HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

Abstract

A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates β-adrenoceptors (β-ARs) which play a key role in the formation of pathogenic blood vessels. Among β-ARs, β3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced β3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of β3-AR transcriptional regulation by HIF-1. Investigation of β3-AR regulation over OIR progression revealed that the expression profile of β3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples β3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the β3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse β3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the β3-AR gene. The additional finding that β3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the β3-AR gene. Our results are indicative of β3-AR involvement in HIF-1-mediated response to hypoxia.

Keywords: ChIP-qPCR; HIF-1 binding site; computational analysis; gene expression; oxygen-induced retinopathy.

Figure 1

Effects of oxygen tension on retinal levels of HIF-1α, VEGF and β3-AR from PD7 to PD17. (A) Schematic diagram of the OIR model including DMOG administration daily from PD7 to PD12. (B) Representative blots showing protein levels of HIF-1α, VEGF and β3-AR as evaluated by Western blot in retinal extracts at different times from normoxic controls or OIR mice without or with DMOG administration. β-actin was used as the loading control. (C–E), Relative densitometric analyses of the protein levels of HIF-1α, VEGF and β3-AR. (F) Retinal mRNA levels of β3-AR at different times from controls or OIR mice untreated or treated with DMOG. * p < 0.05 vs. normoxic controls. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples.

Figure 2

Schematic representation of mouse and human β3-AR genes including their upstream sequences. (A) In the mouse gene, 5 exons (E1–E5; solid boxes) and 4 introns (dashed lines) are depicted. They potentially express up to 6 different alternative mRNAs of which 3 codify for the canonical β3-AR protein (yellow mRNAs) while the other 3 for an alternative β3-AR protein with a different C-terminal sequence (purple mRNAs). The putative transcription-start site (TSS) is indicated by the red arrow. The positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal HBS consensus sequence (underlined sequence 5′-ACGTG-3′). (B) In the human gene, the positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal consensus sequence (underlined sequence 5′-ACGT-3′). The putative TSS is indicated by the red arrow. The highly conserved nucleotides G⁻² and/or C⁺⁵ in the mouse and human HBSs sequence are highlighted in red.

Figure 3

HIF-1α modeling and HIF-1/DNA docking. (A) Root-mean-square (RMS) displacement of protein backbone (black arrow indicates the time at which the stabilization of the protein structure occurs). (B) RMS fluctuation of aminoacid displacement relative to the starting structure and the principal domains of the HIF-1α protein, accordingly colored in (C). (D) HIF-1α protein modelized in its dimeric form showing the correct interaction with the DNA fragment. The two monomers are reported in green and orange respectively, while the DNA fragment is highlighted in blue. The binding site generated by dimerization is better shown in the focus section.

Figure 4

Graphical representation of the 6 HIF-1/DNA models displaying the 3D reconstruction of the HIF-1 docking to each of the nucleotide sequence, including HBS and flanking sequences, and their relative best docking score in kJ/mol.

Figure 5

Direct interactions between HIF-1 and the 6 DNA fragments containing the HBSs as extrapolated from 100 ns molecular dynamics trajectory simulation, expressed as number of contacts within 0.6 nm distance between each other.

Figure 6

Docking analysis of model 1 and model 3. (A) HIF-1/HBS #1 best association complex: full structure and focus on HIF-1-DNA interactions (boxes). (B) HIF-1/HBS #3 best association complex: full structure and focus on HIF-1-DNA interactions (boxes).

Figure 7

HIF-1α interaction with HBS #1 and corresponding β3-AR gene expression at PD12 (from 0 to 12 h of hypoxia) or at PD17. (A) Schematic diagram of the OIR model pointing to the specific times under analysis. (B) Data from HIF-1α chromatin immunoprecipitation and HBS #1-specific qPCR (ChIP-qPCR) represented as fold enrichment relative to IgG input. (C) Corresponding levels of β3-AR mRNA. White bars represent data from retinas of normoxic controls while grey bars represent data from hypoxic mice. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples. * p < 0.05 vs. normoxic controls (n = 6 samples).