Biomedical sciences instrumentation · 2010
The time course of transcriptional regulation in anoxic fibroblasts: implications for carpal tunnel syndrome - biomed 2010.
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Pierce Christine W, Tucci Michelle A, Benghuzzi Hamed A
Аннотация
UNLABELLED: Hypoxia is the initial cellular insult in carpal tunnel syndrome. While much is known about the gross and histological progression of this prevalent disease, little is currently known about its molecular basis. Additionally, some debate presently exists regarding whether the disease occurs as the result of an inflammatory or a fibrotic pathology.
OBJECTIVE: The objective of these investigations was to determine the transcriptional and translational regulation in MRHF fibroblasts subjected to an hypoxic culture environment with respect to HIF-1a and TGF-ss1 mRNA and protein expression.
METHODS: MRHF fibroblasts were cultured under anoxic conditions for various durations up to 72 hours alongside normoxic controls. HIF-1a and TGF-ss1 mRNA levels relative to baseline were determined by real-time PCR. GAP-DH was used as the reference standard for normalization and relative quantification of fold-differences in mRNA expression was determined by the comparative (2[delta][delta]Ct) CT method. Relative differences in protein levels were determined by Western blotting for both HIF-1a and TGF- ss1.
RESULTS: Transcriptional and translational up-regulation of both HIF-1a and TGF-ss1 were observed in response to hypoxic culture conditions. Peak mRNA expression was observed after 15 min of anoxic exposure for both HIF-1a (7.3-fold increase) and TGF-ss1 (3.3-fold increase). In contrast, peak protein levels were observed much later in the course of hypoxia with HIF-1a and TGF-ss1 both demonstrating a significant increase over baseline levels after 2 hours.
CONCLUSION: Both HIF-1a and TGF-ss1 have been shown to regulate the expression of connective tissue growth factor (CTGF). The pronounced and sustained up-regulation of HIF-1a suggests that CTGF expression in hypoxic carpal tissues is predominantly under the early regulatory influence of HIF-1a.
OBJECTIVE: The objective of these investigations was to determine the transcriptional and translational regulation in MRHF fibroblasts subjected to an hypoxic culture environment with respect to HIF-1a and TGF-ss1 mRNA and protein expression.
METHODS: MRHF fibroblasts were cultured under anoxic conditions for various durations up to 72 hours alongside normoxic controls. HIF-1a and TGF-ss1 mRNA levels relative to baseline were determined by real-time PCR. GAP-DH was used as the reference standard for normalization and relative quantification of fold-differences in mRNA expression was determined by the comparative (2[delta][delta]Ct) CT method. Relative differences in protein levels were determined by Western blotting for both HIF-1a and TGF- ss1.
RESULTS: Transcriptional and translational up-regulation of both HIF-1a and TGF-ss1 were observed in response to hypoxic culture conditions. Peak mRNA expression was observed after 15 min of anoxic exposure for both HIF-1a (7.3-fold increase) and TGF-ss1 (3.3-fold increase). In contrast, peak protein levels were observed much later in the course of hypoxia with HIF-1a and TGF-ss1 both demonstrating a significant increase over baseline levels after 2 hours.
CONCLUSION: Both HIF-1a and TGF-ss1 have been shown to regulate the expression of connective tissue growth factor (CTGF). The pronounced and sustained up-regulation of HIF-1a suggests that CTGF expression in hypoxic carpal tissues is predominantly under the early regulatory influence of HIF-1a.