Aleksandra Krstić

Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells

We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-II, upregulation of proautophagic beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3β, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Akt inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis.

Interleukin-6 (IL-6) and low O2 concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)

Low O2 concentration (1%) favors the self‐renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL‐6 influences both stem cells and committed progenitors at 20% O2, we studied its effects in cultures at 1% O2. The pre‐CFC activity in Lin− population of mouse bone marrow was analyzed following 10 days of serum‐free culture in medium (LC1) supplemented with IL‐3 with and without IL‐6, at 20 and 1% O2 and phenotypic differentiation and proliferative history monitored. The IL‐6 receptor expression and initiation of VEGF‐A synthesis were also investigated. At 20% O2, the effects of IL‐6 on pre‐CFC were negligible but effects on CFC were apparent; conversely, at 1% O2, the IL‐6 enhances activity of pre‐CFC but not of CFC. Unlike at 20% O2, at 1% O2 a subpopulation of cells remained Lin− in spite of extensive proliferation. However, the absolute number of Lin− cells, did not correlate with pre‐CFC activity. A relative increase in VEGF transcripts at 1% O2 in presence of IL‐3 alone was enhanced by the addition of IL‐6. IL‐6 enhanced pre‐CFC activity at 1% O2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O2 does not exist in tissues in vivo, further studies in vitro at lower O2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007.

Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2

The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment.

The potential of interleukin-17 to mediate hematopoietic response.

It has long been known that T cells have the potential to modulate hematopoietic response in different ways. More recently, the importance of interleukin (IL)-17-secreting Th17 cells in T-cell-mediated regulation of hematopoiesis was indicated by the line of evidence that IL-17 links T-cell function and hematopoiesis through stimulation of granulopoiesis and neutrophil trafficking. Furthermore, our data demonstrated that IL-17 also affects other cells of hematopoietic system, such as erythroid progenitors, as well as mesenchymal stem cells. In order to better understand the regulatory role of IL-17 in hematopoiesis, molecular mechanisms underlying the effects of IL-17 on hematopoietic and mesenchymal stem cells were also studied.

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

Abstract.  In order to gain more insight into mechanisms operating on the haematopoietic activity of the T‐cell‐derived cytokine, interleukin‐17 (IL‐17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL‐17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL‐6, IL‐10, IGF‐I, IFN‐γ and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL‐17 did not affect granulocyte‐macrophage (CFU‐GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU‐E) progenitors was increased at 48 h, while the number of mature erythroid (CFU‐E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL‐17 also increased IL‐6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin− progenitor cells, demonstrated a slightly enhancing effect of IL‐17 on CFU‐GM and no influence on BFU‐E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL‐17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL‐17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU‐E found at the same time indicate that IL‐17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Signaling Pathways Implicated in Hematopoietic Progenitor Cell Proliferation and Differentiation

The objective of this study was to investigate the signal transduction pathways associated with the clonal development of myeloid and erythroid progenitor cells. The contribution of particular signaling molecules of protein tyrosine kinases (PTKs), mitogen-activated protein (MAP) kinase, and PI-3 kinase signaling to the growth of murine bone marrow colony forming unit–granulocyte-macrophage (CFU-GM) and erythroid (burst forming unit-erythroid [BFU-E] and colony forming unit-erythroid [CFU-E]) progenitors was examined in studies performed in the presence or absence of specific signal transduction inhibitors. The results clearly pointed to different signal transducing intermediates that are involved in cell proliferation and differentiation depending on the cell lineage, as well as on the progenitors’ maturity. Lineage-specific differences were obtained when chemical inhibitors specific for receptor- or nonreceptor-PTKs, as well as for the main groups of distinctly regulated MAPK cascades, were used because all of these compounds suppressed the growth of erythroid progenitors, with no major effects on myeloid progenitors. At the same time, differential involvement of MEK/extracellular signal-regulated kinase (ERK) MAPK transduction pathway was observed in the proliferation and/or differentiation of early, BFU-E, and late, CFU-E, erythroid progenitor cells. The results also demonstrated that phosphatydylinositol (PI)-3 kinase and nuclear factor kappaB (NF-κB) transcriptional factor were required for maintenance of both myeloid and erythroid progenitor cell function. Overall, the data obtained indicated that committed hematopoietic progenitors express a certain level of constitutive signaling activity that participates in the regulation of normal steady-state hematopoiesis and point to the importance of evaluating the impact of signal transduction inhibitors on normal bone marrow when used as potential therapeutic agents.

Low O 2 concentrations enhance the positive effect of IL-17 on the maintenance of erythroid progenitors during co-culture of CD34+ and mesenchymal stem cells

Co-culture of haematopoietic cells with a stromal cell layer does not mimic the physiological, micro-environmental niche, whose major feature is a low oxygen (O2) concentration. Thus, in order to study the effects of IL-17 in a context which better approximates the physiological state, we investigated its effects on cell expansion, colony-forming ability, and the phenotypical profile of normal, human blood CD34+ cells co-cultured for five days with MSC layers at various O2 concentrations (20%, 12.5% and 3% O2. We demonstrated that IL-17 enhances CD34+ and total CFC production during the five days of MSC/CD34+ co-culture. This effect depends upon the O2 concentration, reaching its maximum at 3% O2, and is more pronounced on erythroid progenitors (BFU-E). In addition, the stimulation of IL-6 production by IL-17 in MSC cultures and co-cultures is enhanced by low O2 concentration. The expression of some differentiation markers (CD34, CD13 and CD41) on haematopoietic cells in co-cultures also depends upon the oxygen concentration. Our results strengthen the concept that physiological levels of O2 (mistakenly called hypoxia), should be considered as an important environmental factor that significantly influences cytokine activity.

p38 MAPK signaling mediates IL-17-induced nitric oxide synthase expression in bone marrow cells

The effects of interleukin (IL)-17 on nitric oxide (NO) synthase (NOS) expression, as well as the participation of mitogen-activated protein kinases (MAPKs) in IL-17-mediated effects were examined in murine bone marrow cells. The results demonstrated the ability of IL-17 to upregulate the expression of mRNA for both inducible NOS and constitutive, endothelial NOS isoforms, as well as to enhance the phosphorylation of p38 MAPK. Moreover, both the NOS-inducing effect of IL-17 and the in vitro IL-17-mediated inhibition colony forming unit-erythroid (CFU-E) growth were dependent on p38 MAPK activity. The data demonstrating that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NOS inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME), implied that this effect is mediated through NOS activation. Besides revealing a link between the IL-17, NO, and haematopoiesis, data presented gave an insight into the mechanisms by which IL-17 exerts its modulatory effects on bone marrow cells.

IL-17 and FGF signaling involved in mouse mesenchymal stem cell proliferation.

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.

Interleukin-17 modulates myoblast cell migration by inhibiting urokinase type plasminogen activator expression through p38 mitogen-activated protein kinase.

Interleukin-17 belongs to a family of pro-inflammatory cytokines with pleiotropic effects, which can be associated with several inflammatory diseases of the muscle tissue. Although elevated levels of interleukin-17 have been described in inflammatory myopathies, its role in muscle homeostasis remains to be elucidated. The requirement of the urokinase type plasminogen activator in skeletal myogenesis was recently demonstrated in vivo and in vitro, suggesting its involvement in the regulation of extracellular matrix remodeling, cell migration and myoblast fusion. Our previous results have demonstrated that interleukin-17 inhibits myogenic differentiation of C2C12 myoblasts in vitro concomitantly with the inhibition of cell migration. However, the involvement of urokinase type plasminogen activator in interleukin-17-inhibited myogenesis and migration remained to be analyzed. Therefore, the effect of interleukin-17 on the production of urokinase type plasminogen activator by C2C12 myoblasts was determined in the present study. Our results demonstrated that interleukin-17 strongly inhibits urokinase type plasminogen activator expression during myogenic differentiation. This reduction of urokinase type plasminogen activator production corresponded with the inhibition of cell migration by interleukin-17. Activation of p38 signaling pathway elicited by interleukin-17 mediated the inhibition of both urokinase type plasminogen activator expression and cell migration. Additionally, IL-17 inhibited C2C12 cells migration by causing the cells to reorganize their cytoskeleton and lose polarity. Therefore, our results suggest a novel mechanism by which interleukin-17 regulates myogenic differentiation through the inhibition of urokinase type plasminogen activator expression and cell migration. Accordingly, interleukin-17 may represent a potential clinical target worth investigating for the treatment of inflammatory muscle diseases.