Agnieszka Loboda

HIF-1 and HIF-2 transcription factors — Similar but not identical

Hypoxia inducible factor (HIF)-1 and HIF-2 are heterodimeric transcription factors mediating the cellular response to hypoxia. Recent data indicate that not only ubiquitous HIF-1α, but also more cell-specific HIF-2α, is an important regulator of the hypoxia response. Although both α subunits are highly conservative at protein level, share similar domain structure, heterodimerize with HIF-1β, and bind to the same DNA sequence called hypoxia responsive element (HRE), their effect on the expression of some genes may vary. In this review we stressed the differences between the isoforms, their structure and expression pattern. Moreover, we described diversity of coactivators and proteins which interact with HIFs, and which are responsible for the specificity of their action. Finally, recent data showing link between HIFs and specific microRNA have been presented.

Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism.

The multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) is considered not only as a cytoprotective factor regulating the expression of genes coding for anti-oxidant, anti-inflammatory and detoxifying proteins, but it is also a powerful modulator of species longevity. The vertebrate Nrf2 belongs to Cap ‘n’ Collar (Cnc) bZIP family of transcription factors and shares a high homology with SKN-1 from Caenorhabditis elegans or CncC found in Drosophila melanogaster. The major characteristics of Nrf2 are to some extent mimicked by Nrf2-dependent genes and their proteins including heme oxygenase-1 (HO-1), which besides removing toxic heme, produces biliverdin, iron ions and carbon monoxide. HO-1 and their products exert beneficial effects through the protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis. On the other hand, the disturbances in the proper HO-1 level are associated with the pathogenesis of some age-dependent disorders, including neurodegeneration, cancer or macular degeneration. This review summarizes our knowledge about Nrf2 and HO-1 across different phyla suggesting their conservative role as stress-protective and anti-aging factors.

Heme Oxygenase-1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.

A Novel Role of Hypoxia-Inducible Factor in Cobalt Chloride- and Hypoxia-Mediated Expression of IL-8 Chemokine in Human Endothelial Cells

I n their recent article, Kim et al. (1) demonstrated that activation of hypoxia-inducible factor-1 (HIF-1) transcription factor by hypoxia (1% O2) or CoCl2, a hypoxiamimic, can induce IL-8 mRNA and protein expressions in various human endothelial cells, namely dermal vascular (HDMVEC), pulmonary microvascular (HPMVEC), brain (HBEC), and pulmonary aortic (HPAEC), an observation similar to that of previous, but uncited, investigators (2). The majority of the present experiments has been done in HDMVEC. However, our recently published data indicate that the underlying mechanisms may be more complex. In our experiments done in immortalized HDMVEC line HMEC-1, we have observed that neither hypoxia (3) nor CoCl2 (4) induces IL-8 expression. In fact, the effect of hypoxia (1% O2) on both IL-8 mRNA and protein expression was inhibitory (3). In addition, CoCl2 was not able to enhance the expression of IL-8 (4), although it increased HIF-1 activation and synthesis of vascular endothelial growth factor to the same level as hypoxia (3, 4). Thus, our data point to the other mechanisms of regulation of IL-8 than that demonstrated by Kim et al. Moreover, in another recent article (5), activation of HIF-1 by prolyl hydroxylase inhibitor attenuated IL-8 production in HMEC-1. Interestingly, the negative role of HIF-1 in regulation of IL-8 expression also has been demonstrated recently in several tumor cell lines (6), whereas another study showed that desferoxamine, an activator of HIF-1, decreased TNF-induced IL-8 synthesis in HDMVEC (7). The reasons for such different data are not clear. First, the cell-type specific effect may exist. However, it seems that HDMVEC used by Kim et al., which were provided to them by Drs. Ades and Lawley, might be in fact the same as the HMEC-1 line (8) used by us (3, 4) and others (5, 7). Second, the concentrations of CoCl2 used by Kim et al. to stimulate IL-8 expression were very high, i.e., 0.5-1 mM. Both in our hands (4) and in Ockaili et al. experiments (5), much lower concentrations, i.e., 0.1-0.25 mM, of CoCl2 potently stabilized HIF-1 and activated a hypoxia-responsive element in reporter gene assays. However, such low concentration of CoCl2 did not affect IL-8 production, although it potently enhanced HIF-1dependent vascular endothelial growth factor synthesis by HMEC-1 to the level exerted by hypoxia (3, 4). In fact, 0.25 mM CoCl2 was also not effective in the work of Kim et al. (1). Finally, in our hands, 1% O2 down-regulated IL-8 mRNA expression and protein synthesis after 24 h of culture. In turn, Kim et al. measured IL-8 production only after 6 h of hypoxia (description to Fig. 3 states this time, although in the text the authors mentioned 24 h, recalling the same figure) and observed only an 1.6-fold increase in protein synthesis. Thus, the influence of HIF-1 and hypoxia on IL-8 production is far from clear. Which effects reflect the physiological conditions remain to be elucidated.

Effect of quercetin and its metabolites isorhamnetin and quercetin-3-glucuronide on inflammatory gene expression: role of miR-155

In the present study the effect of quercetin and its major metabolites quercetin-3-glucuronide (Q3G) and isorhamnetin on inflammatory gene expression was determined in murine RAW264.7 macrophages stimulated with lipopolysaccharide. Quercetin and isorhamnetin but not Q3G significantly decreased mRNA and protein levels of tumor necrosis factor alpha. Furthermore a significant decrease in mRNA levels of interleukin 1β, interleukin 6, macrophage inflammatory protein 1α and inducible nitric oxide synthase was evident in response to the quercetin treatment. However Q3G did not affect inflammatory gene expression. Anti-inflammatory properties of quercetin and isorhamnetin were accompanied by an increase in heme oxygenase 1 protein levels, a downstream target of the transcription factor Nrf2, known to antagonize chronic inflammation. Furthermore, proinflammatory microRNA-155 was down-regulated by quercetin and isorhamnetin but not by Q3G. Finally, anti-inflammatory properties of quercetin were confirmed in vivo in mice fed quercetin-enriched diets (0.1 mg quercetin/g diet) over 6 weeks.

Interplay Between Heme Oxygenase-1 and miR-378 Affects Non-Small Cell Lung Carcinoma Growth, Vascularization, and Metastasis

AIMS Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. RESULTS Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. INNOVATION AND CONCLUSION In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers: James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.

HIF-1 versus HIF-2 — Is one more important than the other?

Hypoxia is a state where oxygen availability/delivery is below the level necessary to maintain physiological oxygen tension for a particular tissue. It is well-established that when tissue demand exceeds its oxygen supply, a cascade of intracellular events is activated, with the elevation of the expression of hypoxia-inducible factors (HIFs). As a consequence, the extensive transcriptional response regulating angiogenesis, glucose metabolism, cell growth, metastasis and others processes is induced. The discovery of differences between HIF isoforms has provided new insights into HIFs biology. Importantly, the opposite effects can be exerted by HIF-1 and HIF-2 on the regulation of angiogenic response. Although both isoforms may upregulate the expression of pro-angiogenic vascular endothelial growth factor (VEGF), HIF-1 diminished the expression of interleukin-8 (IL-8) by inhibition of the Nrf2 transcription factor whereas HIF-2 augmented expression of IL-8 in an Nrf2-independent way but via upregulation of SP-1 activity. Moreover, the opposite regulation of c-Myc transcription factor by both HIF isoforms may influence IL-8 regulation. Complexity of effects exerted by both HIF isoforms resulting from the cooperation with other transcription factors should be subjected to intensive investigation especially in the context of pro-and anti-angiogenic therapies.

Heme Oxygenase-1 Inhibits Myoblast Differentiation by Targeting Myomirs

AIMS Heme oxygenase-1 (HMOX1) is a cytoprotective enzyme degrading heme to biliverdin, iron ions, and carbon monoxide, whose expression is induced in response to oxidative stress. Its overexpression has been suggested as a strategy improving survival of transplanted muscle precursors. RESULTS Here we demonstrated that HMOX1 inhibits differentiation of myoblasts and modulates miRNA processing: downregulates Lin28 and DGCR8, lowers the total pool of cellular miRNAs, and specifically blocks induction of myomirs. Genetic or pharmacological activation of HMOX1 in C2C12 cells reduces the abundance of miR-1, miR-133a, miR-133b, and miR-206, which is accompanied by augmented production of SDF-1 and miR-146a, decreased expression of MyoD, myogenin, and myosin, and disturbed formation of myotubes. Similar relationships between HMOX1 and myomirs were demonstrated in murine primary satellite cells isolated from skeletal muscles of HMOX1(+/+), HMOX1(+/-), and HMOX1(-/-) mice or in human rhabdomyosarcoma cell lines. Inhibition of myogenic development is independent of antioxidative properties of HMOX1. Instead it is mediated by CO-dependent inhibition of c/EBPδ binding to myoD promoter, can be imitated by SDF-1, and partially reversed by enforced expression of miR-133b and miR-206. Control C2C12 myoblasts injected to gastrocnemius muscles of NOD-SCID mice contribute to formation of muscle fibers. In contrast, HMOX1 overexpressing C2C12 myoblasts form fast growing, hyperplastic tumors, infiltrating the surrounding tissues, and disseminating to the lungs. INNOVATION We evidenced for the first time that HMOX1 inhibits differentiation of myoblasts, affects the miRNA processing enzymes, and modulates the miRNA transcriptome. CONCLUSION HMOX1 improves the survival of myoblasts, but concurrently through regulation of myomirs, may act similarly to oncogenes, increasing the risk of hyperplastic growth of myogenic precursors.

MicroRNAs as biomarkers of disease onset

MicroRNAs (miRNAs) are small, noncoding RNA molecules with the ability to posttranscriptionally regulate gene expression via targeting the 3′ untranslated region of messenger RNAs. miRNAs are critical for normal cellular functions such as the regulation of the cell cycle, differentiation, and apoptosis, and they target genes during embryonal and postnatal development, whereas their expression is unbalanced in various pathological states. Importantly, miRNAs are abundantly present in body fluids (e.g., blood), which are routinely examined in patients. These molecules circulate in free and exosome encapsulated forms, and can be efficiently detected and amplified by means of molecular biology tools such as real-time PCR. Together with relative stability, specificity, and reproducibility, they are seen as good candidates for early recognition of the onset of disease. Thus, miRNAs might be considered as biomarkers for many pathological states.

Cross-talk between microRNAs, nuclear factor E2-related factor 2, and heme oxygenase-1 in ochratoxin A-induced toxic effects in renal proximal tubular epithelial cells.

SCOPE Ochratoxin A (OTA) is a mycotoxin exhibiting nephrotoxic and potential carcinogenic activity. We investigated the cross-talk between microRNAs, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in ochratoxin A-mediated effects. METHODS AND RESULTS In porcine renal proximal tubular cells, OTA increased expression of profibrotic transforming growth factors β (TGFβ) while concomitantly decreasing expression of Nrf2, HO-1, and erythropoietin. Adenoviral overexpression of Nrf2 counteracted OTA-mediated reduction in HO-1 and erythropoietin expression and cell proliferation as well as increase in reactive oxygen species (ROS) generation and TGFβ expression. Additionally, inhibition of HO activity enhanced whereas adenoviral overexpression of HO-1 reduced expression of TGFβ. Moreover, antioxidants, N-acetyl-cysteine and desferioxamine, prevented OTA-mediated enhancement of ROS generation, and TGFβ expression. Finally, OTA modulated microRNA processing by upregulating LINeage protein 28 and DiGeorge syndrome critical region-8, increasing the total pool of cellular microRNAs and elevating the expression of miR-132 and miR-200c. Inhibition of miR-132 by specific antagomir restored the OTA-driven reduction in Nrf2 expression. Moreover, anti-miR-132 and anti-miR-200c counteracted OTA-mediated decrease in HO-1 levels as well as increase in ROS production and TGFβ expression. CONCLUSION We showed that attenuation of Nrf2 and HO-1 expression through induction of miR-132 and miR-200c by OTA elevates ROS levels and profibrotic TGFβ expression.