Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play important roles in mitogenesis and chemotaxis of endothelial cells. In normal human skin, VEGF is expressed and secreted by epidermal keratinocytes. Emerging data suggest that keratinocyte-derived VEGF targets other cell types besides the dermal endothelial cells. We have recently showed that keratinocytes from human normal skin expressed all five known VEGF receptors and co-receptors (neuropilin 1 and 2). To define the functional significance of VEGFR-2 in epidermis, we examined its role in a keratinocyte cell line, HaCaT cells, in response to VEGF treatment. Expression of VEGFR-2 on HaCaT cells was confirmed at both RNA and protein levels and was regulated by VEGF165 treatment. Treatment of HaCaT cells with VEGF165 induced tyrosine-autophosphorylation of VEGFR-2 and phosphorylation of PLC-gamma and p44/42 MAPK in a time-dependent manner. Preincubation with a neutralizing antibody for VEGFR-2 (MAB3571) completely abrogated these phosphorylation effects. Furthermore, VEGF165 stimulated proliferation and migration of HaCaT cells, and this effect was significantly blocked by a pretreatment with MAB3571. Neutralizing VEGFR-2 in HaCaT cells increased cell adhesion during culture. Our results suggest that VEGFR-2 expressed on HaCaT cells plays a crucial role in VEGF-mediated regulation of cell activity.     

Polypeptides from the Skin of Rana chensinensis Exert the Antioxidant and Antiapoptotic Activities on HaCaT Cells

Studies have shown that frog skin secretes many types of peptides that are good for human skin. In this study, acid and enzymatic extracts of Rana skin peptides (acid/enzymatic Rana skin peptides, ARPs/ERPs) were obtained. The chemical and physical properties of the ARPs and ERPs were identified through UV scanning, HGLC, FRIT, and MS. MTS and flow cytometry were used to test the proproliferative and antiapoptotic effects of the ARPs and ERPs on human immortalized keratinocytes (HaCaT cells). To elucidate the antiapoptotic mechanisms, the mRNA and protein levels of EGF (epidermal growth factor, which enhances stimulation of cellular proliferation in both cells and epithelial tissues) and caspase-3 were evaluated using quantitative RT-PCR. The results indicated that the ARPs and ERPs were extracted from the Rana skin with yields of 0.65% and 0.52%, respectively. Treatment with ARPs (1.6 g/L) and ERPs (0.8 g/L) showed a 1.66-fold (p < 0.001) and 2.1-fold (p < 0.001) enhancement in the proliferation rates of HaCaT cells. The rate of apoptosis decreased by 2.6 fold (p < 0.01) and 3.4 fold (p < 0.01) under the UVB stimulation, respectively, at the same time, the up-regulation of EGF and down-regulation of caspase-3 were found. These results suggested that we can dig into the potential value of ARPs/ERPs in a new field.     

A unified mitochondria mechanistic target of rapamycin acyl-coenzyme A dehydrogenase 10 signal relay modulation for metformin growth inhibition in human immortalized keratinocytes cells

Metformin exhibits antiproliferative and proapoptotic effects in a variety of diseases, characterized by malignant and nonmalignant hyperplastic cells; however, the underlying molecular mechanism of metformin in psoriasis has not been elucidated. In the current study, we found that after metformin treatment the proliferation of human immortalized keratinocytes (HaCaT) was significantly inhibited, while cell apoptosis was increased in a dose-dependent manner, accompanied with enhanced protein expression of acyl-coenzyme A dehydrogenase 10 (ACAD10). Furthermore, mechanism analysis revealed that ACAD10 expression is induced by downregulated activities of mechanistic target of rapamycin 1 (mTORC1) signaling rather than AMP-activated protein kinase signaling. The inactivation of mTORC1 by rapamycin pretreatment or rotenone-induced mitochondrial complex inhibition showed a similar effect because of the metformin treatment on the proliferation and apoptosis of HaCaT keratinocytes. Overexpression of mTORC1 almost reversed the antiproliferation and proapoptosis effects induced by metformin. This study showed that the metformin treatment inhibited HaCaT cells proliferation and promoted apoptosis by affecting the mitochondrial-mTORC1 signaling and elevated the ACAD10 expression. Hence, metformin can be used as a potential therapeutic agent for psoriasis.     

hnRNP A2/B1在人永生化表皮HaCaT细胞凋亡过程中的表达及其调控作用研究

该文以姜黄素诱导人永生化表皮HaCaT细胞凋亡为基础,对hnRNP A2/B1在核基质中的存在、分布及其与细胞凋亡相关基因产物的共定位及相互作用关系进行了研究。蛋白质印迹结果显示,hnRNP A2/B1存在于HaCaT细胞核基质蛋白组分中,在经过姜黄素处理后,表达下调;激光共聚焦显微镜观察显示,hnRNP A2/B1在HaCaT细胞中分别与Fas、p53和Bax等基因产物具有共定位关系,姜黄素处理后其共定位区域出现由核膜或核仁向胞质转移的趋势。GST pull-down实验证实,hnRNPA2/B1分别与Fas、p53和Bax有直接相互作用关系。结果表明,hnRNPA2/B1作为一种核基质蛋白,通过与细胞凋亡相关基因产物的相互作用参与HaCaT细胞的凋亡诱导调控过程,这对深入认识核基质蛋白在细胞凋亡过程中的调控机制具有重要意义。     

Short-time UVA exposure to human keratinocytes instigated polyunsaturated fatty acid without inducing lipid peroxidation

Short-term exposure to ultraviolet A (UVA) radiation can directly injure our skin through inflammatory response and indirectly through oxidative stress, triggering polyunsaturated fatty acid (PUFA) peroxidation in skin cell membrane and formation of DNA adduct, 8-hydroxy-2′-deoxyguanosine (8-OHdG). It is known that UVA exposure leads to photoaging, immunosuppression and skin cancer. However, the changes in PUFA and its oxidized metabolites, and cell cycle after short UVA exposure, are debatable. In this study, human keratinocytes (HaCaT) were exposed to low dose (5?J/cm²) and high dose (20 J/cm²) of UVA and assessed immediately, 8?h, 12?h, and 24?h post-treatment. Both doses showed a transient suppression in S-phase after 8?h of UVA exposure, and G₂/M phase arrest after 12-h UVA exposure in the cell cycle but subsequently returned to normal cycle. Also, no observable DNA damage took place, where 8-OHdG levels were below par after 24-h UVA exposure. A dose of 20 J/cm² UVA stimulated significant amount of arachidonic acid, n-3 docosapentaenoic acid, and docosahexaenoic acid (DHA) but lowered adrenic acid and eicospentaenoic acid after 24-h exposure. Among the 43 oxidized PUFA products determined, enzyme-dependent oxidized PUFAs, namely, 14-hydroxy-DHA (HDoHE) level reduced, and 8- and 13-HDoHE levels elevated significantly in a linear trend with post-treatment time. Out of the nonenzymatic oxidized PUFAs, a significant linear trend with post-treatment time was shown on the reduction of 5-F_2t-Isoprostane (IsoP), 15-F_2t-IsoP, Isofurans, 5-F_3t-IsoP, Neurofurans, and 20-HDoHE. Our observations indicate oxidative stress through short UVA exposure on human keratinocytes did not have detrimental consequences.     

Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.     

Orally active antioxidative copper(II) aspirinate: synthesis, structure characterization, superoxide scavenging activity, and in vitro and in vivo antioxidative evaluations

Ever since it was proposed that reactive oxygen species (ROS) are involved in the pathogeneses of various diseases, superoxide dismutase (SOD)-mimetic complexes have been intensively studied. We prepared copper(II) aspirinate [Cu₂(asp)₄] from Cu(II) and aspirin, which has been in use for many years as an antipyretic, an analgesic, and an anti-inflammatory agent. However, Cu₂(asp)₄ has been found to have additional activities, including anti-inflammatory, antiulcer, anti-ischemic/reperfusion agent, anticancer, antimutagenic, and antimicrobial activities. The activity of copper salicylate [Cu(sal)₂] was also compared with that of Cu₂(asp)₄. The structure of the Cu₂(asp)₄ was determined using X-ray structure analysis. Its SOD-mimetic activity was determined using cytochrome c, electron spin resonance (ESR) spectroscopy, and ESR spin trap methods. The activity of Cu₂(asp)₄ was slightly greater than CuSO₄ and copper acetate [Cu(ace)₂] and slightly less than that of Cu(sal)₂. The in vitro antioxidant activity, evaluated in human epithelial or transformed neoplastic keratinocyte cells, HaCaT, and normal dermal fibroblasts in terms of cell survival following ultraviolet B (UVB) irradiation, was significantly increased in the presence of Cu₂(asp)₄, Cu(sal)₂, and CuSO₄. Further, ROS generation following UVA irradiation in the skin of hairless mice following oral treatment with Cu₂(asp)₄ for three consecutive days was significantly suppressed compared to the vehicle- or Cu(ace)₂-treated mice. On the basis of these results, Cu₂(asp)₄ was observed to be a potent antioxidative compound possessing antioxidative activity in biological systems. In conclusion, Cu₂(asp)₄ is a potent antioxidative agent that may be useful for future treatment of diseases resulting from ROS.     

Probing TGF-β1-induced cytoskeletal rearrangement by fluorescent-labeled silica nanoparticle uptake assay

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial–mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor β1 (TGF-β1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-β1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-β1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-β1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.