Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor.
BACKGROUND
Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.


METHODS
Differentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 microg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.


RESULTS
HIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.


CONCLUSIONS
Expression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1alpha protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.
Westra
Johanna
J
Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Groningen, the Netherlands. j.westra@med.umcg.nl
Brouwer
Elisabeth
E
van Roosmalen
Ingrid A M
IA
Doornbos-van der Meer
Berber
B
van Leeuwen
Miek A
MA
Posthumus
Marcel D
MD
Kallenberg
Cees G M
CG
D001168:Arthritis / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D015415:Biomarkers / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D016209:Interleukin-8 / Q000187:drug effects / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000187:drug effects / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism / Q000503:physiopathology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D015415:Biomarkers; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016209:Interleukin-8; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D020780:Matrix Metalloproteinase 9