Abstract
Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the alpha C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-alpha(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (approximately 64 kDa form) at the 5'- and 3'-untranslated regions (UTR) of the mRNA. The 5'/3'-UTR-dependent mechanism elevates the C-P4H-alpha(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5'-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the approximately 64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3'-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3'-UTR-binding protein, presumably annexin A2. The increased protein level of the approximately 64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-alpha(I) regulation under hypoxia.
Highlights
Position of the extracellular matrix, because collagens constitute the major compound of extracellular matrix proteins
Recent studies revealed that collagen prolyl 4-hydroxylase (C-P4H)-␣(I) mRNA may belong to a subclass of transcripts that are characterized by an increased translational efficiency under hypoxia [11, 12]
Level, and can be explained by a significant decrease of C-P4H-␣(I) mRNA concentration in the translationally inactive RNP fraction. These findings indicate a recruitment of C-P4H-␣(I) mRNAs into polysomes, which supports the view of an enhanced translational control under hypoxia
Summary
Human fibrosarcoma HT1080 (ATCC, passages 16 –21) cells were maintained in Dulbecco’s modified Eagle’s medium (high glucose; PAA Laboratories GmbH), supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 g/ml streptomycin, 15 mM Hepes, and 2 mmol/liter glutamine, at 37 °C, 5% CO2. Cells were maintained in a medium containing 0.4% fetal calf serum for at least 24 h. Measurements started with the application of fresh medium containing 0.4% fetal calf serum. For hypoxic conditions the cells were incubated in a hypoxic chamber (JOUAN IG750). Control cells were incubated under atmospheric oxygen conditions (21% O2, 5% CO2, 37 °C). For RNA and protein isolation, cells were washed with ice-cold phosphate-buffered saline. The RNA was capillary transferred to positively charged nylon membranes (Roche Diagnostics), visualized after ethidium bromide staining to document the relative level of 18 S and 28 S rRNA, and hybridized to digoxigenin-labeled partial C-P4H␣(I) antisense transcripts (1,600 nt, representative for the coding region). The detection was performed using the digoxigenin RNA Labeling Kit (Roche Diagnostics) according to the manufacturer’s protocol. Estimation of mRNA Stability mRNA stability assays were performed as described in Ref. 27
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