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.     

Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expression

Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5-bromo-2'-deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel-induced cell growth appeared enhanced when primary human keratinocytes were co-cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up-modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor-alpha (TGF-alpha) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up-modulation could be required for the nickel-induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment.     

角质形成细胞生长因子受体反义寡核苷酸对HaCaT细胞增殖的影响

目的 研究角质形成细胞生长因子受体反义寡核苷酸(KGFR ASODN)对角质形成细胞生长因子(KGF)促HaCaT细胞增殖效应的抑制作用。方法 HaCaT细胞体外培养,采用绘制生长曲线、MTT实验、集落形成实验研究KGFR ASODN对KGF促增殖效应的抑制作用。采用流式细胞仪研究KGFR ASODN对KGFR表达的影响。结果 反义序列1和反义序列2均可抑制HaCaT细胞的生长速度(P<0.05)。高于0.25μg/mL质量浓度反义序列1和反义序列2均可抑制KGF介导的HaCaT细胞增殖(P<0.05)。高于0.25μg/mL质量浓度反义序列1和反义序列2都可抑制KGF诱导的HaCaT细胞集落形成(P<0.05)。HaCaT细胞存在KGFR基础表达,高于0.25μg/mL质量浓度反义序列1和反义序列2均可抑制HaCaT细胞KGFR表达(P<0.05),抑制率呈浓度依赖性,正义序列核苷酸(S-ODN)对KGFR表达无抑制作用(P>0.05)。结论 KGFR ASODN可有效抑制KGFR表达,进而抑制KGF介导的HaCaT细胞过度增殖。     

Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes

The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.     

Expression of keratinocyte growth factor and its receptor in clear cell acanthoma

The aetiopathogenic mechanism underlying clear cell acanthoma (CCA) is not completely clear and it has been postulated that CCA and psoriasis may have a similar pathogenesis because of the common features shared by the two diseases. As it has been recently demonstrated that in psoriatic lesions the paracrine epithelial growth factors [keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)-7 and FGF-10] are involved in promoting and sustaining the keratinocyte hyperproliferation, the aim of this study was to analyse the expression of KGF on CCA lesions and to search for a role of this growth factor in CCA pathogenesis. Immunohistochemical analysis showed an up-modulation of KGF in CCA, although the immunostaining was variable among the different samples collected. Positive immunoreactivity for KGF was detected mainly on dermal areas where the inflammatory infiltrate was more pronounced suggesting a relationship between lymphocyte activation and KGF up-modulation. Real-time quantitative RT-PCR assay performed on mRNA extracted from formalin-fixed paraffin-embedded CCA and normal skin (NS) samples further demonstrated the overexpression of the KGF/FGF-7 gene in all CCA samples compared with NS. Moreover, the evaluation by immunohistochemistry of KGF receptor distribution, the high-affinity tyrosine kinase receptor for KGF, showed a down-modulation of this receptor, as previously reported in the presence of increased levels of KGF. Taken together these results suggest the inflammatory nature of CCA and further support the hypothesis that this disease may represent, like psoriasis, an inflammatory dermatosis in which KGF up-modulation may be responsible for keratinocyte hyperproliferation and may represent a new common feature of both diseases.     

降钙素基因相关肽对HaCaT细胞表达血管内皮生长因子的调控

目的 探讨降钙素基因相关肽(CGRP)对角质形成细胞表达和分泌血管内皮生长因子(VEGF)的调控.方法 用实时定量PCR(RT-PCR)方法检测CGRP、CGRP1型受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性的抑制剂PD98059、p38MAPK特异性拮抗剂SB203580对HaCaT角质形成细胞表达VEGFmRNA水平的影响;用酶联免疫吸附方法检测HaCaT细胞分泌至培养液中VEGF蛋白的水平.结果 CGRP时间依赖性地促进HaCaT细胞表达VEGFmRNA和分泌VEGF蛋白;CGRP8-37和PD98059均可明显抑制CGRP刺激的HaCaT细胞表达和分泌VEGF,SB203580不能减弱CGRP刺激的VEGF表达和分泌.结论 CGRP可以上调HaCaT细胞表达和分泌VEGF,CGRP1型受体及其相关的ERK1/2信号通路参与其调控.     

Immunohistochemical analysis of keratinocyte growth factor and fibroblast growth factor 10 expression in psoriasis

The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of growth factors, and some studies have reported an increased expression of the insulin-like growth factor 1. Few studies, however, have focused on the possible involvement of the keratinocyte growth factor (KGF/FGF-7) and the fibroblast growth factor 10 (FGF-10/KGF-2), which are secreted by fibroblasts and stimulate keratinocyte proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF-10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF-10 showed that both the growth factors are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T-cell infiltrate and with keratinocyte proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF-10, which in turn contribute to sustain the hyperproliferative status of the keratinocytes.     

角质形成细胞生长因子对HaCaT细胞增殖的影响

目的:研究角质形成细胞生长因子(KGF)对HaCaT细胞增殖的影响,探讨KGF与银屑病表皮过度增殖的关系。方法:利用HaCaT细胞体外培养,采用KGF为增殖诱导剂,四甲基偶氮唑盐比色法(MTT)检测不同浓度的KGF对HaCaT细胞增殖率的影响;检测KGF对细胞生长曲线及细胞集落形成的影响。结果:MTT实验表明,KGF可刺激HaCaT细胞增殖,刺激作用呈浓度依赖性(r=0.981,P<0.05);10 ng/mL KGF作用下HaCaT细胞的生长速度较对照组明显加快(P<0.05),集落形成率较对照组增加(P<0.05),且细胞呈明显的增殖形态,形成的集落较大,集落内细胞多呈星状,胞内染色质丰富。结论:KGF具有显著的促HaCaT细胞增殖作用,银屑病皮损区域KGF表达增加可能是引起表皮过度增殖的重要致病因子。     

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Please cite this paper as: The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Experimental Dermatology 2010; 19 : 865–872. Abstract: Solar lentigines (SLs) are hyperpigmentary lesions presented on sun‐exposed areas of the skin and associated with ageing. The molecular mechanism of SL initiation is not completely understood. Ultraviolet B (UVB) stimulates keratinocytes to produce interlukin‐1 alpha (IL‐1α), which then induces keratinocyte growth factor (KGF) secretion; therefore, we examined their possible roles in the induction of SLs. We found that KGF increases pigment production in both pigmented epidermal equivalents and human skin explants. In addition, UVB exposure increases KGF expression, and KGF treatment induces tyrosinase (TYR) expression in primary melanocytes. The KGF‐induced pigmentary changes were confirmed using pigmented Yucatan swine, and human skins grafted onto immuno‐deficient mice. In both model systems, the topical treatment with KGF, alone or in combination with IL‐1α, resulted in the in vivo formation of hyperpigmentary lesions with increased pigment deposition and elongated rete ridges, which resemble the histological features of human SLs. Preliminary immunohistochemical analysis of human skins showed a moderate increase in KGF, and a strong induction in KGF receptor (KGFR) in SL lesions. In summary, KGF increases pigment production and deposition in vitro and in vivo. Moreover, we show for the first time the in vivo generation of hyperpigmentary lesions with histological resemblance to human SLs and indicate the involvement of KGF/KGFR in the molecular pathology of human SLs.     

Characterization of the expression and function of N-methyl-D-aspartate receptor in keratinocytes

The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.     

Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H(2)O(2) generation

Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal growth factor receptor in primary human keratinocytes. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H(2)O(2). Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal growth factor receptor phosphorylation and H(2)O(2) generation. Third, the structurally different antioxidants n-propyl gallate and N-acetylcysteine inhibited epidermal growth factor receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal growth factor receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal growth factor receptor phosphorylation and activation of extracellular-regulated kinase 1/2. These findings establish the following sequence of events: (1) H(2)O(2) generation, (2) epidermal growth factor receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H(2)O(2) as an important upstream mediator required for ligand-independent epidermal growth factor receptor phosphorylation and downstream signaling.     

Increased expression of the histamine H4 receptor following differentiation and mediation of the H4 receptor on interleukin‐8 mRNA expression in HaCaT keratinocytes

Recent in vivo studies have demonstrated involvement of the histamine H4 receptor in pruritus and skin inflammation. We previously reported that an H4 receptor antagonist attenuated scratching behaviour and improved skin lesions in an experimental model of atopic dermatitis. We also reported the expression of the H4 receptor in human epidermal tissues. In this study, we investigated the expression of H4 receptor mRNA and the function of the receptor in a culture system that mimics in vivo inflammation on the HaCaT human keratinocyte cell line. Increased expression of the H4 receptor was observed in HaCaT cells following differentiation. Treatment of HaCaT cells with histamine and TNFα enhanced the mRNA expression of interleukin (IL)‐8. These increases in expression were significantly inhibited by the H4 receptor antagonist JNJ7777120. Our results indicate that IL‐8 mRNA expression might be enhanced by histamine and TNFα via H4 receptor stimulation in keratinocytes.     

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.     

Nerve growth factor increases the mitogenicity of certain growth factors for cultured human keratinocytes: A comparison with epidermal growth factor

Abstract Newborn foreskin and adult skin keratinocytes (KTs) were cultured in 24-well plates using keratinocyte basal medium (KBM) either alone or supplemented with epidermal growth factor (EGF) or nerve growth factor (NGF), plus one of the following: insulin (INS), insulin-like growth factors (IGF)-l or -2, transforming growth factor alpha (TGFα), basic fibroblast growth factor (bFGF). Culture was maintained until one group of cells reached about 30,000 cells/well, when cells were stained with crystal violet and the extracted dye used to quantify cell numbers. In some cases, cells were subjected to the hexosaminidase assay for enumeration. In KBM alone, EGF, IGF-1, IGF-2 and TGFα were milogenic to newborn KTs. In addition, NGF increased the growth of adult KTs, possibly by mechanisms involving synergy with autocrine growth factors. EGF augmented the growth of newborn cells in the presence of each of the growth factors except TGFα, but adult cells exhibited only additive effects. In the presence of IGF-1 or IGF-2, NGF stimulated the growth of both newborn and adult cells by as much as 150% above purely additive increases in cell numbers. NGF amplifies the effects of most neurotrophic factors that are also KT mitogens and may therefore be significant in psoriatic lesions, where many of these factors are overex-pressed, and in wound healing, in promoting KT growth.     

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。     

Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes

Summary Transformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca²⁺ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca²⁺ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF-receptor gene amplification when extracellular Ca²⁺ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca²⁺ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca²⁺ conditions. At the membrane level, normal keratinocytes and SCC-12F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca²⁺ conditions, but equal mRNA and protein expression. These and previous findings demonstrate that EGF-receptor expression is regulated at the levels of DNA, mRNA and protein as well as by the plasma membrane composition, depending upon the cell type.