliferation. Western blot analysis revealed that NHK expressed B-Raf and Rap-1. Although both 95 kDa and 62 kDa B-Raf isoforms were expressed in subconfluent NHK, only 62 kDa B-Raf was detected in confluent NHK. Transfection of 95 kDa B-Raf into confluent NHK resulted in a cAMP-dependent increase in ERK phosphorylation and cell proliferation. These findings indicate that differential expression of B-Raf isoforms is critical for cAMP-dependent regulation of NHK proliferation that depends on phosphorylation of ERK.     

The participation of proliferative keratinocytes in the preimmune response to sensitizing agents

The aims of this study were to investigate whether keratinocytes are capable of playing a direct preimmune role in the pathophysiology of allergic contact dermatitis (ACD) and to examine to what extent the degree of differentiation might influence this. We measured the ability of sensitizing agents to up-regulate intercellular adhesion molecule 1 (ICAM-1) expression in cultured normal human keratinocytes (NHK) and in the transformed human keratinocyte HaCaT cell line. In proliferative HaCaT cells, following a 24 h exposure, nickel compounds, para-phenylenediamine (pPD) and 1-chloro-2,4-dinitrobenzene produced a concentration-dependent up-regulation of ICAM-1 expression without reducing cell viability, while K₂Cr₂O₇ led to ICAM-1 up-regulation at cytotoxic concentrations, and CrCl₃ was without effect. In NHK, NiSO₄ and pPD induced ICAM-1 expression to a significantly greater extent in proliferative cells than in differentiated cells, where involucrin expression was measured to assess the differentiation state. NiSO₄- or pPD-pretreatment of proliferative HaCaT cells enhanced T-cell binding, which was abolished by neutralizing antibodies to ICAM-1 or CD18. Our investigations concerning the involvement of oxidative stress in the induction of ICAM-1 expression in response to sensitizing agents were inconclusive. The oxidizing agents FeCl₃ and H₂O₂ up-regulated ICAM-1 expression in HaCaT cells but there was no clear relationship between the ability of agents to induce ICAM-1 expression and their ability to alter the levels of reduced glutathione. Although pPD increased interleukin-1α release from NHK, this cytokine was not capable of inducing ICAM-1 expression in NHK. Tumour necrosis factor-α, which does induce ICAM-1 expression in NHK, was not detected in response to pPD, arguing against an autocrine pathway of ICAM-1 induction in response to pPD. In summary, we report the direct interaction of sensitizing agents with keratinocytes leading to the generation of immune signals, particularly by proliferative keratinocytes, suggesting an active role for the proliferative keratinocyte in the pathophysiology of ACD.     

The state of differentiation of cultured human keratinocytes determines the level of intercellular adhesion molecule-1 (ICAM-1) expression induced by gamma interferon.

Inducing the expression of ICAM-1 (CD54) on the surface of epidermal keratinocytes is an important step in initiating leukocyte interaction with the epidermis. We studied the effect of keratinocyte differentiation and of drugs used to treat epidermal inflammation on the induction of this important adhesion molecule. Cell membrane expression of ICAM-1 in cultured human keratinocytes was analyzed using both immunofluorescence and FACS analysis of staining with anti-ICAM-1 monoclonal antibody and was correlated with markers of keratinocyte differentiation. Cell-surface ICAM-1 expression was induced by gamma interferon in all culture conditions, but was significantly greater (p less than 0.014) in cells grown in low-calcium medium ([Ca++] 0.03 mM), and correlated with increased staining for the basal cell keratin K5. The synthetic retinoid Etretin (Ro 10-1670) enhanced the interferon-induced ICAM-1 expression over a wide concentration range (10(-8)-10(-5) M); however, this effect was only seen in the more differentiated cells grown in 0.15 mM and 1.0 mM calcium and not in the cells grown in 0.03 mM calcium. The Etretin effects on intracellular K5 staining paralleled those on cell-surface ICAM-1. Anti-inflammatory glucocorticoids had no effect on ICAM-1 expression in cultured human keratinocytes, even at suboptimal gamma interferon doses (5 U/ml). beta-estradiol, on the other hand, mimicked the Etretin effect, increasing both IFN induction of ICAM-1 expression and K5 staining in more differentiated keratinocytes in 0.15 and 1.0 mM calcium, but not in those in 0.03 mM calcium. Both Etretin and beta-estradiol decreased staining of involucrin, a marker of terminal differentiation, supporting the proposition that in this experimental system these drugs suppress keratinocyte differentiation. The enhanced ICAM-1 induction in keratinocytes with a basal level of differentiation correlates with the in vivo effects of interferon on ICAM-1 and may be a principal determinant in the patterns of ICAM-1 seen in inflammatory skin diseases.     

Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms. Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.     

Serum factors regulate the expression of the proliferation-related genes α5 integrin and keratin 1, but not keratin 10, in HaCaT keratinocytes

Abstract In the highly coordinated programme of gene expression during keratinocyte proliferation and differentiation, ·5 integrin and keratins 1 and 10 (K1/K10) may play important regulatory roles. We were interested in seeing whether, in continuously growing, immortalized HaCaT keratinocytes, similar to normal keratinocytes, the expression of ·5 integrin and K1/K10 was related to cell proliferation and differentiation. After release from cell quiescence the expression of ·5 integrin, both at the mRNA and protein levels, was upregulated in the cells. At the same time, K1/K10 mRNA and protein expression decreased dramatically, while the mRNA for D1 cyclin became detectable, and the cells became highly proliferative. These findings indicate that ·5 integrin and K1/K10 are involved in the regulation of HaCaT proliferation and differentiation, as in normal keratinocytes. However, HaCaT cells are different from normal keratinocytes in their ability to lose K1/K10 expression. There is no evidence that the expression of K1/K10 can be reversed in normal keratinocytes. This ability of dedifferentiation might be a unique feature of HaCaT cells and may be a key component of their immortalized nature. We also found that serum factors regulate mRNA expression of ·5 integrin and K1, but not of K10, in HaCaT cells. This information could be relevant to the understanding of normal epidermal differentiation. Received: 28 August 2000 / Revised: 27 October 2000 / Accepted: 3 February 2001     

sprouty4蛋白对角质形成细胞增殖及分化的影响

 目的:探证sprouty4(SPRY4)蛋白对角质形成细胞增殖及分化的影响。方法：以人永生化表皮细胞株HaCaT细胞作为实验对象,实验组HaCaT细胞采用基因敲降技术进行SPRY4蛋白抑制物的转染,对照组HaCaT细胞不作任何处理。运用实时荧光定量PCR技术（RT-qPCR）检测实验组与对照组中sprouty4蛋白对HaCaT细胞分化指标Involucrin、CK1与CK10的影响,运用CCK-8实验检测HaCaT细胞的增殖功能。结果：RT-qPCR结果显示,实验组HaCaT细胞中SPRY4基因敲降成功,敲降率约为94.75%。与对照组相比,实验组HaCaT细胞中分化指标Involucrin、CK1与CK10表达水平降低;CCK-8实验结果显示,与对照组相比,实验组HaCaT细胞增殖能力增强。结论：SPRY4蛋白表达下降对细胞增殖起到促进作用,对细胞分化起到抑制作用。     

Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal fibroblasts. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of fibroblasts in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and 16, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine factors important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte growth and differentiation and for use in pharmacotoxicology.     

K17反义寡核苷酸对角质形成细胞增殖和K17表达的影响

目的研究脂质体介导角蛋白17(K17)反义寡核苷酸对培养人角质形成细胞增殖和K17表达的影响。方法利用脂质体将人工合成的正义、反义及错配K17寡核苷酸基因片段导入体外培养的人角质形成细胞系HaCaT,应用MTT法检测其对HaCaT细胞增殖的影响,以逆转录聚合酶链反应(RT-PCR)检测K17mRNA水平的变化,并以蛋白质印迹法、免疫荧光细胞化学结合激光扫描共聚焦显微镜检测K17蛋白水平的改变。结果脂质体介导的K17反义寡核苷酸转染HaCaT细胞后,细胞增殖受到明显抑制,同时细胞中K17mRNA和蛋白的表达明显下降,而正义寡核苷酸组、错义寡核苷酸组及空白对照组均无明显变化。结论应用反义技术封闭K17基因,可以阻遏角蛋白K17基因和蛋白的表达,抑制角质形成细胞的体外生长和增殖能力。     

The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes

Keratinocyte growth factor receptor (KGFR = FGFR2-IIIb) is a tyrosine kinase receptor expressed by keratinocytes, which mediates the effects of fibroblast growth factors (FGF). There are contradictory data in the literature regarding the role of FGFR2-IIIb during the proliferation/differentiation programme of keratinocytes. In this study, we aimed to investigate whether overexpression of FGFR2-IIIb may have a role in the regulation of keratinocyte proliferation. We analysed the expression of FGFR2-IIIb in an in vitro HaCaT model system representing different stages of proliferation and differentiation of keratinocytes. Real-time RT-PCR and Western blot analyses demonstrated a correlation between FGFR2-IIIb mRNA and protein expression and the proportion of cells in S/G2/M phase in synchronized HaCaT keratinocytes and thus with proliferation activity (r = 0.96). After treatment with the antipsoriatic drug, dithranol, FGFR2-IIIb is downregulated dose dependently both at mRNA and protein levels. Moreover, when the rate of proliferation is decreased by the lack of cell attachment to the culturing surface, FGFR2-IIIb mRNA (P = 0.0315) and protein expressions were also reduced (P = 0.0242), while a differentiation marker, keratin 10, mRNA (P = 0.0003) and protein levels (P = 0.001) were increased (r = -0.92). Based on our results we conclude that FGFR2-IIIb expression in HaCaT keratinocytes corresponds with the proliferative activation of the cells and is not related to the differentiation programme.