Contributions
Abstract: EP759
Type: E-Poster Presentation
Session title: Hematopoiesis, stem cells and microenvironment
Background
Nitric oxide (NO) is an ubiquitous signaling molecule that interacts with glucocortiocids in response to perturbed tissue homeostasis under hypoxic or inflammatory conditions. Using a mouse model of psychological stress, we have recently shown that chronic stress induces initial erythroid progenitor cell expansion by modulating the production of NO in the bone marrow. However, it remains unknown whether this stress-induced effect requires a NO-glucocorticoid receptor (GR) crosstalk.
Aims
This study was aimed to examine 1) a role of GR in initial stress-induced erythroid progenitor cell response and 2) a possible interaction between GR and NO in the bone marrow during chronic stress.
Methods
Adult male mice were subjected to 2h daily restraint stress for 7 consecutive days. The role of GR was assessed by subcutaneous administration of the GR antagonist mifepristone (RU486), 60 min prior to each restraint procedure. The number of bone marrow erythroid progenitors burst forming units-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) was determined using colony assays. In order to examine a possible GR/NO interplay in the bone marrow during chronic stress, we used a blockade of NO biosynthesis, and mice were randomly assigned to following groups: (1) R - restraint group, exposed to daily restraint stress for 7 consecutive days; (2) L-NAME + R group, received a subcutaneous injection of L-NAME (non-selective nitric oxide synthase inhibitor) 30 min prior daily restraint; (3) L-NAME group, treated with L-NAME only; or (4) control group. To analyze GR activation, we assessed the nuclear translocation of GR by examining the level of GR expression in bone marrow nuclear cell fraction, using Western blot technique, and compared the protein expression levels among experimental groups. Plasma corticosterone levels were determined using a 125I-coupled double antibody radioimmunoassay.
Results
Chronic stress induced a marked increase in the number of BFU-E (two-way ANOVA; F(1,12)=29.996; Bonferroni adjustment, p<0.001) and CFU-E (two-way ANOVA; F(1,12)=28.946; Bonferroni adjustment, p<0.001) progenitors in the bone marrow. Blockade of GR with RU486 prior to daily stress completely abolished the effect of repeated stress on both BFU-E (two-way ANOVA; F(1,12)=27.301; Bonferroni adjustment, p<0.001) and CFU-E cells (two-way ANOVA; F(1,12)=28.168; Bonferroni adjustment, p<0.001). Exposure to chronic stress significantly increased the expression of GR in bone marrow nuclear cell fraction (two-way ANOVA F(1,12)=13.036, P<0.01), and blockade of NO biosynthesis prior to each restraint did not alter stress-induced incresae in nuclear GR protein expression (two-way ANOVA; F(1,12)=0.132; p>0.05). In accordance, analysis of corticosterone levels among experimental groups revealed a highly significant difference in corticosterone concentration regarding the main effect for stress (two-way ANOVA; F(1,12)=159.436, p<0.001), while there was no significant main effect for the blockade of NO biosynthesis (two-way ANOVA; F(1,12) =0.775, p>0.05).
Conclusion
These findings suggest a crucial role for GR in the expansion of bone marrow erythroid progenitors under conditions of chronic stress. Although both GR and NO are required for stress-induced initial erythroid progenitor cell expansion, their interaction within bone marrow microenvironment is not essential for observed effect during chronic stress.
Keyword(s): Bone Marrow, Erythroid progenitor, Nitric oxide
Abstract: EP759
Type: E-Poster Presentation
Session title: Hematopoiesis, stem cells and microenvironment
Background
Nitric oxide (NO) is an ubiquitous signaling molecule that interacts with glucocortiocids in response to perturbed tissue homeostasis under hypoxic or inflammatory conditions. Using a mouse model of psychological stress, we have recently shown that chronic stress induces initial erythroid progenitor cell expansion by modulating the production of NO in the bone marrow. However, it remains unknown whether this stress-induced effect requires a NO-glucocorticoid receptor (GR) crosstalk.
Aims
This study was aimed to examine 1) a role of GR in initial stress-induced erythroid progenitor cell response and 2) a possible interaction between GR and NO in the bone marrow during chronic stress.
Methods
Adult male mice were subjected to 2h daily restraint stress for 7 consecutive days. The role of GR was assessed by subcutaneous administration of the GR antagonist mifepristone (RU486), 60 min prior to each restraint procedure. The number of bone marrow erythroid progenitors burst forming units-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) was determined using colony assays. In order to examine a possible GR/NO interplay in the bone marrow during chronic stress, we used a blockade of NO biosynthesis, and mice were randomly assigned to following groups: (1) R - restraint group, exposed to daily restraint stress for 7 consecutive days; (2) L-NAME + R group, received a subcutaneous injection of L-NAME (non-selective nitric oxide synthase inhibitor) 30 min prior daily restraint; (3) L-NAME group, treated with L-NAME only; or (4) control group. To analyze GR activation, we assessed the nuclear translocation of GR by examining the level of GR expression in bone marrow nuclear cell fraction, using Western blot technique, and compared the protein expression levels among experimental groups. Plasma corticosterone levels were determined using a 125I-coupled double antibody radioimmunoassay.
Results
Chronic stress induced a marked increase in the number of BFU-E (two-way ANOVA; F(1,12)=29.996; Bonferroni adjustment, p<0.001) and CFU-E (two-way ANOVA; F(1,12)=28.946; Bonferroni adjustment, p<0.001) progenitors in the bone marrow. Blockade of GR with RU486 prior to daily stress completely abolished the effect of repeated stress on both BFU-E (two-way ANOVA; F(1,12)=27.301; Bonferroni adjustment, p<0.001) and CFU-E cells (two-way ANOVA; F(1,12)=28.168; Bonferroni adjustment, p<0.001). Exposure to chronic stress significantly increased the expression of GR in bone marrow nuclear cell fraction (two-way ANOVA F(1,12)=13.036, P<0.01), and blockade of NO biosynthesis prior to each restraint did not alter stress-induced incresae in nuclear GR protein expression (two-way ANOVA; F(1,12)=0.132; p>0.05). In accordance, analysis of corticosterone levels among experimental groups revealed a highly significant difference in corticosterone concentration regarding the main effect for stress (two-way ANOVA; F(1,12)=159.436, p<0.001), while there was no significant main effect for the blockade of NO biosynthesis (two-way ANOVA; F(1,12) =0.775, p>0.05).
Conclusion
These findings suggest a crucial role for GR in the expansion of bone marrow erythroid progenitors under conditions of chronic stress. Although both GR and NO are required for stress-induced initial erythroid progenitor cell expansion, their interaction within bone marrow microenvironment is not essential for observed effect during chronic stress.
Keyword(s): Bone Marrow, Erythroid progenitor, Nitric oxide