Features of Intracellular Signal Transduction in Neural Stem Cells under the Influence of Alkaloid Songorine, an Agonist of Fibroblast Growth Factor Receptors.

We performed a comparative in vitro study of the involvement of NF-κB, PI3K, cAMP, ERK1/2, p38, JAKs, STAT3, JNK, and p53-dependent intracellular signaling in the functioning of neural stem cells (NSC) under the influence of basic fibroblast growth factor (FGF) and FGF receptor agonist, diterpene alkaloid songorine. The significant differences in FGFR-mediated intracellular signaling in NSC were revealed for these ligands. In both cases, stimulation of progenitor cell proliferation occurs with the participation of NF-κB, PI3K, ERK1/2, JAKs, and STAT3, while JNK and p53, on the contrary, inhibit cell cycle progression. However, under the influence of songorin, cAMP- and p38-mediated cascades are additionally involved in the transmission of the NSC division-activating signal. In addition, unlike FGF, the alkaloid stimulates progenitor cell differentiation by activating ERK1/2, p38, JNK, p53, and STAT3.

Participation of cAMP-Mediated Signaling Transduction in the Regulation of the Secretory Function of Neuroglia during Ethanol-Induced Neurodegeneration.

We studied the involvement of cAMP and PKA in the regulation of the secretion of neurotrophic growth factors by macro-and microglial cells in the model of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP in the secretion of neurotrophins by intact astrocytes and oligodendrocytes was shown, while PKA does not participate in this process. On the contrary, the inhibitory role of cAMP (implemented via PKA activation) in the production of neurogenesis stimulators by microglial cells under conditions of optimal vital activity was found. Under the influence of ethanol, the role of cAMP and PKA in the production of growth factors by macroglial cells was considerably changed. The involvement of PKA in the cAMP-dependent signaling pathways and inversion of the role of this signaling pathway in the implementation of the neurotrophic secretory function of astrocytes and oligodendrocytes, respectively, directly exposed to ethanol in vitro were noted. Long-term exposure of the nervous tissue to ethanol in vivo led to the loss of the stimulating role of cAMP/PKA signaling on neurotrophin secretion by macroglial cells without affecting its inhibitory role in the regulation of this function in microglial cells.

Psychopharmacological Effects of Stimulation of the Functions of Neural Stem Cells by STAT3 Inhibitor under Conditions of Modeled Ethanol-Induced Encephalopathy.

The psychopharmacological effects of a stimulator of functions of progenitor cells of the nervous tissue STAT3 inhibitor (STAT3 Inhibitor XIV, LLL12) were studied under conditions of modeled alcoholic encephalopathy in C57BL/6 mice. The pharmacological agent corrected the parameters of exploratory behavior (characterizing predominantly cognitive activity) in the experimental animals at the late terms of observation. At the same time, the reproducibility of the conditioned passive avoidance response developed at the beginning of the course STAT3 inhibitor administration decreased. These effects developed against the background of a significant increase in the content of neural stem cells and their proliferative activity in the paraventricular zone of the brain.

The Role of JAK and STAT3 in Regulation of Secretory Function of Neuroglial Cells of Different Types in Ethanol-Induced Neurodegenerationt.

The effects of JAK and STAT3 inhibitors on the production of neurotrophic growth factors by different types of neuroglial cells were studied under conditions of in vitro and in vivo models of ethanol-induced neurodegeneration. It was shown that these signaling molecules do not participate in the secretion of neurotrophins by intact astrocytes and oligodendrocytes. The inhibitory role of JAK in the regulation of this function of microglial cells was revealed. We also revealed significant changes in the role of JAK and the presence of STAT3 specifics within the framework of JAK/STAT signaling in the production of growth factors by various glial elements under the influence of ethanol. Neurodegeneration modeled in vitro led to the appearance of a "negative" effect of STAT3 on the production of neurogenesis stimulants by all types of glial cells. Moreover, the role of STAT3 in oligodendrocytes and microglial cells generally corresponded to that of JAK/STAT signaling. In astrocytes, only selective blockade of STAT3 (but not JAK) led to stimulation of their function. In mice subjected to prolonged peroral alcoholization, the neuroglial responses to the pharmacological regulation of JAK/STAT signaling were different. An inversion of the role of JAK and STAT3 in the production of neurotrophins by oligodendrocytes was noted. In addition, JAK inhibitor did not stimulate secretory function of microglial cells under conditions of prolonged exposure to ethanol in vivo.

The Role of Intracellular Signaling Molecules in the Production of Granulocytic CSF Mononuclear Phagocytes in Stress and Cytostatic Influence.

Under conditions of steady-state hemopoiesis, nuclear factor NF-κB, in contrast to MAP kinase p38, plays an important role in the maintenance of the initial level of secretory activity of monocytes. The increase in the production of G-CSF under stress conditions (10-h immobilization) is mainly regulated by the alternative p38MARK signaling pathway via activation of p38 synthesis. It was shown that under conditions of cytostatic-induced myelosuppression, the production of protein kinase p38 in cells decreases, and it, like NF-κB, is not the main one in the production of hemopoietin by mononuclear phagocytes.

Role of JNK and p53 in Implementation of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue.

We studied the participation of JNK and p53 in the realization of the growth potential of different types of progenitors of the subventricular zone of mouse brain and secretion of neurotrophins by glial cells. The stimulating role of these signaling molecules in mitotic activity and specialization of multipotent neural stem cells was shown. It was found that JNK and p53 do not participate in the regulation of committed neuronal progenitor cells (clonogenic PSA-NCAM+ cells). A dependence of neurotrophic growth factors in individual populations of neuroglia on activity of these protein kinase and transcription factor was revealed. The role of JNK and p53 in astrocytes consists in stimulation of their secretion, and in microglial cells, on the contrary, in its inhibition. The secretory neurotrophic function of oligodendrogliocytes is not associated with JNK and p53 activity.

Involvement of Signaling Cascades in Granulocytopoiesis Regulation under Conditions of Cytostatic Treatment.

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Hemostimulating Effects of c-Jun N-Terminal Kinase (JNK) Inhibitor during Cytostatic Myelosuppression and Mechanisms of Their Development.

The hemostimulating effects of c-Jun N-terminal kinase (JNK) inhibitor were examined on the mouse model of myelosuppression provoked by 5-fluorouracil. Blockade of JNK during postcytostatic period accelerated recovery of granulomonocytopoiesis and erythropoiesis. It also increased the content of neutrophilic granulocytes and erythroid cells in the hematopoietic tissue and elevated the counts of neutrophils and reticulocytes in the peripheral blood. The development of these phenomena resulted from elevated content and up-regulated functional activity of bone marrow hematopoietic progenitors associated with the direct action of JNK inhibitor on these progenitors and enhanced secretion of hemopoietins by stromal elements of the hematopoiesis-inducing microenvironment.

Participation of cAMP/PKA-Mediated Signaling Pathways in Functional Activity of Regeneration-Competent Cells in the Nervous Tissue under Conditions of Ethanol-Induced Neurodegeneration.

We studied the involvement of cAMP/PKA signaling in the realization of the growth potential of neural progenitors and secretion of neurotrophic growth factors by glial elements under conditions of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP and PKA in cell cycle progression of the neural progenitor cells and in production of neurotrophins by the cells in nervous tissue under the optimal conditions to vital activity was demonstrated. Ethanol inverted the role of cAMP/PKA signaling pathways in determination of the proliferation-differentiation status of neural stem cells. Selective blockade of adenylate cyclase or PKA in neural stem cells increased the rate of their division against the background of relative decrease in differentiation rate. In addition, cAMP/PKA signaling does not longer participate in neurotrophin production by glial cells in neurodegeneration. These findings suggest that inhibitors of activity/expression of adenylate cyclase and PKA can be considered as possible drugs with regenerative activity for the treatment of nervous system pathologies provoked by alcohol.

Role of MAPK ERK1/2 and p38 in the Realization of Growth Potential of Various Types of Regeneration-Competent Cells in Mouse Neural Tissue during Ethanol-Induced Neurodegeneration In Vitro.

Ethanol-induced neurodegeneration was modeled in vitro to study the roles of ERK1/2 and p38 in realization of the growth potential of neural progenitor cells and secretion of neurotrophic growth factors by glial elements. Addition of the neurotoxic dose of C2H5OH (65 mM) to the culture medium abolished the effects of specific ERK1/2 and p38 inhibitors on the formation of colonies (neurospheres) and proliferative activity of neural CFU in cultured cells derived from paraventricular region of the mouse brain. The study established that these protein kinases are not implicated in ethanol-induced stimulation of the formation of neural CFU, differentiation of neural progenitors, and synthesis of humoral functional regulators of neural CFU by glial cells.

Functional State of Various Types of Regeneration-Competent Cells in the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The in vitro and in vivo models of ethanol-induced neurodegeneration were used to evaluate the content and functional activity of various types of regeneration-competent cells in subventricular zone of the cerebral hemispheres in C57Bl/6JY mice. In nervous tissue culture, ethanol (65 mM) produced no effect on formation of neurospheres. When administered per os in a daily dose of 3 g/kg for 8 weeks, ethanol produced no effect on the number of neural CFU in situ. In both cases, ethanol reduced proliferative activity of neural CFU. Long-term administration of ethanol in vivo suppressed differentiation of neural stem cells and decreased the number of committed precursors (neural cluster-forming units) in the subventricular zone of cerebral hemispheres. In vitro application of ethanol stimulated secretion of humoral growth factors by the cluster-forming neural glial cells. In contrast, in vivo administration of ethanol suppressed this secretion.

Role of Signaling Molecules in the Regulation of Granulocytopoiesis during Stress-Inducing Stimulation.

The role of signaling molecules in synthesis of humoral regulators of granulocytopoiesis by the hematopoietic microenvironmental cells during stress was analyzed using specific inhibitors. The major role in stimulation of the synthesis of granulocytic CSF during stressful stimulation is played by PI3K/Akt signaling cascade. Nuclear transcription factor NF-κB plays an auxiliary role in the regulation of functional activity of the bone marrow mononuclears. However, this factor affects the synthesis of granulocytic CSF by CD4+ cells of the bone marrow in response to stressful stimulation. Different degree and specific character of involvement of the signaling proteins in the regulation of the production of humoral factors determining colony-stimulating activity are explained by changes in functional state of monocyte-derived macrophages in different periods of stress response.

Particular Role of JAK/STAT3 Signaling in Functional Control of Mesenchymal Progenitor Cells.

The role of JAK/STAT3-mediated signaling pathway in the realization of the growth potential of mesenchymal precursor cells was examined in vitro. The stimulating role of JAKs and STAT3 towards proliferating activity of progenitor cells and their different role in the regulation of differentiation of the progenitor elements were demonstrated. Inhibitors of JAKs and STAT3 reduced the yield of fibroblast CFU and their mitotic activity. Blockade of JAKs accelerated and selective inactivation of STAT3 decelerated differentiation of progenitor cells.

Role of JAK1, JAK2, and JAK3 in Functional Stimulation of Mesenchymal Precursor Cells by Alkaloid Songorine.

We studied the role of some JAK in the effect of diterpene alkaloid songorine on realization of the growth potential of mesenchymal precursor cells. The participation of JAK1, JAK2, and JAK3 in stimulation of proliferation of the precursor cells was demonstrated. Specific inhibitors of these JAK reduced the yield of fibroblast CFU and the rate of their division. Inhibition of JAK2 against the background of songorine treatment increased the rate of precursor differentiation.

Role of NF-κB, PI3K, MAPK/ERK 1/2, and p38 in Erythropoietin Production by Bone Marrow Nuclears under Conditions of Immobilization Stress.

The involvement of the studied signal cascades in the regulation of erythropoietin production by bone marrow nuclears under conditions of immobilization stress depends on the type of the hemopoiesis-inducing microenvironment cells and the period of blood system reaction to stress exposure. Secretory activity of monocytes is regulated mainly by PI3K improving cell resistance to disturbances. The functional role of signal cascades involved in the production of erythropoietin by T cells is determined by the stage of the common adaptation syndrome.

Role of PI3K, MAPK/ERK 1/2, and p38 in Production of Erythropoietic Activity by Bone Marrow Cells after Blood Loss.

The leading role in the regulation of erythropoietic activity of adherent bone marrow cells under conditions of post-hemorrhagic anemia is played by classical MAP kinase pathway (ERK pathway). Erythropoietin is not the decisive factor in the formation of erythropoietic activity of adherent cells. PI3K, MAPK/ERK 1/2, and p38-signaling proteins are not the main regulators of local production of erythropoietin after 30% loss of circulating blood volume.

Implication of JAK1, JAK2, and JAK3 in the Realization of Proliferation and Differentiation Potential of Mesenchymal Progenitor Cells In Vitro.

Involvement of individual JAK kinases in the realization of growth potential of mesenchymal progenitor cells was examined in vitro. Important role of JAK2 and JAK3 in determining the initial level of mitotic activity of progenitor cells was established. The yield of fibroblast CFUF was suppressed under the effect of specific inhibitors of JAK kinases. Blockade of JAK3 increased the rate of progenitor element differentiation. JAK1 had no effect on proliferation and differentiation status of progenitor cells.

Role of PI3K, ERK, and p38 Signaling Pathways in the Production of Humoral Erythropoiesis Regulators under Normal Conditions.

PI3- and MAP-kinase signaling pathways duplicate and interchange each other in production of agents that determine total erythropoietic activity under conditions of balanced erythropoiesis. The alternative p38-dependent MAP-kinase pathway is the major regulator of erythropoietic activity of adherent bone marrow cells. Blockade of PI3K and p38 signaling pathways stimulated production of erythropoietin by cells that do not produce it constitutively.

Role of JNK and Contribution of p53 to the Realization of the Growth Potential of Mesenchymal Precursor Cells under the Effect of Fibroblast Growth Factor.

The role of JNK-mediated signal pathway and participation of p53 transcription factor in stimulation of mesenchymal precursor cell function by the fibroblast growth factor was studied. The levels of fibroblast colony- and cluster formation and proliferative activity of mesenchymal precursors increased in response to JNK and p53 specific inhibitors. JNK and p53 blockers did not change the rate of fibroblast growth factor-induced progenitor element differentiation.

Role of JNK and Contribution of p53 into Growth Potential of Mesenchymal Progenitor Cells In Vitro.

Specific JNK and p53 inhibitors stimulated the formation of fibroblast colonies (CFU-F) and clusters (ClFU-F) and increased proliferative activity of mesenchymal progenitor cells. No effects of inhibitors of JNK and p53 on differentiation of progenitor elements were revealed.