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. 2008 Mar-Apr;14(3-4):141-9.
doi: 10.2119/2007-00011.Krasnov.

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Peter Krasnov  1 , Tatyana Michurina, Michael A Packer, Yuri Stasiv, Naoki Nakaya, Kateri A Moore, Kenneth E Drazan, Grigori Enikolopov
Affiliations

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Peter Krasnov et al. Mol Med. 2008 Mar-Apr.

Abstract

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.

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Figures

Figure 1
Figure 1
NOS expression in bone marrow cells. (A) Q-PCR analysis of NOS isoform expression. mRNA expression of each NOS isoform was determined in cultured stromal cells (SC) and hematopoietic cells (HC) using Q-PCR and isoform-specific primers. Data are presented as a ratio of expression level of NOS isoform in stromal cells to that in bone marrow. Data were analyzed using t test; *P < 0.005, **P < 0.0001, #P < 0.005. (B) Western blotting analysis of NOS isoform expression. Lysates of bone marrow (BM), primary bone marrow culture (PBMC), SC, HC, human endothelial cells (HE), and brain (BR) (latter two as control and reference) were analyzed by electrophoresis and blotting using antibodies against individual NOS isoforms and β-actin (loading control).
Figure 2
Figure 2
NOS expression in the fetal liver. (A) Fetal liver is enriched in nNOS. RNA expression of NOS isoforms was examined by Q-PCR in liver at 14 days of embryonic development (e14) and postnatal day 1 (p1) and in adult liver. Data for each NOS isoform are presented as ratio of expression level of NOS isoform in e14 and p1 liver to that in adult liver. Data were analyzed using t test; *P < 0.05, **P < 0.005, #P < 0.005. (B) nNOS expression in the fetal liver. Expression of nNOS was determined in sections of e14 (a), p1 (b), and adult (c) liver using monoclonal antibodies against nNOS. (d, e) Negative controls (omitting primary antibody) for e14 (d) and adult (e) liver. (C) Western blotting analysis of NOS isoform expression in the liver. Lysates of fetal liver at embryonic day 14 (Fl-e14), fetal liver at perinatal day 1 (Fl-p1), adult liver (AL), human endothelial cells (HE), macrophage (M), and brain (BR) (latter three as control and reference) were analyzed by electrophoresis and blotting using antibodies against individual NOS isoforms.
Figure 3
Figure 3
Expression of nNOS correlates with the supporting ability of stromal cell lines. (A) nNOS expression in a set of fetal liver-derived stromal cell lines with different supporting capability for hematopoietic stem cells. mRNA expression for each NOS isoform was determined by Q-PCR. Data are presented as a ratio of expression level of NOS isoform in a stromal line to that in murine bone marrow. Data were analyzed using t test; *P < 0.02. (B) Western blotting analysis of NOS expression in fetal liver-derived stromal cell lines. Lysates of stromal cells, bone marrow, and brain (as control and reference) were analyzed after electrophoresis using antibodies against NOS isoforms. (C) LTC-IC assay. The number of early hematopoietic progenitors was determined by cultivating hematopoietic cells on the AFT024 stromal cell layer for 4 weeks and replating in methylcellulose for colony formation. Data are presented as the number of LTC-IC per 1 × 104 bone marrow cells taken for analysis. Data were analyzed using t test; *P < 0.02 (D) nNOS expression in selected stromal cell lines. mRNA expression of NOS isoforms in several stromal lines differing in their ability to support hematopoietic stem cells and in cultured stromal cells from fetal liver (FLS) determined by Q-PCR. Data are presented as a ratio of expression level of NOS isoform in a line to that in murine bone marrow. Data were analyzed using t test; *P < 0.05, **P < 0.001.
Figure 4
Figure 4
Hematopoietic progenitors in bone marrow and spleen of NOS-knockout mice. Number of CFCs was determined for the bone marrow (BM) of wild-type (WT) and nNOS-, eNOS-, and iNOS-knockout mice and in the spleen of wild-type and nNOS-knockout mice. For each NOS knockout, data are presented as a ratio of frequency (number of CFCs per 104 cells) (A) or total number of CFCs per femur in knockouts to those in wild-type mice (B). Data were analyzed using t test; *P < 0.05.
Figure 5
Figure 5
Hematopoietic progenitors and mesenchymal stem cells in the NOS knockouts. Number of CFCs (A), hematopoietic cells (B), and stromal cells (C) per culture after cultivation of bone marrow cells from wild-type (WT) and NOS-knockout mice and spleen cells from wild-type and nNOS knockouts. Data are presented as a ratio of colonies or cells per culture in knockouts to those in wild-type mice. Data were analyzed using t test; *P < 0.05. (D) Addition of SNAP during cultivation decreases the number of hematopoietic progenitors. Bone marrow cells were cultivated in the Myelocult media with or without 250 μM SNAP for the indicated number of days, and the number of committed progenitors was determined using the CFC assay. The data are presented as a ratio of colonies per 104 cells after cultivation to the number of colonies in the starting culture (input), i.e., determined at day 0. Similar results were obtained when the CFC number per culture was calculated. Data were analyzed using t test; *P < 0.01, **P < 0.001, #P < 0.01, ##P < 5 × 10−7. (E) Number of CFC-Fs per 0.5 × 106 cells in the bone marrow of wild-type and nNOS-knockout mice. Data were analyzed using t test; *P < 0.05.
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