Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro , the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 m m . In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.     

Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells

Background

Environmental and endogenous stresses to skin are considered causative reasons for skin cancers, premature ageing, and chronic inflammation. Screening of substances with preventive and/or curative properties is currently based on mechanistic studies of their effects towards stress-induced responses in skin cell cultures.

Objective

We compared effects of plant polyphenols (PPs) on the constitutive, UVA-, LPS-, or TNF-alpha-induced inflammatory responses in cultured normal human epidermal keratinocytes (NHEK) and immortalized HaCaT cells.

Methods

Representatives of three classes of PPs, flavonoids, stilbenoids, and phenylpropanoids were studied. Their effects on mRNA were determined by qRT-PCR; protein expression was assayed by Western blot and bioplexed ELISA; phosphorylation of Akt1, ERK1/2, EGFR, and NFkappaB was quantified by intracellular ELISA or Western blot.

Results

PPs or their combination with UVA or LPS induced strong up-regulation of stress responses in HaCaT but not in NHEK. In addition, compared to NHEK, HaCaT responded to TNF-alpha with higher synthesis of MCP-1, IP-10 and IL-8, concomitant with stronger NFkappaB activation. PPs down-regulated the chemokine release from both cell types, although with distinct effects on NFkappaB, Akt1, ERK, and EGFR activation.

Conclusion

Results of pharmacological screenings obtained by using HaCaT should be cautiously considered while extending them to primary keratinocytes from human epidermis.     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

Abstract: In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N -thioacetylsphingosine (C₂-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N -acetylsphingosine (C₂-Cer=O), as well as the new compound C₂-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC₅₀) of 20 μg/ml and 10 μg/ml, respectively, whereas FS-5 has been potent with an IC₅₀>40 μg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.     

中波紫外线辐射对原代及永生化角质形成细胞损伤能力的比较研究

目的：观察原代角质形成细胞和永生化HaCaT角质形成细胞对中波紫外线照射的反应差异。方法：采用不同剂量中波紫外线(UVB)照射上述两种细胞，评估UVB对细胞作用的时间及剂量效应，以倒置光学显微镜观察细胞受损程度，MTT法检测细胞活性。结果：UVB照射后，细胞损伤程度均随照射剂量加大而加重；另经24h、48h及72h孵育后，对两种细胞进行不同时间段的细胞活性(MTT)比值比较(72h／24h，72h／48h)：分别为原代角质形成细胞(1．16和1.63)和HaCaT细胞(0．96和0．91)。MTT结果显示低剂量紫外线照射时，细胞损伤恢复可发生在照射后48h；高剂量紫外线照射后，两种细胞的损伤程度均随孵育时间延长而加重。结论：原代角质形成细胞抵抗紫外线照射损伤的能力较强，而HaCaT细胞相对较易受损。     

Induction of apoptosis in human HaCaT keratinocytes

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-γ caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of ‘nonapoptotic programmed cell death’. Dexamethasone, tumor necrosis factor-α, transforming growth factor-β, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A ₂ analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body. Received: 28 November 1995     

Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF‐α and IL‐1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF‐α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.     

Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT

Please cite this paper as: Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT. Experimental Dermatology 2010. Abstract: Vitamin A is a pivotal regulator of differentiation and growth of developing and adult skin. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes through retinoic acid nuclear receptors. Here, we present evidence that other vitamin A derivates – retinol and retinal – are also capable of functioning as regulators of gene expression in the keratinocyte cell line HaCaT. We have shown that all‐trans retinol (ATRol) and all‐trans retinal (ATRal) are capable of modulating gene expression in keratinocytes, which is not because of vitamin A metabolism in the cells, and retinol and retinal modulate gene expression through nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Based on the data, we propose that ATRol and all‐trans retinal, in addition to all‐trans retinoic acid, can function as important regulators of gene expression manifesting their effect through the nuclear receptors RARs and RXRs.     

Pathways involved in proliferating,senescent and immortalized keratinocyte cell death mediated by two different TRAIL preparations

Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non-neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non-tagged version, NT-Apo2L/TRAIL, and a leucine zipper fusion protein, LZ-Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT-Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ-Apo2L/TRAIL; NT-Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ-Apo2L/TRAIL; LZ-Apo2L/TRAIL but not NT-Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence--a biological state previously observed to be resistant to UV-light-induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF-B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl-2 or Bcl-x(L) (survival factors that reduce susceptibility to UV-light-induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ-Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV-light mediated by Bcl-2/Bcl-x(L), as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun-induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF-kappaB signaling, while sparing adjacent normal keratinocytes.     

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers., tolerogens and irritants

Abstract To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensi-tizer (2,4-dinitrofiuorobenzene, 3-n-penladecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Intcrleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, in-terlcukin-8 production was not detectable in unstimulatcd normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immuno-genic or inflammatory signals in man.     

Biological effects and metabolism of 9-cis-retinoic acid and its metabolite 9,13-di-cis-retinoic acid in HaCaT keratinocytes in vitro: comparison with all-trans-retinoic acid

Abstract 9-cis-Retinoic acid (9cRA), a geometric isomer of all-trans-retinoic acid (atRA), is an endogenous high-affinity ligand for retinoid X receptors and retinoic acid receptors activating them with high potency. 9,13-di-cis-Retinoic acid (9,13dcRA) has been described as a major plasma metabolite of 9cRA. In this study, the biological activity and the metabolism of 9cRA and 9,13dcRA were investigated and compared with those of atRA in a retinol-free culture system of HaCaT keratinocytes. 9cRA exhibited a slightly weaker activity overall than atRA in inhibiting cell proliferation, inducing cellular retinoic acid binding protein II (CRABP II) mRNA levels and upregulating cytokeratin 19 expression. 9,13dcRA regulated HaCaT keratinocyte activity only at the highest concentration tested (10^–6 M). In cultures of HaCaT keratinocytes with atRA and 9cRA, rapid intracellular accumulation of atRA was observed within 2 h, and atRA levels were higher with atRA treatment than with 9cRA treatment. 9,13dcRA remained relatively stable in the medium with intracellular 9,13dcRA levels below the level of detection. Taken together, 9cRA seems to be slightly less potent than atRA in regulating the biological activity of HaCaT keratinocytes, while its metabolite 9,13dcRA is effectively inactive at biologically relevant concentrations. Our data suggest a prodrug/drug relationship between 9cRA and atRA in human keratinocytes. 9,13dcRA seems to be a weaker prodrug of atRA or an inactive metabolic derivative. Received: 6 July 2000 / Revised: 8 September 2000 / Accepted: 25 October 2000     

Expression of thermosensitive two-pore domain K+ channels in human keratinocytes cell line HaCaT cells

Recent studies have shown that keratinocytes can sense temperature via thermo-transient receptor potential (TRP) channels. It is not known whether other thermosensitive ion channels such as TREK-1, TREK-2 and TRAAK (TREKs/TRAAK) that are members of the two-pore domain K(+) (K(2P)) channel family are expressed in human keratinocytes. Here, we identified the expression of TREKs/TRAAK in human keratinocytes-derived cell line HaCaT cells using RT-PCR, immunocytochemistry, Western blot analysis and patch-clamp technique. RT-PCR showed that all six K(2P) channels tested (TASK-1, TASK-3, TREK-1, TREK-2, TRAAK and TASK-2) were expressed in HaCaT cells, as well as in skin and dorsal root ganglion (DRG) of rat. The expression of TREKs/TRAAK mRNA identified by RT-PCR was further studied at the protein level. Using anti-TREK-1, -TREK-2 and -TRAAK, bands of approximately 46, approximately 60 and approximately 43 kDa, respectively, were observed at plasma membrane of HaCaT cells. Immunostaining also showed that TREK-1, TREK-2 and TRAAK were expressed in all area of cells including plasma membrane. Whole-cell K(+) currents recorded from HaCaT cells were activated by arachidonic acid and heat. These results suggest that TREKs/TRAAK channels could act as thermosensors in human keratinocytes.     

High density of beta2-adrenoceptors in a human keratinocyte cell line with complete epidermal differentiation capacity (HaCaT)

Summary A non-tumorigenic keratinocyte cell line with complete epidermal differentiation capacity (HaCaT) was used in radioligand binding experiments to determine the number of beta-adrenoceptors. Intact cells were saturated with ³H-labelled (–)CGP-12177 (CGP), a hydrophilic non-selective beta-adrenergic antagonist as radioligand. In order to investigate the beta-adrenergic subtype selectivity, displacement experiments were performed with different antagonists and agonists. Binding of CGP to keratinocytes has been shown to be reversible and saturable and to have high affintiy (B _max=114.0±8.8 fmol/10⁷ cells with 6866 receptors/cell, K _D=0.095±0.017 nmol/l; n=11). Betaadrenergic antagonists inhibited binding yielding monophasic displacement curves. IC₅₀-values (nmol/l) were: propranolol (non-selective) 1.68; CGP-12177 (non-selective) 1.08; ICI 118,551 (beta₂-selective) 2.92; bisoprolol (beta₁-selective) 1230; and CGP-20712 (beta₁-selective) 24980. Agonists displaced CGP in the order isoprenaline> adrenaline>noradrenaline. We conclude that HaCaT cells express a high density of beta₂-adrenoceptors providing a good model system to study adrenergic receptor mechanisms under reproducible experimental conditions in keratinocytes.Part of this work was presented at the Meeting of the European Society of Dermatological Research (ESDR), Turin, June 9–12, 1990     

空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响

目的 探讨空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响。方法 收集北京市2015—2016年采暖季雾霾天气PM2.5,制备成混悬液。将HaCaT细胞分为空白组（仅用细胞培养基）、对照组（不加PM2.5,其他处理同实验组）和实验组［100 ~ 400 mg/L（细胞形态学观察和增殖实验用50 ~ 800 mg/L）PM2.5混悬液处理］处理24 h后,倒置显微镜下观察细胞形态变化;CCK8法检测细胞存活率;流式细胞仪检测细胞周期分布及细胞凋亡;Western 印迹法检测细胞周期蛋白A2（cyclin A2）及细胞周期蛋白依赖性激酶1（CDK1）的表达水平。结果 随着PM2.5浓度升高,HaCaT细胞形态逐渐发生改变,细胞数目逐渐减少。与对照组（100 ± 4.95）%相比,50 mg/L PM2.5组HaCaT细胞存活率无明显变化（P＞0.05）,100、200、400、800 mg/L PM2.5组细胞存活率［分别为（91.77 ± 2.04）%、（80.01 ± 1.57）%、（57.80 ± 1.56）%、（21.98 ± 0.86）%］均显著下降（P＜0.05）。流式细胞仪检测显示,与对照组相比,PM2.5组（100、200、400 mg/L）S期细胞比例逐渐增高,G2/M期细胞比例逐渐降低,差异均有统计学意义（P＜0.05）。Western印迹法显示,100、200、400 mg/L PM2.5组cyclin A2、CDK1蛋白表达均较对照组有所降低,尤其以200 mg/L组降低最明显,差异均有统计学意义（P＜0.05）。100、200、400 mg/L PM2.5组细胞总凋亡率分别为（9.98 ± 0.21）%、（12.56 ± 0.74）%、（16.74 ± 1.48）%,与对照组（6.24 ± 0.17）%相比,差异均有统计学意义（P＜0.05）。结论 PM2.5可能通过下调cyclin A2、CDK1表达水平诱导HaCaT细胞发生S期阻滞,从而抑制细胞增殖,促进HaCaT细胞凋亡。     

地塞米松及甲氨蝶呤上调HaCaT细胞结合珠蛋白的表达

目的:探讨地塞米松及甲氨蝶呤对人角质形成细胞系HaCaT细胞合成结合珠蛋白(Hp)的影响。方法:采用RT-PCR方法检测Hp mRNA表达水平,ELISA方法检测Hp浓度。结果:地塞米松或甲氨蝶呤刺激HaCaT24h后,HaCaT细胞表达Hp增加;但较短刺激时间或较低刺激浓度时,HaCaT细胞表达Hp水平无明显变化。结论:地塞米松及甲氨蝶呤能够上调HaCaT细胞Hp的合成,这种作用具有时间依赖性及剂量依赖性的趋势。     

人角质形成细胞株HaCaT细胞表皮生长因子受体内化和下调的研究

目的 了解表皮生长因子受体（EGFR）信号转导与负反馈调节的分子生物学机制。方法 采用免疫沉淀、免疫印迹、间接免疫荧光结合激光扫描共聚焦显微镜等技术，研究HaCaT和CHOwt细胞株中多种配体诱导的EGFR内化和下调情况。结果 免疫印迹检测表明，表皮生长因子（EGF）与肝素结合的EGF（HB-EGF）可引起EGFR总量的快速下调，转化生长因子α（TGFα）及Heregulin则未明显促进受体的降解。EGF、HB-EGF和TGFα处理HaCaT细胞较长时间后活化型受体数量亦不同程度减少。间接免疫荧光染色显示，未处理细胞的EGFR主要分布在胞膜，胞质亦见少量分布。经10 min EGF处理后EGFR聚集成斑状结构（HaCaT细胞明显），并形成内吞体（CHOwt细胞明显），而此时EGFR总量尚未发生明显改变。经过4 h EGF处理后，HaCaT和CHOwt细胞内EGFR信号明显减弱，说明此时受体已经下调。结论 不同配体对HaCaT细胞的EGFR总量及活化型受体的下调作用不同。HaCaT和CHOwt细胞EGFR内化和下调的信号转导机制可能存在差异。     

Cyclic AMP as a negative regulator of DNA synthesis in FRSK cells, a fetal rat epidermal cell line

In FRSK cells, a cell line derived from fetal rat epidermal cells, cyclic AMP-elevating agents forskolin (10 microM) and cholera toxin (10 ng/ml) increased cellular cyclic AMP content and suppressed [3H] thymidine incorporation. These effects of forskolin were enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM). Dibutyryl cyclic AMP (1 mM), an analog of cyclic AMP, decreased not only basal but also both tumor promoter 12-O-tetradecanoylphorbol-13-acetate- and epidermal growth factor-stimulated [3H] thymidine incorporation. From these results, we suggest that cyclic AMP may be a negative regulatory factor of DNA synthesis in FRSK cells.