Abstract
Prolyl-4-hydroxylase alpha(I) (P4Halpha(I)) is the rate-limiting subunit for P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we have characterized the human P4Halpha(I) promoter for transcription factors and DNA elements regulating P4Halpha(I) expression. Using a progressive deletion cloning approach, we have constructed pGL3-P4Halpha(I) recombinant plasmids. We have identified a positive regulatory region at the positions of bp -184 to -97 responsible for approximately 80% of the P4Halpha(I) promoter efficiency. Three E-boxes were located within this region, and the E-box at position bp -135 explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using chromatin immunoprecipitation assay. Suppression of USF1 and/or USF2 using specific short interference RNA resulted in a significant reduction in P4Halpha(I) promoter activity, and overexpressed USF1 or USF2 increased P4Halpha(I) promoter activity significantly. Although transforming growth factor beta1 increased the USF1/USF2-E-box binding and P4Halpha(I) promoter activity, this up-regulatory effect can be largely prevented by USF1/USF2-specific short interference RNA. On the other hand, cigarette smoking extracts, which have been shown to suppress P4Halpha(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4Halpha(I) promoter activity. Furthermore, the E-box on the P4Halpha(I) promoter appeared to indiscriminately bind with either USF1 or USF2, with a similar outcome on the promoter efficiency. In conclusion, our study shows that USF1/USF2 plays a critical role in basal P4Halpha(I) expression, and both positive (transforming growth factor beta1) and negative (cigarette smoking extract) regulators appear to influence the USF-E-box interaction and affect P4Halpha(I) expression.
Highlights
Dation of mainly collagens and elastins, which are in turn regulated by other molecules, e.g. fibrillin
A deletion up to bp Ϫ97 resulted in an ϳ80% decrease in the transcription activity. These results suggest that the basal transcription of the prolyl 4-hydroxylase (P4H)␣(I) gene was positively regulated by a region located between bp Ϫ184 and Ϫ97
These results suggest that the transcription of the P4H␣(I) gene was positively regulated by the USF1/USF2, either one of which would be efficient for P4H␣(I) expression
Summary
Dation of mainly collagens and elastins, which are in turn regulated by other molecules, e.g. fibrillin. P4H is an intracellular enzyme required for the synthesis and formation of all known types of collagen It catalyzes the formation of hydroxyproline from proline residues located in repeating Xaa-ProGly triplets in the procollagens during post-translational processing and is the process essential for folding the procollagen polypeptide chains into stable triple helical molecules [6]. Relatively little data is available for factors affecting P4H expression and activity, P4H may be regulated by nitric oxide, tumor necrosis factor ␣, transforming growth factor  (TGF), insulin-like growth factor 1,  fibroblast growth factor, certain cytokines, and cigarette smoking [15,16,17,18,19,20,21,22] Environmental stimuli, such as hypoxia, can induce P4H␣(I) expression at both transcriptional and post-transcriptional levels, which results in excess accumulation of collagen, a process relevant to fibrosis [23, 24]. Ϫ580 to Ϫ560 Ϫ480 to Ϫ457 Ϫ417 to Ϫ394 Ϫ320 to Ϫ298 Ϫ271 to Ϫ251 Ϫ184 to Ϫ164 Ϫ145 to Ϫ125
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
