Nuclear localization of the mitochondrial factor HIGD1A during metabolic stress

Abstract

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.

Figure 1. HIGD1A is a HIF-1 target localized to mitochondria under physiological conditions, but localizes to the nucleus during pathological stress in MEFs.

(A) RT-PCR analysis of Higd1a mRNA expression in wild-type and Hif-1α^−/− MEFs cultured under 20% O₂ (N, normoxia) or 2% O₂ (H, hypoxia). (B) Immunoblot analysis of HIGD1A protein expression in wild-type and Hif-1α^−/− MEFs, as well as wild-type and Hif-1/2α^−/− TSCs cultured under 20% O₂ (N, normoxia) or 2% O₂ (H, hypoxia). (C) Immunoblot analysis of HIGD1A protein expression in Hif-1/2α^−/− TSCs stably expressing GFP, HIF-1α, HIF-2α or DNA-binding domain deficient versions of each (HIF-1αΔb and HIF-2αΔb). (D) Immunofluorescence microscopy of endogenous HIGD1A in control MEFs indicated a mitochondrial localization pattern during physiological hypoxia (2% O₂), while more severe hypoxia (1% oxygen) coupled with glucose starvation (Ischemia) triggered its nuclear localization. Complex IV subunit 2 immunoreactivity was used as a marker of mitochondria. Nuclei are identified with DAPI staining. (E) Immunofluorescence microscopy of endogenous HIGD1A indicated that HIGD1A exhibited a nuclear localization pattern following exposure to the DNA damaging agent Etoposide. (F) Live cell immunofluorescence microscopy of HIGD1A-GFP fusion protein indicated that prior to Etoposide exposure, HIGD1A protein is extranuclear, with nuclear accumulation observed as early as 2 hours following drug exposure, and increasing throughout the duration of the experiment.

Figure 2. HIGD1A interacts with AIF and its nuclear localization is dependent on BAX and BAK.

(A) Immunofluoresce confocal laser scanning microscopy of HIGD1A-GFP overexpressing MEFs indicated a co-localization of HIGD1A with AIF in mitochondria in the absence of Etoposide, with both proteins localizing to the nucleus following exposure to Etoposide (40 µM). Quantitation of the relative nuclear localization HIGD1A in the presence of etoposide versus control (- etoposide) in wild-type MEFs. * = p<0.05 (student’s t-test) (B) Confocal cross sections in xy (top left), yz (right), and xz (bottom) revealed co-localization of HIGD1A and AIF in nuclei of Etoposide exposed MEFs. (C i) Immunoblot analysis of fractionated cell extracts (C = cytoplasm, M = mitochondria, N = nucleus) obtained from MEFs overexpressing HIGD1A or GFP alone (as control) show that cells treated with Etoposide contain greater levels of HIGD1A-GFP fusion protein in the nucleus as compared to untreated control cells. GAPDH was expressed in cytoplasmic as well as mitochondrial fractions under control conditions, translocating to the nucleus following Etoposide exposure in both control and GFP:HIGD1A expressing MEFs. Histone H3 was used as a nuclear marker, Complex IV subunit II (Comp. IV) was used as a mitochondrial marker. (C ii) Immuno-precipitation assays with HIGD1A-GFP fusion protein or control GFP expressing MEFs with an anti-GFP antibody revealed specific interaction between HIGD1A and AIF in vitro. Two other mitochondrial factors, BNIP3 and VDAC, did not bind HIGD1A. (D) Immunofluorescence microscopy of Bax/Bak^−/− MEFs revealed diminished nuclear localization of AIF and HIGD1A following exposure to Etoposide (40 µM) Quantitation of the relative nuclear localization HIGD1A in the presence of etoposide versus control (- etoposide) in Bax/Bak ^−/− MEFs.

Figure 3. HIGD1A localizes to the nucleus in the setting of human neonatal hypoxic-ischemic encephalopathy (HIE) in vivo.

(A) Schematic depiction of a coronal section through a human neonatal brain highlighting the subventricular zone (SVZ). (B) The SVZ of brains obtained from infants with HIE exhibited increased levels of the hypoxia marker CA9 compared with non-HIE control brains. (C)Immunofluorescence microscopy indicated low-level, extra-nuclear localization of endogenous HIGD1A in control human neonatal brains. Endogenous HIGD1A levels are increased in regions of human neonatal brains of infants who suffered HIE. Arrows indicate nuclear localization of endogenous HIGD1A in each. Experimental observations were made at least three times, and in vivo patient data are representative of three cases.

Figure 4. HIGD1A localizes to the nucleus in the setting of murine myocardial infarction (MI) in vivo.

Top panel is a representative H&E stain of a mouse heart subjected to MI highlighting the area of infarct as well as regions distal to it where tissue was analyzed. As seen, in areas distal to the infarct, HIGD1A and AIF are expressed in a primarily extranuclear distribution. In the area of infarct, however, HIGD1A and AIF exhibit a much more diffuse localization that clearly includes nuclei (arrows).

Figure 5. Nuclear localization of HIGD1A in mouse models of human breast cancer xenografts.

(A) Top panel is a representative H&E stained slide of a human breast cancer xonograft indicating the perinecrotic region surrounding the necrotic core, as well as areas distal to the necrosis. Immunofluorescence microscopy analysis indicated that HIGD1A and the hypoxia marker CA9 were only minimally expressed distal to the region of necrosis, whereas both were highly expressed in the peri-necrotic region. (B) Perinecrotic regions contained predominantly nuclear (white arrows) localized HIGD1A, whereas areas distal to tumor necrotic regions had predominantly extranuclear HIGD1A. (B) Immunofluorescence microscopy of human gliobastoma xenografts demonstrating predominantly extranuclear HIGD1A before administration of Bevacizumab (pre-Bevacizumab), whereas after administration of Bevacizumab (post-Bevacizumab), HIGD1A becomes predominantly nuclear as indicated by white arrows.

Figure 6. Nuclear localization of HIGD1A in response to Bevacizumab in human glioblastoma xenografts as well as glioblastoma patient biopsies.

(A) Immunofluorescence microscopy of human glioblastoma xenografts showing HIGD1A expression and localization before (pre) and after (post) Bevacizumab treatment. White arrows indicate nuclear HIGD1A. (B) Immunofluorescence microscopy of paired human patient gliobastoma biopsies showing CA9 (hypoxia marker) and HIGD1A expression and localization before (pre) and after (post) treatment with the anti-angiogenic agent, Bevacizumab (Avastin). As indicated, HIGD1A was induced and predominantly nuclear in human glioblastoma samples after administration of Bevacizumab to patients. Lower levels of HIGD1A was expressed before treatment. As indicated in the inset HIGD1A localization to the nucleus is pronounced in glioblastoma after treatment with Bevacizumab (white arrows).