NO and TNF-alpha released from activated macrophages stabilize HIF-1alpha in resting tubular LLC-PK1 cells.
Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1alpha (subjected to protein stability regulation) and constitutively expressed HIF-1beta. Besides hypoxia, diverse agonists are identified that stabilize HIF-1alpha during normoxia. Here we used a coculture system of RAW 264.7 macrophage cells and tubular LLC-PK(1) cells to establish that lipopolysaccharide- and interferon-gamma-stimulated but not resting macrophages elicited HIF-1alpha accumulation in LLC-PK(1) cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application of tumor necrosis factor-alpha (TNF-alpha)-neutralizing antibodies, we identified NO and TNF-alpha as signaling molecules. Working in concert, NO and TNF-alpha have a stronger response when allowed direct cell-to-cell contact instead of contact with only the cell supernatant of activated macrophages. We show that signal transmission by NO with TNF-alpha in LLC-PK(1) cells is mediated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is blocked by wortmannin or dominant-negative forms of PI3-K as well as protein kinase B. We conclude that NO and TNF-alpha, derived from activated macrophages, provoke HIF-1alpha stabilization in LLC-PK(1) cells under normoxic conditions, which underscores HIF-1alpha stabilization due to intercellular regulation.
Zhou
Jie
J
Department of Cell Biology, Faculty of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany.
Fandrey
Joachim
J
Schümann
Jens
J
Tiegs
Gisa
G
Brüne
Bernhard
B
D000818:Animals; D002450:Cell Communication / Q000502:physiology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007249:Inflammation / Q000378:metabolism / Q000503:physiopathology; D018836:Inflammation Mediators / Q000378:metabolism; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018836:Inflammation Mediators; D009569:Nitric Oxide; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha