A HIF1α Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas

Pheochromocytomas are neural crest–derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1α. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1α activity in tumors

Polarized blazar X-rays imply particle acceleration in shocks

Most of the light from blazars, active galactic nuclei with jets of magnetized plasma that point nearly along the line of sight, is produced by high-energy particles, up to around 1 TeV. Although the jets are known to be ultimately powered by a supermassive black hole, how the particles are accelerated to such high energies has been an unanswered question. The process must be related to the magnetic field, which can be probed by observations of the polarization of light from the jets. Measurements of the radio to optical polarization—the only range available until now—probe extended regions of the jet containing particles that left the acceleration site days to years earlier1,2,3, and hence do not directly explore the acceleration mechanism, as could X-ray measurements. Here we report the detection of X-ray polarization from the blazar Markarian 501 (Mrk 501). We measure an X-ray linear polarization degree ΠX of around 10%, which is a factor of around 2 higher than the value at optical wavelengths, with a polarization angle parallel to the radio jet. This points to a shock front as the source of particle acceleration and also implies that the plasma becomes increasingly turbulent with distance from the shock

X-ray Polarization Observations of BL Lacertae

Blazars are a class of jet-dominated active galactic nuclei with a typical double-humped spectral energy distribution. It is of common consensus the Synchrotron emission to be responsible for the low frequency peak, while the origin of the high frequency hump is still debated. The analysis of X-rays and their polarization can provide a valuable tool to understand the physical mechanisms responsible for the origin of high-energy emission of blazars. We report the first observations of BL Lacertae performed with the Imaging X-ray Polarimetry Explorer ({IXPE}), from which an upper limit to the polarization degree $\Pi_X<$12.6\% was found in the 2-8 keV band. We contemporaneously measured the polarization in radio, infrared, and optical wavelengths. Our multiwavelength polarization analysis disfavors a significant contribution of proton synchrotron radiation to the X-ray emission at these epochs. Instead, it supports a leptonic origin for the X-ray emission in BL Lac.Comment: 17 pages, 5 figures, accepted for publication in ApJ

A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas.

Pheochromocytomas are neural crest-derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1alpha. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1alpha activity in tumors

HIF1α Attenuates SDHB Levels

<div><p>(A) HIF1α expression was induced by treatment of mouse pheochromocytoma MPC 9/3L cells with 150 μM cobalt chloride for the indicated times. SDHB expression decreased in treated cells. Glut1 indicates increased activity of HIF1α, and β-actin was used as a loading control.</p> <p>(B) Transient expression in HEK293 cells of a HIF1α double mutant PA (P402A/P564A) that is resistant to VHL-mediated degradation reduced expression of SDHB.</p> <p>(C) A2058 cell lines stably expressing HIF1α shRNA do not show change in SDHB after cobalt chloride exposure, while SDHB is downregulated in control GFP shRNA cells treated with cobalt chloride.</p> <p>(D) Proposed model of HIF1α and SDHB interregulation. HIF1α downregulates SDHB, which leads to complex II dysfunction. High succinate levels resulting from loss of complex II, in turn, inhibit prolyl hydroxylase (PHD) activity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-b19" target="_blank">19</a>]. Non-hydroxylated HIF1α is resistant to VHL-mediated targeting for degradation and can therefore activate downstream genes, such as angiogenic factors. “E3 complex” indicates the E3 ubiquitin ligase complex for which VHL is the substrate recognition factor.</p></div

Low Expression of SDHB Is a General Feature of Cluster 1 Tumors

<div><p>(A) Expression of SDHB protein in pheochromocytomas with <i>SDHB</i> or <i>SDHD</i> mutations. Western blot analysis of SDHB of whole cell lysates from primary tumors was performed as described in Methods. Lane 1 is normal adrenal medulla used as control and lanes 2–6 are tumors 140, 158, 136, 58, and 220, respectively, from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-g001" target="_blank">Figure 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-t001" target="_blank">Table 1</a>. β-actin was used as a loading control.</p> <p>(B) SDHB expression segregates with cluster membership. Cluster 2 tumors, comprising MEN2, NF1, and other sporadic tumors, are shown in lanes 2–4 (tumors 105, 91, and 196, respectively, from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-g001" target="_blank">Figure 1</a>). Cluster 1 contains tumors with <i>VHL</i> and <i>SDHB</i> mutations and a subset of sporadic samples (lanes 5–7 are tumors 16, 85, 101, and 152, respectively, from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-g001" target="_blank">Figure 1</a>). Lane 1 is normal adrenal medulla. β-actin was used as a loading control.</p> <p>(C) Immunostaining of SDHB protein in pheochromocytomas or paragangliomas with various genetic backgrounds. A MEN2-related pheochromocytoma is shown on the top row, followed by tumors with mutations in <i>NF1, SDHB, SDHD,</i> and <i>VHL</i> genes. Corresponding hematoxylin/eosin staining is shown on the left.</p></div

Unsupervised Analysis of Pheochromocytomas Links Tumors with <i>VHL</i> and <i>SDHB</i> or <i>SDHD</i> Mutations

<p>Unsupervised hierarchical clustering identifies two major clusters in pheochromocytomas: Cluster 1 contains VHL (V), SDHD (D), and SDHB (B) tumors; Cluster 2 contains MEN2 (M) and NF1 (N) pheochromocytomas. Multiple tumors from seven independent unclassified families with recurrent pheochromocytoma (numbered 1–7) and also sporadic tumors (S) are distributed between the two clusters. Letters or numbers on the first row indicate the various tumor classes, as described above. The second row identifies tumor location as adrenal (A) or extra-adrenal (E). Mutations were later detected in samples marked with an asterisk, guided by cluster distribution (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010008#pgen-0010008-t001" target="_blank">Table 1</a> for details).</p