Jelena Kocic

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 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.

Rac1 modulates TGF-β1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes

Transforming growth factor‐β1 (TGF‐β1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant‐negative N17TRac1 abolished TGF‐β1‐induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase‐9 (MMP9) production to levels comparable to those induced by TGF‐β1, while N17TRac1 was inhibitory. TGF‐β1‐mediated EMT involves the expression of the E‐cadherin repressor Snail1, regulated by the Rac1 and mitogen‐activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK‐dependent, as the MEK inhibitor PD98059 decreased TGF‐β1‐induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF‐β1‐mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.

SMAD3 is essential for transforming growth factor-β1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes.

Transforming growth factor-β1 (TGF-β1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-β1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-β1 signalling was interfered by the chemical inhibitor of the TGF-β1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-β1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-β1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-β1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-β1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-β1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-β1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-β1 in transformed 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.

New ruthenium(II) complexes with N-alkylphenothiazines: Synthesis, structure, in vivo activity as free radical scavengers and in vitro cytotoxicity

Three new complexes of the general formula L[RuCl(3)(DMSO)(3)] (1-3), where L = chlorpromazine hydrochloride, trifluoroperazine dihydrochloride or thioridazine hydrochloride, were prepared and characterized by elemental analysis and spectroscopic methods (FT-IR, UV-Vis, (1)H NMR and (13)C NMR). In addition, the crystal structure of the complex 2 containing trifluoroperazine dihydrochloride was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P2(1)/n, with a = 10.4935(7) A, b = 18.6836(12) A, c = 19.9250(13) A, beta = 98.448(2) degrees, V = 3864.0(4) A(3). The structure was refined to the agreement factors of R = 4.79%, R(w) = 11.23%. The effect of three different doses (0.4, 4.5 and 90.4 microM/kg bw) of complex 2 on superoxide dismutase (SOD) and catalase (CAT) activity was investigated under physiological conditions. Influence on nitrite production (NO(2)(-)) and the level of erythrocytes malondialdehyde (MDA) in rats blood was also evaluated. Complex 2 did not affect the CAT enzyme activity in vivo and did not cause the hydroxyl radicals production. In the 0.4 and 4.5 microM/kg bw doses it showed almost the same or lower SOD activity and nitrite levels, while the dose of 90.4 microM/kg bw significantly increased these parameters. Finally, the cytotoxicity of complexes were assayed in four human carcinoma cell lines MCF-7, MDA-MB-453 (breast carcinoma), SW-480 (colon adenocarcinoma) and IM9 (myeloma multiple cells). Antiproliferative activity in vitro with low IC(50) during 48 h of treatment was observed.

SKIP is required for TGF-β1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor‐β1 (TGF‐β1) potently induces the epithelial‐mesenchymal transition (EMT) during tumoral progression. Although Sky‐interacting protein (SKIP) regulates TGF‐β1‐induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF‐β1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS‐S, Smad3 activation decreased, along with an inhibition of TGF‐β1‐induced EMT, and the cells were sensitized to the TGF‐β1‐dependent inhibition of proliferation. Also, AS‐S cells showed a weaker migration and invasion response. Moreover, TGF‐β1‐induced urokinase‐type plasminogen activator expression was inhibited, concomitantly with a TGF‐β1‐independent increment of the plasminogen‐activator inhibitor‐1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF‐β1 in transformed cells.

Transforming growth factor-β superfamily, implications in development and differentiation of stem cells

Abstract Transforming growth factor-β (TGF-β) family members, including TGF-βs and bone morphogenetic proteins (BMPs), play important roles in directing the fate of stem cells. In embryonic stem cells, the TGF-β superfamily participates in almost all stages of cell development, such as cell maintenance, lineage selection, and progression of differentiation. In adult mesenchymal stem cells (MSCs), TGF-βs can provide competence for early stages of chondroblastic and osteoblastic differentiation, but they inhibit myogenesis, adipogenesis, and late-stage osteoblast differentiation. BMPs also inhibit adipogenesis and myogenesis, but they strongly promote osteoblast differentiation. The TGF-β superfamily members signal via specific serine/threonine kinase receptors and their nuclear effectors termed Smad proteins as well as through non-Smad pathways, which explain their pleiotropic effects in self-renewal and differentiation of stem cells. This review summarizes the current knowledge on the pleiotropic effects of the TGF-β superfamily of growth factors on the fate of stem cells and also discusses the mechanisms by which the TGF-β superfamily members control embryonic and MSCs differentiation.

Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice

Aim:  The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)‐17 and the specific role of nitric oxide (NO) in IL‐17‐induced effects in the process of haematopoiesis.