Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy.
Megakaryocytes (MKs) undergo an endomitotic cell cycle, leading to polyploidy. We examined the expression of the flavoproteins and oxidative stress-promoting enzymes, NADPH oxidases (Nox's), in MKs because of their known role in promoting the cell cycle. Although the expression of Nox isoforms varies between cell types, they are induced at the mRNA level by mitogenic stimuli. Western blotting or reverse transcription-polymerase chain reaction of purified mouse MKs isolated from thrombopoietin (TPO)-treated bone marrow (BM) cultures indicated high expression of Nox1, a weak expression of Nox4, and no significant expression of Nox2. Immunofluorescence of freshly isolated MKs confirmed strong expression of Nox1 in one-third of MKs, whereas Nox1 staining was detected in nearly all MKs in TPO-stimulated BM cultures. Treatment of mouse BM cultures with Nox inhibitors resulted in accumulation of MKs with low DNA content levels and significant reduction of higher ploidy MKs. Purified, Nox-inhibited MKs showed a notable decrease in the level of the G(1) phase cyclin E, a cyclin associated with MK polyploidy, and its up-regulation restored most of the effect of Nox inhibitors. Hence, this study shows the expression of Nox isoforms in MKs and highlights a potential role of flavoproteins in promoting polyploidization in this lineage.
McCrann
Donald J
DJ
Department of Biochemistry, Whitaker Cardiovascular Institute, Boston University School of Medicine, MA 02118, USA.
Eliades
Alexia
A
Makitalo
Maria
M
Matsuno
Kuniharu
K
Ravid
Katya
K
D000818:Animals; D001853:Bone Marrow / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D007527:Isoenzymes / Q000096:biosynthesis; D008533:Megakaryocytes / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D018345:Mice, Knockout; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis*; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011003:Ploidies; D013926:Thrombopoietin / Q000494:pharmacology; D046509:Tissue Culture Techniques
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D013926:Thrombopoietin; D009247:NADH, NADPH Oxidoreductases; C488842:Cybb protein, mouse; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C422943:Nox4 protein, mouse