Casein kinase 1 regulates human hypoxia-inducible factor HIF-1

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.J Cell Sc

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Understanding complexity in the HIF signaling pathway using systems biology and mathematical modeling

Hypoxia is a common micro-environmental stress which is experienced by cells during a range of physiologic and pathophysiologic processes. The identification of the hypoxia-inducible factor (HIF) as the master regulator of the transcriptional response to hypoxia transformed our understanding of the mechanism underpinning the hypoxic response at the molecular level and identified HIF as a potentially important new therapeutic target. It has recently become clear that multiple levels of regulatory control exert influence on the HIF pathway giving the response a complex and dynamic activity profile. These include positive and negative feedback loops within the HIF pathway as well as multiple levels of crosstalk with other signaling pathways. The emerging model reflects a multi-level regulatory network that affects multiple aspects of the physiologic response to hypoxia including proliferation, apoptosis, and differentiation. Understanding the interplay between the molecular mechanisms involved in the dynamic regulation of the HIF pathway at a systems level is critically important in defining new appropriate therapeutic targets for human diseases including ischemia, cancer, and chronic inflammation. Here, we review our current knowledge of the regulatory circuits which exert influence over the HIF response and give examples of in silico model-based predictions of the dynamic behaviour of this system

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

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Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/ immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation

A Dialogue between the Hypoxia-Inducible Factor and the Tumor Microenvironment

The hypoxia-inducible factor is the key protein responsible for the cellular adaptation to low oxygen tension. This transcription factor becomes activated as a result of a drop in the partial pressure of oxygen, to hypoxic levels below 5% oxygen, and targets a panel of genes involved in maintenance of oxygen homeostasis. Hypoxia is a common characteristic of the microenvironment of solid tumors and, through activation of the hypoxia-inducible factor, is at the center of the growth dynamics of tumor cells. Not only does the microenvironment impact on the hypoxia-inducible factor but this factor impacts on microenvironmental features, such as pH, nutrient availability, metabolism and the extracellular matrix. In this review we discuss the influence the tumor environment has on the hypoxia-inducible factor and outline the role of this factor as a modulator of the microenvironment and as a powerful actor in tumor remodeling. From a fundamental research point of view the hypoxia-inducible factor is at the center of a signaling pathway that must be deciphered to fully understand the dynamics of the tumor microenvironment. From a translational and pharmacological research point of view the hypoxia-inducible factor and its induced downstream gene products may provide information on patient prognosis and offer promising targets that open perspectives for novel “anti-microenvironment” directed therapies

Lamin B receptor: Interplay between structure, function and localization

Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophobic segments that span the membrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have been attributed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contributing to the shape of interphase nuclear architecture, while its transmembrane domains exhibit sterol reductase activity. Mutations within the transmembrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and Greenberg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic behavior have not yet been fully unraveled. Here, we provide an overview of the current knowledge of the interplay between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR. © 2017 by the authors. Licensee MDPI, Basel, Switzerland

Specific inhibition of hif activity: Can peptides lead the way?

Reduced oxygen availability (hypoxia) is a characteristic of many disorders including cancer. Central components of the systemic and cellular response to hypoxia are the Hypoxia Inducible Factors (HIFs), a small family of heterodimeric transcription factors that directly or indirectly regulate the expression of hundreds of genes, the products of which mediate adaptive changes in processes that include metabolism, erythropoiesis, and angiogenesis. The overexpression of HIFs has been linked to the pathogenesis and progression of cancer. Moreover, evidence from cellular and animal models have convincingly shown that targeting HIFs represents a valid approach to treat hypoxia-related disorders. However, targeting transcription factors with small molecules is a very demanding task and development of HIF inhibitors with specificity and therapeutic potential has largely remained an unattainable challenge. Another promising approach to inhibit HIFs is to use peptides modelled after HIF subunit domains known to be involved in protein–protein interactions that are critical for HIF function. Introduction of these peptides into cells can inhibit, through competition, the activity of endogenous HIFs in a sequence and, therefore also isoform, specific manner. This review summarizes the involvement of HIFs in cancer and the approaches for targeting them, with a special focus on the development of peptide HIF inhibitors and their prospects as highly-specific pharmacological agents. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Vitamin D and Hypoxia: Points of Interplay in Cancer

Vitamin D is a hormone that, through its action, elicits a broad spectrum of physiological responses ranging from classic to nonclassical actions such as bone morphogenesis and immune function. In parallel, many studies describe the antiproliferative, proapoptotic, antiangiogenic effects of calcitriol (the active hormonal form) that contribute to its anticancer activity. Additionally, epidemiological data signify the inverse correlation between vitamin D levels and cancer risk. On the contrary, tumors possess several adaptive mechanisms that enable them to evade the anticancer effects of calcitriol. Such maladaptive processes are often a characteristic of the cancer microenvi-ronment, which in solid tumors is frequently hypoxic and elicits the overexpression of Hypoxia-Inducible Factors (HIFs). HIF-mediated signaling not only contributes to cancer cell survival and proliferation but also confers resistance to anticancer agents. Taking into consideration that calcit-riol intertwines with signaling events elicited by the hypoxic status cells, this review examines their interplay in cellular signaling to give the opportunity to better understand their relationship in cancer development and their prospect for the treatment of cancer. © 2022 by the authors. Licensee MDPI, Basel, Switzerland