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Effects of streptolysin o on extracellular matrix gene expression in normal human epidermal keratinocytes.

ABSTRACT: ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05's mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hours, followed by RNA extraction and cRNA production. Gene expression profiling utilized microarrays containing nucleic acid probes for 113 extracellular matrix (ECM) genes. Microarrays yielded 6 upregulated and 4 downregulated genes with ?2-fold changes and p<0.05 in t-tests. Quantitative real-time polymerase chain reactions (qPCR) were used to verify gene regulation. Upregulated genes of interest were VCAN (formerly CSPG2, encoding versican), CD44 (encoding hyaluronan receptor), ICAM1 (encoding intercellular adhesion molecule-1) and CTGF (encoding connective tissue growth factor). All four upregulated genes encode proteins involved in promoting keratinocyte migration and proliferation. Downregulated genes of interest were MMP9 (encoding matrix metalloproteinase 9) and SPP1 (encoding osteopontin). ML-05 may enhance wound healing through the expression of specific genes encoding proteins capable of promoting keratinocyte migration, proliferation, and other activities related to maintaining ECM structure and function.

SUBMITTER: Mamber SW

PROVIDER: S-EPMC3315172 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Effects of streptolysin o on extracellular matrix gene expression in normal human epidermal keratinocytes.

Mamber Stephen W SW Gurel Volkan V Rhodes Ryan G RG McMichael John J

Dose-response : a publication of International Hormesis Society 20110314 4

ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05's mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hou ...[more]

PMID: 22461761

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Project description:There is limited information on region-specific gene expression in the human corneal stroma. In this study, we aimed to investigate the expression profile of the extracellular matrix and adhesion molecules in the normal corneal stroma using laser capture microdissection (LCM) and molecular techniques.Frozen sections of human cornea without ocular disease were used to isolate the central and peripheral corneal stromal keratocytes by LCM. RNA was extracted from LCM-captured tissues and the RT2 Profiler PCR Arrays were used to examine the expression profile of extracellular matrix and adhesion molecules in the central and peripheral stroma. Real-time quantitative PCR was used to quantify gene expression. Proteomic and western blotting (WB) analyses were performed to confirm gene expression at protein level. Function association network was generated via the web tools String and Cytoscape.The gene expression profiling demonstrated that 35 out of the 84 extracellular matrix and adhesion molecules represented in the array were expressed in stromal keratocytes. Among them, 24 genes were not previously described in the corneal stroma. Two genes were found more abundantly expressed in the central stroma than in the periphery: TGFBI, COL6A2 (p < 0.05). ADAMTS13 was detected only in the central stroma. Proteomics and WB analysis confirmed the expression of 10 genes. Functional analysis revealed that most identified genes were presented in a core cluster that had multiple and strong associations with other genes.This study identified genes not previously described in the corneal stroma, revealed regional differences in gene expression between central and peripheral stroma, and also detected some interesting candidate genes that may play important roles in corneal function. These observations serve as the foundation to further investigate the molecular and cellular mechanisms regulating the pathogenesis of regional corneal stromal disorders such as keratoconus.

Project description:BACKGROUND: Epidemiological data show that the incidence of carcinomas in humans is highly dependent on age. However, the initial steps of the age-related molecular oncogenic processes by which the switch towards the neoplastic state occurs remain poorly understood, mostly due to the absence of powerful models. In a previous study, we showed that normal human epidermal keratinocytes (NHEKs) spontaneously and systematically escape from senescence to give rise to pre-neoplastic emerging cells. METHODS: Here, this model was used to analyze the gene expression profile associated with the early steps of age-related cell transformation. We compared the gene expression profiles of growing or senescent NHEKs to post-senescent emerging cells. Data analyses were performed by using the linear modeling features of the limma package, resulting in a two-sided t test or F-test based on moderated statistics. The p-values were adjusted for multiple testing by controlling the false discovery rate according to Benjamini Hochberg method.The common gene set resulting of differential gene expression profiles from these two comparisons revealed a post-senescence neoplastic emergence (PSNE) gene signature of 286 genes. RESULTS: About half of these genes were already reported as involved in cancer or premalignant skin diseases. However, bioinformatics analyses did not highlight inside this signature canonical cancer pathways but metabolic pathways, including in first line the metabolism of xenobiotics by cytochrome P450. In order to validate the relevance of this signature as a signature of pretransformation by senescence evasion, we invalidated two components of the metabolism of xenobiotics by cytochrome P450, AKR1C2 and AKR1C3. When performed at the beginning of the senescence plateau, this invalidation did not alter the senescent state itself but significantly decreased the frequency of PSNE. Conversely, overexpression of AKR1C2 but not AKR1C3 increased the frequency of PSNE. CONCLUSIONS: To our knowledge, this study is the first to identify reprogrammation of metabolic pathways in normal keratinocytes as a potential determinant of the switch from senescence to pre-transformation.

Dataset Information

Effects of streptolysin o on extracellular matrix gene expression in normal human epidermal keratinocytes.

Publications

Effects of streptolysin o on extracellular matrix gene expression in normal human epidermal keratinocytes.

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