FIH-1-Mint3 Axis Does Not Control HIF-1α Transcriptional Activity in Nucleus Pulposus Cells*

Background: Cells of the hypoxic nucleus pulposus (NP) require tightly regulated HIF-1 signaling for proper function. Results: Although overexpressed FIH-1 can regulate HIF-1 activity in NP cells, silencing endogenous FIH-1 results in no change in HIF-1 target gene expression. Conclusion: FIH-1 does not represent a major mechanism of controlling HIF-1-dependent transcription in NP cells. Significance: This study describes a physiological adaptation of NP cells. The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxia-sensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1α-C-TAD and HIF-2α-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

[1]  D. Mukhopadhyay,et al.  The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme , 2014, PloS one.

[2]  R. Kahn,et al.  Recruitment of the Mint3 Adaptor Is Necessary for Export of the Amyloid Precursor Protein (APP) from the Golgi Complex* , 2013, The Journal of Biological Chemistry.

[3]  Y. Toyama,et al.  Prolyl Hydroxylase 3 (PHD3) Modulates Catabolic Effects of Tumor Necrosis Factor-α (TNF-α) on Cells of the Nucleus Pulposus through Co-activation of Nuclear Factor κB (NF-κB)/p65 Signaling* , 2012, The Journal of Biological Chemistry.

[4]  G. Kapur,et al.  Hypoxia decreases podocyte expression of slit diaphragm proteins , 2012, International journal of nephrology and renovascular disease.

[5]  I. Shapiro,et al.  Tonicity enhancer binding protein (TonEBP) and hypoxia‐inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF‐1α degradation , 2012, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[6]  Y. Toyama,et al.  Expression of Prolyl Hydroxylases (PHDs) Is Selectively Controlled by HIF-1 and HIF-2 Proteins in Nucleus Pulposus Cells of the Intervertebral Disc , 2012, The Journal of Biological Chemistry.

[7]  I. Shapiro,et al.  HIF‐1α and HIF‐2α degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc , 2012, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[8]  Rachelle S. Singleton,et al.  Quantitative Mass Spectrometry Reveals Dynamics of Factor-inhibiting Hypoxia-inducible Factor-catalyzed Hydroxylation , 2011, The Journal of Biological Chemistry.

[9]  P. V. van Diest,et al.  Subcellular FIH-1 expression patterns in invasive breast cancer in relation to HIF-1α expression , 2011, Cellular Oncology.

[10]  I. Shapiro,et al.  Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins. , 2011, Arthritis and rheumatism.

[11]  T. Albert,et al.  Hypoxia activates the notch signaling pathway in cells of the intervertebral disc: implications in degenerative disc disease. , 2011, Arthritis and rheumatism.

[12]  P. Ratcliffe,et al.  Differential Sensitivity of Hypoxia Inducible Factor Hydroxylation Sites to Hypoxia and Hydroxylase Inhibitors* , 2011, The Journal of Biological Chemistry.

[13]  B. Yan,et al.  Prevention of apoptosis by the interaction between FIH1 and Bax , 2011, Molecular and Cellular Biochemistry.

[14]  P. V. van Diest,et al.  Expression of nuclear FIH independently predicts overall survival of clear cell renal cell carcinoma patients. , 2010, European journal of cancer.

[15]  R. Blantz,et al.  Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade. , 2010, American journal of physiology. Renal physiology.

[16]  U. Schlötzer-Schrehardt,et al.  Factor inhibiting HIF limits the expression of hypoxia-inducible genes in podocytes and distal tubular cells. , 2010, Kidney international.

[17]  I. Shapiro,et al.  Hypoxia-inducible factor regulation of ANK expression in nucleus pulposus cells: possible implications in controlling dystrophic mineralization in the intervertebral disc. , 2010, Arthritis and rheumatism.

[18]  D. Peet,et al.  The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism. , 2010, Cell metabolism.

[19]  M. Seiki,et al.  Mint3 Enhances the Activity of Hypoxia-inducible Factor-1 (HIF-1) in Macrophages by Suppressing the Activity of Factor Inhibiting HIF-1* , 2009, The Journal of Biological Chemistry.

[20]  D. Peet,et al.  Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1). , 2009, The international journal of biochemistry & cell biology.

[21]  W. Kaelin.  The von Hippel–Lindau tumour suppressor protein: O2 sensing and cancer , 2008, Nature Reviews Cancer.

[22]  P. Bedossa,et al.  Overexpression of the Oxygen Sensors PHD-1, PHD-2, PHD-3, and FIH Is Associated with Tumor Aggressiveness in Pancreatic Endocrine Tumors , 2008, Clinical Cancer Research.

[23]  B. Buchholz,et al.  HIF activation protects from acute kidney injury. , 2008, Journal of the American Society of Nephrology : JASN.

[24]  Yan Zeng,et al.  HIF‐1α Is a Regulator of Galectin‐3 Expression in the Intervertebral Disc , 2007 .

[25]  Kristina M. Cook,et al.  Asparaginyl Hydroxylation of the Notch Ankyrin Repeat Domain by Factor Inhibiting Hypoxia-inducible Factor* , 2007, Journal of Biological Chemistry.

[26]  D. Mukhopadhyay,et al.  Protein Kinase C-Mediated Modulation of FIH-1 Expression by the Homeodomain Protein CDP/Cut/Cux , 2007, Molecular and Cellular Biology.

[27]  T. Albert,et al.  Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. , 2007, American journal of physiology. Cell physiology.

[28]  P. Ratcliffe,et al.  Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH) , 2006, Proceedings of the National Academy of Sciences.

[29]  T. Albert,et al.  Nucleus pulposus cells express HIF‐1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment , 2006, Journal of cellular biochemistry.

[30]  D. Heinegård,et al.  Extracellular matrix in disc degeneration. , 2006, The Journal of bone and joint surgery. American volume.

[31]  L. Setton,et al.  Mechanobiology of the intervertebral disc and relevance to disc degeneration. , 2006, The Journal of bone and joint surgery. American volume.

[32]  K. Stenmark,et al.  Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression. , 2006, Molecular biology of the cell.

[33]  J. Cleveland,et al.  Two transactivation mechanisms cooperate for the bulk of HIF‐1‐responsive gene expression , 2005, The EMBO journal.

[34]  Patrick D. Sutphin,et al.  Coordinate Regulation of the Oxygen-Dependent Degradation Domains of Hypoxia-Inducible Factor 1α , 2005, Molecular and Cellular Biology.

[35]  K. Kivirikko,et al.  Catalytic Properties of the Asparaginyl Hydroxylase (FIH) in the Oxygen Sensing Pathway Are Distinct from Those of Its Prolyl 4-Hydroxylases* , 2004, Journal of Biological Chemistry.

[36]  J. Pouysségur,et al.  HIF prolyl‐hydroxylase 2 is the key oxygen sensor setting low steady‐state levels of HIF‐1α in normoxia , 2003, The EMBO journal.

[37]  K. Kivirikko,et al.  Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor* , 2003, Journal of Biological Chemistry.

[38]  W. Jelkmann,et al.  Intracellular localisation of human HIF-1α hydroxylases: implications for oxygen sensing , 2003, Journal of Cell Science.

[39]  J. Deisenhofer,et al.  Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Peet,et al.  FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. , 2002, Genes & development.

[41]  P. Ducheyne,et al.  Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2 , 2002, Cell and Tissue Research.

[42]  T. Tabira,et al.  X11L2, a new member of the X11 protein family, interacts with Alzheimer's beta-amyloid precursor protein. , 1999, Biochemical and biophysical research communications.

[43]  T. Südhof,et al.  Mint 3: a ubiquitous mint isoform that does not bind to munc18-1 or -2. , 1998, European journal of cell biology.

[44]  S. Bhattacharya,et al.  An essential role for p300/CBP in the cellular response to hypoxia. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[45]  L. Kauppila,et al.  Ingrowth of blood vessels in disc degeneration. Angiographic and histological studies of cadaveric spines. , 1995, The Journal of bone and joint surgery. American volume.

[46]  J. Glowacki,et al.  Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes. , 1994, The Journal of clinical investigation.