The response of c-jun/AP-1 to chronic hypoxia is hypoxia-inducible factor 1 alpha dependent

Abstract

Hypoxia (low-oxygen tension) is an important physiological stress that influences responses to a wide range of pathologies, including stroke, infarction, and tumorigenesis. Prolonged or chronic hypoxia stimulates expression of the stress-inducible transcription factor gene c-jun and transient activation of protein kinase and phosphatase activities that regulate c-Jun/AP-1 activity. Here we describe evidence obtained by using wild-type and HIF-1 alpha nullizygous mouse embryonic fibroblasts (mEFs) that the induction of c-jun mRNA expression and c-Jun phosphorylation by prolonged hypoxia are completely dependent on the presence of the oxygen-regulated transcription factor hypoxia-inducible factor 1 alpha (HIF-1 alpha). In contrast, transient hypoxia induced c-jun expression in both types of mEFs, showing that the early or rapid induction of this gene is independent of HIF-1 alpha. These findings indicate that the c-jun gene has a biphasic response to hypoxia consisting of inductions that depend on the degree or duration of exposure. To more completely define the relationship between prolonged hypoxia and c-Jun phosphorylation, we used mEFs from mice containing inactivating mutations of critical phosphorylation sites in the c-Jun N-terminal region (serines 63 and 73 or threonines 91 and 93). Exposure of these mEFs to prolonged hypoxia demonstrated an absolute requirement for N-terminal sites for HIF-1 alpha-dependent phosphorylation of c-Jun. Taken together, these findings suggest that c-Jun/AP-1 and HIF-1 cooperate to regulate gene expression in pathophysiological microenvironments.

FIG. 1.

(A) Hypoxia Hx) induces the immediate-early accumulation of c-jun mRNA independently of the presence of HIF-1α. Autoradiograph of a Northern blot of total RNA from wild-type and HIF-1α null mEFs incubated under aerobic conditions (air [5% CO₂]) or exposed to hypoxia (pO₂ ≤ 1%) for the indicated times. The blot was probed sequentially for c-jun and 28S rRNA transcripts. This finding is representative of three independent experiments. (B) The early hypoxia-inducible accumulation of c-jun mRNA is dependent on serum (10% FBS). Histogram of relative amounts of c-jun mRNA detected by quantitative RT-PCR amplification of total RNA harvested at the indicated times. For details, see Materials and Methods.

FIG. 2.

Hypoxia (Hx) rapidly induces the accumulation of HIF-1α protein in wild-type cells. Autoradiographs of immunoblots of total protein from wild type and HIF-1α null mEFs showing the accumulation of HIF-1α in wild-type cells exposed to hypoxia (pO₂ ≤ 1%) for the indicated times. In these studies and in all others, hypoxic cells were harvested for protein assays under anoxic conditions. For details, see Materials and Methods.

FIG. 3.

The delayed accumulation of c-jun mRNA induced by prolonged hypoxia (Hx) is dependent on the presence of HIF-1α. Autoradiograph of a Northern blot of total RNA from wild-type and HIF-1α null mEFs incubated under aerobic conditions (air [5% CO₂]) or exposed to hypoxia (pO₂ ≤ 0.01%) for the indicated times. The blot was probed sequentially for the mRNAs for c-jun and 28S rRNA. This finding is representative of three independent experiments.

FIG. 4.

Phosphorylation of c-Jun in response to prolonged hypoxia (Hx). (A) Immunoblot of total protein from wild-type and HIF-1α null mEFs harvested under aerobic conditions or following exposure to hypoxia (pO₂ ≤ 0.01%) for the indicated times. The blot was probed with an anti-phospho-c-Jun antibody (KM-1). This immunoblot is representative of multiple independent experiments. (B) Immunoblots of total protein from wild-type mEFs harvested under aerobic conditions or following exposure to hypoxia (pO₂ ≤ 0.01%) for the indicated times. Replicate blots were probed with the KM-1 antibody (top panel) or with an antibody that recognizes nonphosphorylated c-Jun (bottom panel; antibody H79). (C) Immunoblot of total protein from spontaneously immortalized wild-type or HIF-1α null mEFs exposed to hypoxia (pO₂ ≤ 0.01%) for 8 h. The blot was probed with the KM-1 antibody. (D) Immunoblots of protein from serum-starved wild-type and HIF-1α null mEFs incubated under aerobic conditions (air [5% CO₂]) or exposed to hypoxia (pO₂ ≤ 0.01%) for the indicated times. Aerobic cells and cells to be exposed to hypoxia were incubated in medium containing no serum for 18 h before the study was performed. The blot was probed with the KM-1 antibody.

FIG. 5.

(A) Immunoblot of total protein from wild-type mEFs harvested under aerobic conditions (left panel) or following exposure to hypoxia (Hx) (pO₂ ≤ 0.01%; right panel) for 8 h. The same total protein sample from hypoxic cells was probed with different anti-c-Jun antibodies on one membrane. For details, see Materials and Methods. (B) Top panel: immunoblot of total protein from aerobic (two identical cultures) and hypoxic (pO₂ ≤ 0.01%; 8 h) wild-type mEFs and mEFs containing the knock-in alleles c-jun (S63A S73A) or c-jun (T91A T93A). Control cells from each genotype were exposed to EGF (50 ng/ml, 15 min). Wild-type cells were also exposed to anisomycin (10 μg/ml, 1 h). Middle panel: Ponceau red staining of total protein on the immunoblot shown above. The position of a marker having an electrophoretic mobility within the range appropriate for c-Jun phospho-isoforms (50.4 kDa; lanes 2 and 12 from the left; Bio-Rad Laboratories, Hercules, Calif.) is indicated by an arrow. Bottom panel: replicate immunoblot of total protein from aerobic and hypoxic wild-type mEFs and mEFs containing the knock-in alleles c-jun (S63A S73A) or c-jun (T91A T93A). The blot was probed with the H79 antibody that recognizes nonphosphorylated c-Jun.

FIG. 6.

Autoradiograph of a Northern blot of total RNA from wild-type mEFs and mEFs containing the knock-in alleles c-jun (S63A S73A) or c-jun (T91A T93A) incubated under aerobic conditions (air [5% CO₂]) or exposed to hypoxia (Hx) (pO₂ ≤ 0.01%) for the indicated times. The blot was probed sequentially for c-jun and 28S rRNA transcripts.