Study of transcriptional effects of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line.
ABSTRACT: Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks. HaCaT were obtained from Cell Lines Service (Eppelheim, Germany) and grown in DMEM medium (PAN biotech, Aidenbach, Germany) supplemented with 10% FBS (Life Technology, Grand Island, NY, USA), L-glutamine and non-essential amino acid. Before the treatment HaCaT cells were cultured in serum-free medium for 12hrs. HaCaT were treated with MSU (1mg/ml) vs DMEM control for 12hrs then submitted to RNA extration and gene expression profiling. Triplicate experiments were performed: HaCaT control (n=3), MSU-treated (n=3).
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks. Overall design: HaCaT were obtained from Cell Lines Service (Eppelheim, Germany) and grown in DMEM medium (PAN biotech, Aidenbach, Germany) supplemented with 10% FBS (Life Technology, Grand Island, NY, USA), L-glutamine and non-essential amino acid. Before the treatment HaCaT cells were cultured in serum-free medium for 12hrs. HaCaT were treated with MSU (1mg/ml) vs DMEM control for 12hrs then submitted to RNA extration and gene expression profiling. Triplicate experiments were performed: HaCaT control (n=3), MSU-treated (n=3).
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5µg/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide Two experimental groups: 1. The treatment group was sub-cultured up to passage 22 to establish a chronic exposure state. 2. The passage control group was also sub-cultured up to 22 passages but with no exposure to arsenic trioxide. 4 technical replicates with 3 replicates making a total of 8X3 =24 samples HaCat Cell untreated (passage control): 1. H1_H001, H1_H002, H1_H003 2. H2_ H004, H2_H005, H2_H006 3. H3_ H007, H3_H008, H3_H009 4. H4_ H010, H4_H011, H4_H012 HaCat Cell treated with 0.5µg/ml of arsenic trioxide: 5. A1_H013, A1_H014, A1_H015 6. A2_H016, A2_H017, A2_H018 7. A3_H019, A3_H020, A3_H021 8. A4_H022, A4_H023, A4_H024 Cell Type: Human Skin Keratinocyte: 1.5 ×105 HaCaT cells were cultured in 7.5 ml of complete DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin, streptomycin in T-25 culture plate. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 ºC. The treatment groups were exposed to 0.5µg/mL As2O3 (equivalent to LC 0.5), and passaged at 90% confluent. Total RNA was extracted from 4 technical replicates of unexposed HaCaT cells and HaCaT cells chronically exposed to arsenic trioxide up to passage 22 using RNA STAT-60 (TEL-TEST, INC, Friendswood, TX, USA).
Project description:Oxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in a variety of human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT keratinocyte cell culture model, we here aimed at investigating the cellular and transcriptional response to periodic, low dose oxidative challenge over three months. Chronic redox stress was generated by frequent incubation with cold physical plasma treated cell culture medium. Using mRNA microarray technology, we found both acute ROS stress responses as well as numerous adaptions on the transcriptional level and over several weeks of redox challenge. This included an altered expression of 260 genes that function in inflammation and redox homeostasis, such as, signaling molecules, cytokines, and anti-oxidant enzymes. Apoptotic signaling was affected to a minor extend, especially in p53 down-stream targets. Strikingly, the anti-apoptotic heat shock protein HSP27 was strongly upregulated. These results suggest a variety of adaptive responses relating involved a number of cellular processes elicited by frequent redox stress over several months. They may help to better understand inflammatory responses in redox related diseases and possibly allow uncovering new biomarkers of ROS-stress. Microarrays were used to analyze and investigate the biological effects of repeated exposure of cold physical plasma on human HaCaT keratinocytes. Using an argon plasma jet kinpen, regulated transcripts were analyzed and further described in Schmidt et al. (submittes): “Long-term exposure to cold plasma-generated ROS –an in vitro model for redox-related diseases of the skin”. HaCaT keratinocytes exposed to plasma treated medium - time course
Project description:Transcriptional profiling in HACAT cells using a whole human genome array; HACAT cells treated with si RNA against Keap 1 or a scrambled si RNA sequence (Scram) vs HACAT cells mock transfected with lipofectamine (reference control) Experiment Overall Design: 2 biological replicates, 2 technical (dye swap) replicates per treatment.
Project description:Constitutive activation of EGFR- and NF-kB-dependent pathways is a hallmark of cancer, yet signaling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced upon human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signaling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signaling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial-mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-kB activity that transmits pro-survival and invasive signals through EGFR signaling. We used microarrays to detail the global programme of gene expression upon BCL-3 overexpression We used two experimental conditions, namely HaCat cells infected with a control lentivirus as well as HaCat cells infected with a BCL-3 expressing construct. Both experimental conditions were in triplicates.
Project description:We employed human HaCaT cells as a model system to identify cellular proteins that accompany SDS-induced toxicity based on a proteomic approach. HaCaT human keratinocyte cell line were treated with a non-cytotoxic dose of SDS (25 µg/ml, as determined by the MTT assay and microscopically examination) for 48 h. The altered abundance of proteins from HaCaT keratinocytes exposed to SDS was analyzed by LC-MS/MS approach and quantified using Progenesis LC software. The abundance of 217 proteins (which were identified by multiple peptides, ≥ 2) was altered in keratinocytes exposed to SDS; in which 131 proteins had increased abundance while 86 proteins was down regulated. The Pathview map of 131 up-regulated proteins was built and enhancement of glycolysis/gluconeogenesis was found.
Project description:Proprionibacterium acnes is a Gram positive bacterium found ubiquitously on human skin, where it is typically considered to assume a commensal relationship with its host. However, it is also closely associated with the skin condition acne vulgaris. More controversially, it has a postulated involvement in infections of implanted prosthetic devices and has been isolated from malignant prostate tissues. The role of P. acnes in these pathologies remains undetermined, although both bacterial and host factors are implicated. By microarray analysis, we have identified fundamental differences in the global transcriptional profiles of keratinocyte and prostate cells to P. acnes infection. Notably, P. acnes infection of the keratinocyte cell line, HaCaT, elicited a robust, but acute inflammatory response. By contrast, the inflammatory response of the prostate cell line, RWPE1, was delayed and persisted for longer times. Immunofluorescence and electron microscopy revealed higher numbers of internalized P. acnes bacteria in RWPE1 cells, which could still be detected intracellularly three weeks post infection. This contrasted with HaCaT cells, which P. acnes largely failed to invade. Moreover, P. acnes induced a delayed, sustained activation of NFκB in RWPE1 cells, which was absent in HaCaT cells. Further characterization of the host-cell response to infection revealed that the intermediate filament protein, vimentin, was a key determinant of P. acnes invasion, persistence and inflammatory profile in RWPE1 cells. siRNA mediated knock down of vimentin in RWPE1 cells attenuated bacterial invasion and the inflammatory response to infection; similarly, overexpression of vimentin in HaCaT cells increased bacterial invasion. We conclude that vimentin-dependent host-tissue tropism, in part, determines P. acnes invasion and inflammatory capacity. This could contribute to P. acnes pathology at non-skin infection sites. Microarray experiments were performed as dual-color hybridizations. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color-swap dye-reversal was performed.