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. 2012 Oct 26;287(44):37134-44.
doi: 10.1074/jbc.M112.352872. Epub 2012 Aug 28.

Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes

Ellinoora Aro  1 , Richa Khatri, Rita Gerard-O'Riley, Laura Mangiavini, Johanna Myllyharju, Ernestina Schipani
Affiliations

Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes

Ellinoora Aro et al. J Biol Chem. .

Abstract

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α(2)β(2) tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.

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Figures

FIGURE 1.
FIGURE 1.
RT-PCR of total RNA extracted from primary mouse epiphyseal growth plate chondrocytes cultured for 10 days under normoxic conditions. The products of amplification of mRNAs coding for the proα1 chain of type II collagen (Col2a1), aggrecan, vascular endothelial growth factor A (Vegfa), glucose transporter 1 (Glut1), and the C-P4H α(I) and α(II) subunits (P4ha1 and P4ha2, respectively) are shown.
FIGURE 2.
FIGURE 2.
Quantification of Hif-1a, Hif-2a, Vhl, P4ha1, P4ha2, Vegfa, Glut1, Epo, and Col2a1 mRNAs in control and HIF-1α (A), HIF-2α (B), and VHL (C) knock-down chondrocytes. Primary chondrocytes were isolated from growth plates of newborn Hif-1af/f, Hif-2af/f, and Vhlf/f mice, cultured in a monolayer and transduced on day 1 after plating with adenoviruses producing either β-galactosidase (black columns) or Cre recombinase (gray columns) to generate control and HIF-1α, HIF-2α, and VHL knock-down chondrocytes, respectively. On day 10 the cells were exposed to hypoxia for 8 h. Data are given as means ± S.D. (error bars; triplicates of one representative experiment are shown). Statistical differences were calculated as ***, p < 0.01; **, p < 0.05.
FIGURE 3.
FIGURE 3.
Quantification of the efficiency of deletion of the Hif-1a, Hif-2a, and Vhl genes by analysis of genomic DNA. Primary chondrocytes were isolated from growth plates of newborn Hif-1af/f, Hif-2af/f, and Vhlf/f mice, cultured in a monolayer and transduced on day 1 after plating with adenoviruses producing either β-galactosidase (black columns) or Cre recombinase (gray columns). Genomic DNA was isolated on day 10. Error bars, S.D. ***, p < 0.01.
FIGURE 4.
FIGURE 4.
Staining of recombinant human and mouse C-P4H-I and C-P4H-II tetramers with anti-human C-P4H α(I) and α(II) antibodies. A, protein lysates from insect cells expressing human (H) or mouse (M) C-P4H α(I) or α(II) subunits together with PDI/β were run on 8% PAGE under nondenaturing conditions followed by Coomassie Blue staining and subjected to C-P4H activity measurements. B, equal amounts of the human and mouse C-P4H-I and C-P4H-II were run on 8% PAGE under nondenaturing conditions and analyzed by Western blotting using an anti-human C-P4H α(I) or α(II) antibody. The arrows indicate C-P4H α2β2 tetramers (A) and individual isoenzymes (B). The lanes in A were regrouped from different parts of the same gel.
FIGURE 5.
FIGURE 5.
Analysis of C-P4H-I and C-P4H-II protein expression in control and Hif-1a (A), Hif-2a (B), and Vhl (C) floxed chondrocytes. The Hif-1af/f, Hif-2af/f, and Vhlf/f chondrocytes were cultured in a monolayer and transduced on day 1 after plating with adenoviruses producing either β-galactosidase or Cre recombinase. On day 10 the cells were exposed to 1% O2 for 8 or 24 h, or the culture was continued in normoxia. Total cell lysates were analyzed by 8% nondenaturing PAGE followed by Western blotting with antibodies against the C-P4H α(I) and α(II) subunits. The accumulation of HIF-1α (A and C) and HIF-2α (B) was studied by 8% SDS-PAGE followed by Western blotting with an antibody recognizing HIF-1α or HIF-2α, respectively. In the latter case the lysates from cells cultured in 1% O2 for 24–72 h were pooled for analysis. α-Tubulin or β-actin was used as a loading control. The lanes in the C-P4H-II and HIF-1α panel in C were regrouped from different parts of the same Western blot.
FIGURE 6.
FIGURE 6.
Analysis of C-P4H activity in Hif-1af/f, Hif-2af/f, and Vhlf/f chondrocytes transduced with adenoviruses producing either β-galactosidase (black columns) or Cre recombinase (gray columns). On day 10 after transduction the cells were exposed to 1% O2 for 8, 24, 48, and 72 h, or the culture was continued in normoxia. This C-P4H activity assay measures the formation of 4-hydroxy[14C]proline in a [14C]proline-labeled substrate consisting of nonhydroxylated procollagen polypeptide chains. Data are given as means ± S.D. (error bars; triplicates of at least three independent experiments). Statistical differences were calculated as ***, p < 0.01.
FIGURE 7.
FIGURE 7.
Analysis of the activity of purified recombinant human C-P4H-I in 21% O2 and 1% O2. The amount of enzyme increased 6-fold (6×) in the assays carried out in 1% O2 relative to that present in the reactions carried out in 21% O2. This C-P4H activity assay measures the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate.
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