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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 5, September, 2014

GENERAL PATHOLOGY AND PATHOPHYSIOLOGY

Involvement of NF-κB-Dependent Signaling and p38 MAPK Signaling Pathway in the Regulation of Hemopoiesis during Restrain StressA. M. Dygai, V. V. Zhdanov, G. N. Zyuz’kov, E. V. Udut, L. A. Miroshnichenko, E. V. Simanina, A. V. Chaikovskii, L. A. Stavrova, M. G. Danilets, V. I. Agafonov, and T. Yu. Khrichkova

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 5, pp. 557-561, May, 2014Original article submitted December 22, 2013

Protein kinase p38 was shown to contribute to the increase in production of granulocyte CSF by microenvironmental cells under conditions of restraint stress. Stimulation of colony-forming activity was not accompanied by the increase in maturation of clonogenic structures in the bone marrow granulocytic stem. This process was realized with the involvement of NF-κB-dependent signaling and p38 MAPK signaling pathway. Our study showed that the p38 MAPK signaling pathway plays an important role in the regulation of granulocytopoiesis, but not of erythropoiesis.Key Words: signaling pathways; transcription factor NF-κB; MAPK p38; immobilization; granulocyte colony-stimulating factor

E. D. Goldberg Research Institute of Pharmacology, Siberian Division

of the Russian Academy of Medical Sciences, Tomsk, Russia. Address for correspondence: mmu@pharm.tsu.ru. A. M. Dygai

The exposure to various extreme factors that possess myeloinhibitory activity (cytostatics and ionizing ra-diation) and do not induce hypoplasia of the hemo-poietic tissue (stress, infl ammation, and blood loss) is accompanied by consecutive activation of individual components in the cascade regulatory mechanism for hemopoiesis. The major components of a regulatory system for hemopoiesis include the central neuroendo-crine mechanisms, cells of the hemopoiesis-inducing microenvironment (HIM), secreted cytokines, and components of the intercellular matrix, which regulate proliferation and differentiation of hemopoietic cells [4,6]. Experiments on the model of restraint stress (RS) revealed a signifi cant increase in the production of cytokines that improve colony-forming function of

the bone marrow [1,3]. Signaling cascades (STAT-, MAPK-, and PI3K/Akt-signaling cascades, including the trigger of nuclear factor NF-κB) have an impor-tant role in the production of these substances [9-14]. However, little is known about the basic principles for the formation, transduction, and transformation of humoral signals into variations of proliferation and differentiation of precursor cells under the infl uence of extreme factors. The role of various elements of signal transduction in the regulation of hemopoiesis under stress conditions should be studied in details.

Here we studied the role of NF-κB-dependent sig-naling and MAPK cascade in the regulation of hemo-poiesis on the model of RS.

MATERIALS AND METHODS

Experiments were performed on male (CBA×C57Bl/6)F1 mice (n=24) aging 2 months and weighing 20-22 g.

DOI 10.1007/s10517-014-2611-0

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The animals were obtained from the Department of Experimental Biological Models (E. D. Goldberg Re-search Institute of Pharmacology).

The animals were subjected to RS by gentle fi xa-tion in the supine position for 10 h [7]. The bone mar-row was sampled before (baseline, intact mice) and 3 days after RS [2].

Culture methods were used to study the direct ef-fects of a transcription factor NF-κB inhibitor aurothi-omalate and MAPK p38 inhibitor SB203580 (Calbio-chem) on the production of granulocyte CSF (G-CSF) and erythropoietin by adherent and nonadherent HIM cells. The in vitro working concentrations of inhibitors were estimated previously (50 μM aurothiomalate and 300 μM SB203580). The conditioned medium of bone marrow cells was obtained by incubation of adherent and nonadherent myelokaryocytes in a liquid culture medium at 37oC, 5% CO2, and 100% humidity for 24 h [2]. The contents of G-CSF and erythropoietin in conditioned media were measured by ELISA with R&D Systems and Biomerica, Inc. kits.

The number of committed precursor cells for gran-ulocytopoiesis and erythropoiesis in the bone marrow was studied in vitro by cloning of myelokaryocytes in a semisolid culture medium. The maturation inten-sity of granulocyte and erythroid precursor cells was evaluated from the index of maturation (ratio between the counts of clusters and colonies grown in the same well) [2]. Precursor cells were cultured under stan-dard conditions (methylcellulose medium MethoCult M3334 with erythropoietin; and methylcellulose me-dium MethoCult GF M3001 with granulocyte-macro-phage CSF; StemCell Technologies). Otherwise, these cells were cultured under specifi ed conditions with the following inhibitors at working concentrations: 50 μM aurothiomalate, 300 μM SB203580, 30 μM 2’,5’-dide-oxyadenosine (adenylate cyclase inhibitor), 50 μM LY 294002 (PI3K inhibitor), 0.02 μM Inhibitor IV (IKK-2 inhibitor), and 100 μM PD 98059 (MEK 1/2 inhibitor) (all inhibitors were from Calbiochem).

The results were analyzed by methods of variation statistics (Student’s t test and nonparametric Mann–Whitney U test).

RESULTS

A screening assay (inhibitory analysis) was performed to evaluate the role of MAPK- and PI3K/Akt-signaling cascades in the regulation of major cell functions. Ad-dition of inhibitors of PI3K, adenylate cyclase, IKK-2, and MEK 1/2 to the culture medium had no effect on the yield of granulocyte CFU (CFU-G). However, the inhibition of MAPK p38 and nuclear transcription fac-tor NF-κB was followed by a decrease in the number of CFU-G in the suspension of bone marrow cells from

intact animals (Table 1). These results indicate that p38 and NF-κB play a role in the regulation of proliferation and differentiation of progenitor cells under conditions of well-balanced hemopoiesis. Changes in the blood system induced by various extreme factors, as well as the mechanisms for these variations are nonspecifi c and similar. Taking into these data and results of our study, we hypothesized that signaling proteins serve as a com-ponent of the adaptive response during stress-induced hyperplasia of bone marrow hemopoiesis.

Previous studies have shown that myelokaryo-cyte count in the hemopoietic tissue of mice increases on day 6-7 after 10-h immobilization. These changes were mainly related to an increase in the number of cells in the erythroid and granulocytic hemopoietic stem, which resulted in the development of reticulo-cytosis, erythrocytosis, neutrophilia, and monocytosis in the peripheral blood [1,3].

Stimulation of granulocytopoiesis and erythropoi-esis was associated with an increase in colony-forming activity of the bone marrow. The yield of CFU-G and erythroid CFU was elevated on day 3-5 of the study [1,3]. Moreover, stimulation of colony formation is preceded by an increase in the production of IL-1 and IL-3 by adherent and nonadherent cells of the bone marrow during restraint stress. These substances in-duce the production of G-CSF by endothelial cells, macrophages, and fi broblasts. This hemopoietin is es-sential for the in vivo formation of granulocyte colo-nies [5]. Overproduction of proinfl ammatory cytokines IL-1, IL-6, and TNF-α can contribute to the decrease in erythropoietin level [8]. Therefore, we measured the content of hemopoietins in conditioned media of bone marrow cells after RS.

ELISA showed that G-CSF production by nonad-herent myelokaryocytes increases signifi cantly after RS (by 12 times in comparison with intact animals; Fig. 1, b). However, G-CSF production by adherent TABLE 1. Effect of Inhibitors on CFU-G Content in the

Sus pension of Nonadherent Bone Marrow Cells from

(CBA×C57Bl/6)F1 Mice (M±m)

Inhibitor CFU-G

Without inhibitor 23.00±2.12

p38 17.33±0.56*

NF-κB 18.57±0.11*

Pi3k 27.56±3.92

IKK2 13.86±6.09

cAMP 13.86±6.09

MEK 1/2 18.57±5.82

Note. *p<0.05 in comparison with cells from intact mice not treated

with the inhibitor.

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myelokaryocytes remained practically unchanged on day 3 after RS (Fig. 1, a). It was probably related to the late involvement of resident macrophages and stromal cells of the bone marrow in the reaction of he-mopoietic tissue to extreme factors [1,3]. Erythropoie-tin level in supernatants of adherent and nonadherent myelokaryocytes from stressed animals did not differ from that in intact mice (Fig. 1, c, d).

Addition of a protein kinase p38 inhibitor to the culture of nonadherent bone marrow cells from mice of the RS group was followed by a signifi cant decrease in G-CSF content in conditioned media (69.56±15.37 vs. 330.74±34.40 pg/ml in the absence of this inhibi-tor; Fig. 1, b). The inhibition of nuclear transcrip-tion factor had no effect on hemopoietin production by nonadherent bone marrow myelokaryocytes from stressed mice (Fig. 1, b). The increase in G-CSF ac-tivity of supernatants from adherent myelokaryocytes after the inhibition of signaling pathways under ex-treme conditions was similar to that observed in intact animals (Fig. 1, a).

Culturing of bone marrow cells from treated mice with test inhibitors was followed by the decrease in colony-forming activity. The maturation index of com-mitted precursor cells for granulocytopoiesis remained unchanged under these conditions (Fig. 2, a, b). There-fore, the NF-κB-dependent cascade and alternative protein kinase signaling pathway do not determine differentiation of hemopoietic precursor cells at this stage of RS-induced changes in the hemopoietic tissue.

The inhibition of NF-κB-dependent signaling and p38 MAPK signaling pathway had little effect on erythropoietin production by HIM cells (i.e., erythron system) after RS (Fig. 1, c, d). The in vitro inhibition of these signaling pathways did not modulate colony-forming activity and maturation intensity of erythroid precursor cells from the bone marrow of stressed ani-mals (Fig. 2, c, d).

A change in colony-forming properties of the bone marrow after treatment with inhibitors probably illustrate the involvement of NF-κB-dependent signal-ing and p38 MAPK signaling pathway in proliferative

Fig. 1. Contents of G-CSF (a, b) and erythropoietin (c, d) in conditioned media of adherent (a, c) and nonadherent myelokaryocytes (b,

d) from intact and stressed (CBA×C57Bl/6)F1 mice. Here and in Fig. 2: light bars, culturing without the inhibitor; shaded bars, culturing in

the presence of SB203580; dark bars, culturing in the presence of aurothiomalate.

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activity of CFU-G under stress conditions. Taking into account the dynamics of G-CSF level in supernatants from nonadherent HIM cells after addition of a p38 inhibitor, it can be concluded that hemopoietin pro-duction by mobile subpopulations of macrophages [1] during RS is a p38-dependent process. Therefore, the regulation of granulocytopoiesis during RS involves the alternative protein kinase signaling pathway. Functional activity of the erythroid stem under extreme conditions is probably regulated by other cascade reactions.

This work was supported by the grant of the Presi-dent of the Russian Federation for Young PhD Ccien-tists (MD-893.2013.7).REFERENCES 1. E. D. Goldberg, A. M. Dygai, and V. V. Zhdanov, Role of the

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Fig. 2. Number of CFU-G (a) and CFU-E colonies (c) and intensity of their differentiation (ClFU-G/CFU-G, b; and ClFU-E/CFU-E, d) in the

bone marrow from intact and stressed (CBA×C57Bl/6)F1 mice.

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