HIF-1alpha induces cell cycle arrest by functionally counteracting Myc

Abstract

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1alpha, a basic helix-loop-helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1alpha for hypoxia-induced growth arrest and activation of p21(cip1), a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21(cip1) activation has been elusive. Here we demonstrate that HIF-1alpha, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21(cip1). The HIF-1alpha antagonism is mediated by displacing Myc binding from p21(cip1) promoter. Neither HIF-1alpha transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1alpha. In keeping with its antagonism of Myc, HIF-1alpha also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1alpha pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

Figure 1

HIF-1α expression is sufficient to induce cell cycle arrest by activating p21^cip1. (A) HCT116 wild-type and p21^−/− variant were analyzed for BrdU incorporation after adenoviral infection, as indicated. The original data are presented, together with the percentage of each phase in the inlet. CON, no infection. (B) HCT116 wild-type and p21^−/− cells were subjected to hypoxia (H) for 8 or 16 h prior to cell cycle analysis. The percentage of each phase is shown in bar graphs. N, normoxia. (C, D) Adenovirus-infected cells were assayed for mRNA (C) and protein (D) levels of specified genes by RT–PCR and Western blot, respectively. Both endogenous HIF-1α and ΔODD mRNA levels were determined in the same PCR reactions, and β-actin levels served as loading controls. (E, F) Hypoxic induction of p21^cip1 expression was examined at mRNA (E) and protein levels (F). hTERT mRNA levels were also included as a control (see below).

Figure 2

HIF-1α induction of p21^cip1 and G₁ arrest is independent of its DNA-binding and transcriptional activity. (A) Transcriptional activity of HIF-1α, ΔODD, and ΔODD plus C800V or LCLL mutations was determined in Cos-7 cells in a HIF-1-mediated reporter system (Huang et al, 1996). Relative luciferase units (RLU) are presented with means plus standard errors from three independent experiments. (B, D) HCT116 cells were assayed for VEGF expression by RT–PCR (B) or VEGF secretion by ELISA (D) after adenoviral infection as indicated. DFO, desferrioxamine. (C, E) ΔODD LCLL mutant was analyzed for its effects on p21^cip1 expression at mRNA (C) and protein (E) levels. (F) Effect of the ΔODD LCLL mutant on cell cycle profile was determined as above, with the percentage of each phase indicated in the inlet. (G–I) ΔODD and its R27G mutant were assayed in Cos-7 cells for HIF-1-mediated reporter activity (G) and Western analysis of protein levels (H), and in HCT116 cells for induction of cell cycle arrest (I). CON, transfected with pcDNA3.

Figure 3

HIF-1α counteracts Myc activity. (A) Effect of ΔODD on Myc transcriptional activity was examined with an hTERT luciferase reporter in HCT116 cells. The graph represents three independent experiments in duplicate. (B) Myc target genes, as indicated, were analyzed by RT–PCR after adenoviral infection. c-myc mRNA levels were also determined. (C) After transfection with c-myc siRNA (siRNA), HCT116 cells were infected with Ad-ΔODD and analyzed with RT–PCR for Myc target gene expression, as indicated. CON, no siRNA. Neg, negative control siRNA.

Figure 4

HIF-1α displaces Myc binding from p21^cip1 promoter and overrides Myc target gene expression. (A) After Ad-ΔODD infection of HCT116 cells, chromatin immunoprecipitation assays were performed with indicated antibodies at the top. Both the proximal and distal regions of p21^cip1 promoter were analyzed. Input, genomic DNA prior to immunoprecipitations. (B) Normoxic (N) and hypoxic (H) HCT116 cells were stained by immunofluorescence with anti-Myc antibody (in red) and antibodies against HIF-1α, p21^cip1, or hTERT (in green). Only merged images are presented. The cells in panel iii were treated with doxorubicin (Dox). (C) Myc expression levels from 100 individual cells in (B) were plotted in dots along the x-axis against those of HIF-1α, p21, or hTERT in the y-axis. Cells expressing both proteins are depicted in dots along the diagonal lines.

Figure 5

HIF-1α forms a weak complex with Myc in vivo. (A, B) ΔODD and Myc, as indicated, were co-expressed in 293 cells. Co-immunoprecipitations with antibodies against HA (α-HA IP) or Myc (α-Myc IP) were performed, followed by Western analyses detecting co-immunoprecipitates (marked with arrow heads), as well as immunoprecipitates. Input, whole-cell extracts subjected to direct Western analysis. (C) Omission of antibody (−Ab) and use of normal immunoglobulin (IgG) were included as controls for an anti-Myc co-immunoprecipitation. (D) The interaction of endogenous HIF-1α and Myc in HCT116 cells was shown in an anti-HIF-1α co-immunoprecipitation, followed by anti-Myc Western blotting.

Figure 6

The N-terminal HIF-1α interacts with Myc and induces cell cycle arrest. (A) A schematic representation of HIF-1α. Structural domains are indicated at the bottom (Huang and Bunn, 2003), and the relevant codon numbers at the top. (B, C) HIF-1α mutants (amino acids 1–400, 1–329, and 1–167) were transfected into 293 cells, and analyzed by co-immunoprecipitation assays to determine the minimum requirement for Myc binding. (D, E) The N-terminal HIF-1α (1–400 and 1–167) was examined for its ability to activate p21^cip1 promoter in Cos-7 cells (D), and to alter cell cycle in HCT116 cells (E). CON, transfected with pcDNA3.

Figure 7

An HIF-1α–Myc pathway regulating hypoxia-responsive genes lacking the canonical HRE. Apart from activating the classic hypoxia-inducible genes such as erythropoietin (EPO), VEGF, and Glut-1, HIF-1α functionally counteracts Myc, thereby overriding Myc target gene expression. The upregulated genes are in red, and the downregulated in green.