Arsenite and arsenate activate extracellular signal-regulated kinases 1/2 by an epidermal growth factor receptor-mediated pathway in normal human keratinocytes

BACKGROUND: Inorganic arsenic is an environmental contaminant and is associated with the increased risk of human skin cancer. Arsenic has been reported to activate or inhibit a variety of cellular signalling pathways which has effects on cell growth, differentiation and apoptosis. However, the molecular mechanisms of these arsenic-induced biological effects are not completely understood. OBJECTIVES: To understand the molecular basis for the mode of action of arsenicals, we examined the effect of arsenite and arsenate on the activation of mitogen-activated protein kinases (MAPK) and the upstream signalling cascade in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to arsenite or arsenate. Western blot analysis was performed to determine the activation of extracellular signal-regulated kinases (ERK) 1/2, c-jun N-terminal kinases (JNK), p38, and MAPK or ERK kinases (MEK) 1/2. Epidermal growth factor receptor (EGFR) tyrosine phosphorylation and recruitment of its adaptor proteins, Shc and Grb2, to EGFR were detected by immunoprecipitation and Western blot analysis. RESULTS: Both arsenicals activated ERK1/2, which are most highly activated in response to mitogenic stimulation, in addition to JNK and p38, which show greater activation in response to cellular stresses. The kinetics of ERK1/2 activation differed from those of JNK and p38 activation. Both arsenicals transiently activated ERK1/2 prior to JNK and p38 activation. MEK1/2, upstream kinases of ERK1/2, were also activated by arsenicals with similar time kinetics to that of ERK1/2 activation. To investigate a signalling pathway leading to activation of MEK1/2-ERK1/2, we examined the tyrosine phosphorylation of EGFR and Shc adapter protein. Both arsenicals stimulated tyrosine phosphorylation of EGFR and Shc. After arsenical treatment, Shc immunoprecipitates contained coprecipitated EGFR and Grb2, suggesting that both arsenicals induce the assembly of EGFR-Shc-Grb2 complexes. Both the EGFR inhibitor tyrphostin AG1478 and anti-EGFR blocking antibody markedly attenuated ERK1/2 activation induced by arsenicals, but did not affect JNK and p38 activation. CONCLUSIONS: Our data indicate that both arsenite and arsenate activate the EGFR-Shc-Grb2-MEK1/2-ERK1/2 signalling cascade in NHEK.     

蛋白激酶B和丝裂原活化蛋白激酶在烟酸保护的HaCaT细胞抵抗中波紫外线损伤中的作用

目的 探讨烟酸保护由UVB诱导的角质形成细胞损伤的胞内信号传导分子机制。 方法 UVB照射和烟酸处理HaCaT细胞,TUNEL法检测细胞凋亡,Western印迹检测蛋白激酶B（Akt）/丝裂原活化蛋白激酶（MAPK）通路相关蛋白Akt、P38、JNK、ERK1/2的磷酸化水平变化。ELISA检测细胞分泌内皮素1（ET-1）及碱性成纤维细胞生长因子（bFGF）的水平。结果 Western印迹结果表明,UVB照射和烟酸处理HaCaT细胞后,p-Akt、p-P38、p-JNK、p-ERK1/2蛋白在60 min内都显著激活（P < 0.01）。烟酸预处理后的HaCaT细胞再经UVB照射,可以发现p-Akt、p-P38、p-ERK1/2信号分子在2 h内激活更显著（P < 0.01）。３个抑制剂加UVB照射组较3个单独抑制剂组ET-1、bFGF表达降低,差异均有统计学意义,其中LY294002组、SB203580组ET-1、bFGF水平最低;烟酸预保护的抑制剂处理组HaCaT细胞在UVB照射后,LY294002和U0126组没有出现ET-1、bFGF水平回升,SB203580组bFGF水平出现回升。结论　Akt信号分子在烟酸保护的HaCaT细胞抵抗UVB损伤中起一定的调控作用。     

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm(2)) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min--2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Ultraviolet B irradiation increases keratin 1 and keratin 10 expressions in HaCaT keratinocytes via TRPV1 activation and ERK phosphorylation

In this study, we characterized the effect of ultraviolet B (UVB) irradiation with or without epidermal growth factor (EGF) on the regulation of keratinocyte differentiation under physiological concentration of Ca²⁺ (1.8 mM). In addition, growth factor deprivation used to measure signal transduction and kinase phosphorylation in many studies is physiologically unreal. Therefore, 1% of serum was also included in all experiment. We found that UVB irradiation Ca²⁺ dependently induced morphological differentiation and increased keratin 1 and 10 (K1/K10) expressions. Both were inhibited by treatment of cells with EGF. In quiescent cells, phosphorylation of ERK was stimulated by acute EGF treatment, while it rapidly desensitized in chronic EGF treatment or 1% serum exposure. UVB irradiation‐induced keratinocyte differentiation required Ca²⁺ influx through TRPV1. Ca²⁺‐dependent phosphorylation of ERK was responsible for the expression of K1/10. Cotreatment of cells with EGF during UVB irradiation inhibits the UVB irradiation‐induced differentiation by desensitizing ERK phosphorylation.     

Additive effect of heat on the UVB-induced tyrosinase activation and melanogenesis via ERK/p38/MITF pathway in human epidermal melanocytes

Heat is known as an environmental factor that causes significant skin pigmentation, but its effects on melanogenesis have been poorly studied. It has been shown that mitogen-activated protein kinase (MAPK) is involved in ultraviolet B (UVB) and stress-induced melanogenesis in melanocytes. In this study, we investigated the effects of heat and UVB, on melanocyte melanogenesis, differentiation, and MAPK phosphorylation. The results showed that heat (1 h at 40 °C for 5 days) increased cell dendrites, enlarged cell bodies, and induced extracellular signal-regulated kinases (ERK)/p38/MITF activation but did not influence melanogenesis of human epidermal melanocytes from skin phototype III. UVB irradiation (20 mJ/cm² for 5 days) induced melanogenesis and c-jun N-terminal kinases (JNK)/p38/MITF/tyrosinase activation in melanocytes from skin phototype III. UVB combined with heat resulted in much more significant tyrosinase activation and melanogenesis as compared with UVB alone in melanocytes from skin phototype III. Furthermore, heat treatment and UVB irradiation induced JNK, ERK, and p38 activation but not melanogenic and morphological changes in melanocytes from skin phototype I. These findings suggested that heat promoted melanocyte differentiation, probably via heat-induced ERK/p38/MITF/activation. Furthermore, heat had an additive effect on the UVB-induced tyrosinase activation and melanogenesis. These results provide a new clue for dermatologists for the treatment of hypopigmented skin disease with heat combined with UVB irradiation.     

Rottlerin: a multifaced regulator of keratinocyte cell cycle

Abstract:  In this study we showed that Rottlerin (also called Kamala or Mallotoxin), a natural product purified from Mallotus phillippinensis , is a potent suppressor of human keratinocytes (HaCaT cell line) proliferation. Following Rottlerin treatment, Thymidine incorporation into DNA and re-epithelialisation in a scratch wound model was decreased. At the molecular level, Rottlerin hampered the NFkB activation process, causing loss of cyclin D1 and promoting, in a PKCδ-dependent pathway, ERK activation, which, in turn induced the cell cycle inhibitor p21 Cip1/Kip1. The NFkB-dependent drop in cyclin D1, along with the PKCδ/ERK-dependent induction of p21 Cip1/Kip1, is responsible for growth arrest. These results open the way to further investigation on the Rottlerin therapeutic potential against keratinocyte hyper-proliferative disorders.     

Erythroid differentiation regulator 1 (Erdr1) is a proapototic factor in human keratinocytes

Skin is constantly exposed to physical and chemical stressors. The exposure of keratinocytes to ultraviolet B (UVB) irradiation causes epidermal damage via induction of apoptosis. Erythroid differentiation regulator 1 (Erdr1) modulates growth and survival of cells under various stressful conditions, but the function of Erdr1 in human keratinocyte apoptosis has not been investigated so far. Here, we investigated the effect of Erdr1 on UVB-induced apoptosis in human keratinocytes and also examined the underlying regulatory mechanism. First, Erdr1 expression was detected in human primary keratinocytes and normal human skin tissues. Expression of Erdr1 was enhanced in human keratinocytes following UVB irradiation. Knock-down of Erdr1 led to resistance to UVB-induced apoptosis. Also, Erdr1 overexpression increased UVB-induced apoptosis and induced caspase-3 activation. Furthermore, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 significantly reduced Erdr1 expression following UVB irradiation. These results indicate that UVB induces Erdr1 via a MAPK-dependent mechanism. Taken together, these findings suggest that Erdr1 has a role as a proapoptotic factor in human keratinocytes and acts via ERK and p38 MAPK pathways. Therefore, Erdr1 may be a potential therapeutic target to reduce apoptosis in keratinocytes in conditions such as psoriasis and skin cancer.     

Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1/2 and JNK pathways

Background Basic fibroblast growth factor (bFGF, FGF‐2) has been described as a multipotent cytokine that regulates cell growth as well as differentiation, matrix composition, chemotaxis, cell adhesion and migration in numerous cell types. It is known that bFGF stimulates proliferation of cultured fibroblasts. However, the detailed mechanism of fibroblast proliferation induced by bFGF in vitro still remains to be elucidated. Objectives We investigated the precise effects of bFGF on fibroblast proliferation and the signalling pathways responsible for bFGF‐induced proliferation in cultured human dermal fibroblasts (HDFs). Methods HDFs were cultured with bFGF in the presence or absence of specific inhibitors against MAPK signalling pathways including ERK, JNK and p38. The number of cells was counted and immunoblotting findings were examined for the activation of ERK1/2 and JNK. Furthermore, the inhibitory effects of ERK1, ERK2 and JNK1 were proven by the transfection of siRNA. Results bFGF increased the number of HDFs in a dose‐ and time‐dependent manner. The bFGF‐induced proliferation was suppressed by the MEK inhibitors PD98059 and U0126, and the JNK inhibitor SP600125. bFGF increased the phosphorylation levels of ERK1/2 and JNK1. Treatment with ERK1, ERK2 or JNK1 siRNA significantly inhibited bFGF‐induced proliferation. Conclusions This study indicates that ERK1/2 and JNK pathways play an important role in the bFGF‐mediated effect in HDFs. This study also suggests that controlling ERK1/2 and/or JNK signalling may therefore be a new therapeutic approach for the treatment of chronic and untreatable skin ulcers.     

P38 and JNK signal pathways are involved in the regulation of phlorizin against UVB‐induced skin damage

Phlorizin is well known to inhibit sodium/glucose cotransporters in the kidney and intestine for the treatment of diabetes, obesity and stress hyperglycaemia. However, the effects of phlorizin against ultraviolet B (UVB) irradiation and its molecular mechanism are still unknown. We examined the effects of phlorizin on skin keratinocyte apoptosis, reactive oxygen species (ROS) production, pro‐inflammatory responses after UVB irradiation and the changes of some signal molecules by in vitro and in vivo assay. We observed that phlorizin pretreatments inhibited HaCaT cell apoptosis and overproduction of ROS induced by UVB. Phlorizin also decreased the expression of UVB‐induced pro‐inflammatory cytokines, such as interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) at the mRNA level. Topical application of phlorizin on UVB‐exposed skin of nude mice prevented the formation of scaly skin and erythema, inhibited the increase of epidermal thickness and reduced acute inflammation infiltration in skin. Additionally, PCR, Western blot and immunohistochemical data showed that phlorizin reversed the overexpression of cyclooxygenase‐2 (Cox‐2) induced by UVB irradiation both in vitro and in vivo. The activation of p38 and JNK mitogen‐activated protein kinases (MAPK) after UVB irradiation was also inhibited by phlorizin. These findings suggest that phlorizin is effective in protecting skin against UVB‐induced skin damage by decreasing ROS overproduction, Cox‐2 expression and the subsequent excessive inflammation reactions. It seemed that p38 and JNK MAPK signal pathways are involved in the regulation of the protective function of phlorizin.     

UVB应激性衰老成纤维细胞分泌细胞外基质对HaCaT细胞ERK信号的影响

目的 探讨UVB诱导的衰老（UVB-SIPS）成纤维细胞分泌的细胞外基质（ECM）对HaCaT细胞增殖的影响以及细胞外信号调节激酶（ERK）信号途径的作用。方法 采用辐射诱导人皮肤成纤维细胞衰老,分别制备由未衰老和衰老成纤维细胞分泌的ECM包被的培养皿（分别定为PRE-ECM组、SCIP-ECM组）。以空白培养皿（NON-ECM组）为参照,在HaCaT细胞接种于培养皿后,采用MTT法和流式细胞法检测其细胞增殖、细胞周期等指标的变化;免疫印迹法分析ERK活化水平。干预性研究ERK信号通路对上述功能的影响。结果 三组中,SCIP-ECM组可诱导最强烈的ERK1/2的活化。采用U0126抑制ERK信号活化可完全抑制衰老成纤维细胞来源的ECM的促细胞增殖效应。阻断HaCaT细胞的ERK活化,NON-ECM组、PRE-ECM组和SCIP-ECM组S + G2/M期细胞的比例明显下降,分别由处理前37.40%、41.34%和43.31%减少至29.41%、36.48%到39.96%。结论 UVB-SCIP成纤维细胞分泌的ECM通过刺激ERK磷酸化促进HaCaT细胞增殖。     

中波紫外线对体外培养HaCaT细胞MAPK信号通路的影响

目的 探讨中波紫外线照射后不同时间点体外培养的HaCaT细胞MAPK信号通路受调控效应。 方法 1.5、4.5、7.5、10、20、30、50 mJ/cm2中波紫外线照射HaCaT细胞后8 h,对照组细胞除不进行UVB照射外,其他处理同各剂量UVB组,蛋白免疫印迹法测定ERK1/2、JNK和p38蛋白表达及磷酸化水平;50 mJ/cm2中波紫外线照射HaCaT细胞,蛋白免疫印迹法测定照射后2、4、8、12 h 4个时间点,细胞ERK1/2、JNK和p38蛋白表达及磷酸化水平。使用Quantity One软件计算目的条带吸光度,以相应蛋白条带的吸光度值与GAPDH蛋白内参条带吸光度值的比值表示相对蛋白含量。 结果 7.5、10、20、30、50 mJ/cm2 UVB照射HaCaT细胞后8 h,ERK1/2、JNK磷酸化水平明显上调（P < 0.05）,20、30、50 mJ/cm2 UVB照射HaCaT细胞后p38磷酸化水平上调显著（P < 0.05）,且都在50 mJ/cm2照射后效应最为显著（P < 0.05）。50 mJ/cm2 UVB照射HaCaT细胞后8 h,ERK1/2磷酸化产物水平出现上调,在处理后的12 h,与对照组（10.277 ± 0.320）比较,差异有统计学意义（44.844 ± 2.023,P < 0.05）,而p38和JNK磷酸化产物水平在照射后2 h即开始上调（P < 0.05）,4、8、12 h,p38和JNK与对照相比,UVB照射后虽存在着显著地磷酸化水平差异（P < 0.05）,但随时间推移JNK磷酸化产物水平这种上调的趋势逐渐弱化（P < 0.05）。 结论 在50 mJ/cm2中波紫外线照射后的HaCaT细胞中,ERK1/2、p38、JNK这3种MAPK信号通路产生了活化效应,具有一定的时间效应差异。     

Activation of PKCδ and p38δ MAPK during okadaic acid dependent keratinocyte apoptosis

There is substantial interest in identifying agents that differentially activate keratinocyte differentiation versus apoptosis. Okadaic acid (OA) is a tumor promoter in mouse skin that also stimulates apoptosis of murine keratinocytes. OA also enhances human keratinocyte differentiation; however, the impact of OA treatment on apoptosis in these cells has not been examined. We show that OA promotes normal human keratinocyte apoptosis as evidenced by increased accumulation of cells having sub-G1/S DNA content, decreased mitochondrial integrity, increased annexin V binding, increased cytoplasmic cytochrome c level, and increased procaspase 3 and PARP cleavage. Cyclin A, cyclin D1, cdk2, cdk4, p53 and p21 levels are reduced. These changes are associated with release of the PKCδ catalytic domain and increased phosphorylation of PKCδ-T₅₀₅-responses consistent with PKCδ activation. In contrast, phosphorylation of PKCδ-Y₃₁₁ is not increased. The apoptotic response is enhanced in OA treated cells in the presence of p38δ, a PKCδ target. OA treatment selectively activated p38δ, and OA-dependent apoptosis is not inhibited by treatment with the p38α/β inhibitor, SB203580. These findings are consistent with the idea that the response is mediated by p38δ. Our data indicate that OA is an agent that regulates both keratinocyte differentiation and apoptosis, and that this regulation is mediated via activation of a PKCδ/p38δ signaling cascade.     

Baicalein increases keratin 1 and 10 expression in HaCaT keratinocytes via TRPV4 receptor activation

In this study, we characterized the effect of baicalein on the regulation of keratinocyte differentiation and proliferation, which are abnormal in atopic dermatitis or psoriasis. Treatment of HaCaT keratinocytes with 10 μm baicalein slightly inhibited cell growth, caused morphological differentiation and increased expression of keratins 1 and 10 (K1/K10) without affecting ROS generation, cytochrome c release or apoptosis. Baicalein treatment caused growth arrest in G₀/G₁ phase and also induced Ca²⁺ influx via TRPV4 receptor activation. Phosphorylation of ERK, Akt and p38 MAPK, but not JNK, was increased by baicalein, and inhibition of phosphorylation of ERK, but not that of Akt or p38 MAPK, blocked the baicalein‐induced increase in K1/K10 expression, suggesting that ERK activation is involved in this increase. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ influx by pharmacological inhibition or silencing of the TRPV4 receptor did not affect growth arrest, ROS generation or apoptosis, but inhibited baicalein‐induced ERK phosphorylation and K1/K10 expression. Thus, baicalein treatment increases differentiation, and decreases proliferation, of keratinocytes. The mechanism of differentiation of keratinocytes is distinct from that of proliferation, the former being Ca²⁺ dependent and the latter Ca²⁺ independent.     

p38 Mitogen-Activated protein kinase mediates dual role of ultraviolet B radiation in induction of maturation and apoptosis of monocyte-derived dendritic cells

Although ultraviolet B (UVB) induces apoptosis and functional perturbations in dendritic cells (DC), for example, Langerhans cells (LC), it also stimulates some LC into maturation after irradiation in vivo. To analyze its reciprocal effects on DC, we elucidated the direct effect of UVB on DC in vitro using human monocyte-derived DC (MoDC). UVB from 50 to 200 J per m2 stimulated the maturation of MoDC with (1) augmented expression of CD86 and HLA-DR, (2) enhanced production of IL-1beta, IL-6, IL-8, and TNF-alpha at both the mRNA and protein levels, and (3) enhanced allostimulatory capacity on a per-cell basis, whereas the exceeded doses induced apoptotic cell death. Western-blot analysis of MoDC after UVB demonstrated a concentration-dependent phosphorylation of p38- and c-JUN N-terminal kinase (JNK)-mitogen-activated protein kinases (MAPK), but not that of extracellular signal-regulated kinases. p38 MAPK-inhibitor, SB203580, inhibited both UVB-induced maturation and apoptosis of MoDC. Interestingly, MoDC that had undergone apoptosis exhibited an augmented expression of HLA-DR without upregulation of CD86 antigen, suggesting their tolerogenic phenotype. Thus, our study revealed a dual effect of UVB, to stimulate maturation or to induce apoptosis in MoDC, depending on the dosage, via p38 MAPK pathway.