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.

Role of MAPK ERK1/2 and p38 in the Regulation of Secretory Functions of Different Populations of Neuroglia in Ethanol-Induced Neurodegeneration.

We studied the participation of ERK1/2 and p38 in secretion of neurotrophic growth factors by various types of neuroglia under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. The inhibitory role of these protein kinases in the production of neurotrophins by intact astrocytes and the absence of their participation in the regulation of functions of oligodendrocytes and microglial cells were shown. Under conditions of ethanol neurotoxicity, the role of ERK1/2 and p38 in the production of growth factors by glial elements was significantly changed. Neurodegeneration modeled in vitro led to inversion of the role of both protein kinases in the secretion of neurotrophins by astroglia and inhibition of the cytokine-synthesizing function of oligodendrocytes and microglial cells by ERK1/2 and p38. In mice receiving ethanol per os for a long time (as well as in cells in vitro exposed to ethanol), mitogen-activated kinases stimulated the function of astrocytes and inhibited the production of growth factors by microglial cells. At the same time, chronic alcoholization was accompanied by the appearance of the stimulating role of ERK1/2 and p38 in the implementation of the secretory function by oligodendrocytes.

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.

Specific Features of Intracellular Signal Transduction in the Regulation of Functions of Neural Stem Cells and Committed Neuronal Progenitors.

We studied the role of NF-κB-, cAMP/PKA-, JAKs/STAT3-, ERK1/2-, p38-, JNK- and p53- mediated signaling pathways in the realization of the growth potential of neural stem cells and committed neuronal progenitors under in vitro conditions. The method of pharmacological blockade with selective inhibitors of individual signaling molecules revealed some principal differences in their role in the determination of the proliferation and differentiation status of progenitor cells of different classes. Analysis of the peculiarities of intracellular signaling in cells and comparison of the role of its individual elements attest to the prospects of developing new drugs with neuroregenerative activity based on STAT3 inhibitors or JNK activators. These modulations of activity of signaling molecules can stimulate the realization of the growth potential of committed neuronal progenitors and neutral stem cells, respectively. The blockade of STAT3 and an increase in the content of phosphorylated forms of JNK had no "negative" effects on the functioning of multipotent neural stem cells and committed neuronal progenitors, respectively.

The Role of NF-κВ in the Realization of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The role of NF-κВ in the realization of the growth potential of neural progenitor cells from the subventricular area of cerebral hemispheres and secretion of neurotrophins by glial elements was studied under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. It was found that this transcription factor does not participate in the regulation of mitotic activity of neural stem cells and neuronal-committed progenitors under optimal conditions and under the influence of ethanol in vitro. At the same time, NF-κВ suppresses differentiation/maturation of neural progenitor cells. Long-term peroral administration of ethanol to mice was accompanied by the inhibitory influence of NF-κВ on proliferation of progenitor cells. Blockade of NF-κВ in neural stem cells and committed neuronal precursors in animals with neurodegeneration induced cell cycle progression in these elements. The involvement of NF-κВ in the secretory function of astrocytes and oligodendrogliocytes was established. Inactivation of the nuclear transcription factor reduced the production of neurotrophins, in particular, in the case of ethanol exposure. At the same time, no changes in the function of microglia were noted.

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.

Specific Roles of JAKs and STAT3 in Functions of Neural Stem Cells and Committed Neuronal Progenitors during Ethanol-Induced Neurodegeneration.

Peculiar roles of JAKs and STAT3 in realization of growth potential of various types of progenitor cells in neural tissue were examined during ethanol-induced neurodegeneration modeled both in vitro and in vivo. During in vitro action of C2H5OH, these signal molecules exerted the opposite effects on mitotic activity of multipotent neural stem cells and committed neural progenitors (the clonogenic PSA-NCAM+ cells). The JAKs and STAT3 inhibitors down-regulated the rate of neural stem cell division (proliferative activity) but up-regulated such activity of the committed neural progenitors. A long-term in vivo exposure of mice to ethanol inversed the roles of JAKs and STAT3 in determination of proliferative status of neural stem cells and eliminated involvement of JAKs in functional control over the committed progenitors of neurons. The data attest to much promise of STAT3 inhibitors in treatment of ethanol-induced CNS diseases as the remedies that stimulate realization of growth potential in multipotent neural stem cells and committed neural progenitors.

Responses of Blood System to Doxorubicin/Docetaxel Chemotherapy in Patients with Breast Cancer.

We studied the effects of combined chemotherapy with doxorubicin/docetaxel on erythroid and granulocytic hematopoietic lineages with particular attention focused on their recovery in patients with stages III-IV breast cancer. Intensification of differentiation of erythroid and granulocytic CFU (even under conditions of their suppressed proliferation) provided the increase in the content of mature and morphologically differentiated elements in the bone marrow and peripheral blood. High proliferative activity of erythroid and granulomonocytic precursors resulted from enhanced production of hematopoiesis-stimulating activities by microenvironment elements.

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.

Peculiarities of Intracellular Signal Transduction in the Regulation of Functions of Mesenchymal, Neural, and Hematopoietic Progenitor Cells.

The role of NF-κB, cAMP/PKA, JAKs/STAT3, ERK1/2, p38, JNK, and p53 signaling pathways in the realization of growth potential of mesenchymal, neural, erythroid, and granulomonocytic progenitor cells were examined in vitro. Using selective blockers of signaling molecules, we revealed some principal distinctions of their involvement in determination of proliferation-differentiation status of the progenitor cells of different functional classes. The most salient peculiarities were observed in the roles of cAMP/PKA, JNK, and JAKs/STAT3 signaling pathways in the control of functions of various types of the regeneration-competent elements. The specific features of intracellular signaling revealed in histogenetically and functionally different progenitor cells attest to visibility of differentiated pharmacological stimulation of regeneration in individual tissues and prospectiveness in the development of targeted remedies for regenerative medicine based on modifiers of activity of the intracellular signaling molecules.

Strategy of Pharmacological Regulation of Intracellular Signal Transduction in Regeneration-Competent Cells.

The study focuses on the development of principally novel priority-oriented healthcare strategy - targeted therapy in regenerative medicine known as Strategy of Pharmacological Control over Intracellular Signal Transduction in Regeneration-Competent Cells. It implies selective action of promising drugs on specific key elements in the signaling cascade responsible for functional activity of various progenitor cells (including stem cells) and elements of tissue microenvironment. The results of pioneer studies are described that were aimed on revealing the peculiarities in signal transduction and the role of distinct signaling molecules (the potential targets) in the control of cell cycle and other functions of progenitor elements and regulatory cells of different types. The models of some pathological states were employed to demonstrate the possibility of effective implementation of the advanced pharmacotherapeutic concept. The developed theoretical and applied platform can be used to launch synthesis of principally novel preparations with regenerative activity.

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.

Mechanisms of Granulocytopoiesis Recovery Stimulation with Filgrastim in Breast Cancer Patients Receiving Chemotherapy.

We studied myelotoxicity of modern schemes of chemotherapy for breast cancer (docetaxel/doxorubicin and cyclophosphamide/doxorubicin/5-fluorouracil) towards granulocytopoiesis, the mechanisms determining the differences of hematological effects of these schemes, and the efficiency of correction of the observed changes with granulocyte CSF (filgrastim). Granulocytopoiesis stimulation with filgrastim during the treatment with docetaxel/doxorubicin combination was more pronounced than during cyclophosphamide/doxorubicin/5-fluorouracil therapy. The observed differences were found at all levels of granulocyte lineage organization (central and peripheral), which is related to different effects of the cytostatic substances used in the proposed protocols on the structures controlling hemopoiesis.

Role of JAK/STAT3 Signaling in Functional Stimulation of Mesenchymal Progenitor Cells with Alkaloid Songorine.

JAK/STAT signaling pathway was examined comparatively during realization of growth potential of mesenchymal progenitor cells stimulated with diterpene alkaloid songorine or fibroblast growth factor. The stimulating role of JAKs and STAT3 on the mitotic activity and differentiation of progenitor cells cultured with songorine was revealed. Under these conditions, the study demonstrated suppression of fibroblast colony formation against the background of reduced number of actively proliferating CFU-fibroblasts and a drop of differentiation index of progenitor cells induced by pan-JAKs and STAT3 inhibitors. The observed changes were in almost complete agreement with the character of functional reactions of the progenitor elements in response to blockade of JAKs and STAT3 with fibroblast growth factor. In addition, blockade of JAKs with this factor enhanced the differentiation rate of the progenitor cells.