4-Thiazolidinone-based derivaTives rescue Tna α-inhibiTed osTeoblasT differenTiaTion in mouse mesenchymal precursor cells

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease of yet unknown etiology. Tumor necrosis factor α (TNFα) is recognized as a regulatory substance that plays a central role in RA development and progression. On the other side, the bone morphogenetic protein (BMP) and Wnt signaling pathways are key mechanisms that induce and support cartilage and bone formation and maintenance. Previous studies showed that the pro-inflammatory cytokines TNFα and interleukin 1β (IL-1β) are central players in the inhibition of activity of skeletogenesis. The aim of this study was to evaluate the anti-inflammatory activity of novel 4-thiazolidinone-based derivatives towards TNFα–induced pro-inflammatory effects during bone formation. We performed in vitro evaluation of functional effects of 4-thiazolidinones denoted as Les-4368, Les-4370, Les-3882 and Les-3288 that were used in different doses (0.02, 0.1, 0.3 and 1.0 μM) on the TNFα-mediated inhibition of the BMP-induced osteoblast differentiation in mouse mesenchymal precursor (stem) cells of C2C12 line. Treatment of these cells with TNFα completely inhibited their myogenic differentiation, as well as strongly inhibited the BMP-induced osteogenesis. Strikingly, the treatment of C2C12 cells with Les-4368 and Les-3882 rescued the osteoblast differentiation from negative control of TNFα, and, moreover, converted this cytokine from the inhibitor of osteogenesis into its stimulator. Western-blot analysis of Inhibitory κBα (I-κBα) degradation was used to elucidate a mechanism of the anti-inflammatory effects. Les-3882 was more active, and it stimulated osteoblast differentiation at low dose (0.1 μM), presumably, via modulation of the NF-κB signaling pathway.


Rheumatoid arthritis (RA) is an autoimmune inflammatory disease of yet unknown etiology. Tumor necrosis factor α (TNFα) is recognized as a regulatory substance that plays a central role in RA development and progression. On the other side, the bone morphogenetic protein (BMP) and Wnt signaling pathways are key mechanisms that induce and support cartilage and bone formation and maintenance. Previous studies showed that the pro-inflammatory cytokines TNFα and interleukin 1β (IL-1β) are central players in the inhibition of activity of skeletogenesis. The aim of this study was to evaluate the anti-inflammatory activity of novel 4-thiazolidinone-based derivatives towards TNFα-induced pro-inflammatory effects during bone formation.
We performed in vitro evaluation of functional effects of 4-thiazolidinones denoted as Les-4368, Les-4370, Les-3882 and Les-3288 that were used in different doses (0.02, 0.1, 0.3 and 1.0 μM) on the TNFα-mediated inhibition of the BMP-induced osteoblast differentiation in mouse mesenchymal precursor (stem) cells of C2C12 line.Treatment of these cells with TNFα completely inhibited their myogenic differentiation, as well as strongly inhibited the BMP-induced osteogenesis.Strikingly, the treatment of C2C12 cells with Les-4368 and Les-3882 rescued the osteoblast differentiation from negative control of TNFα, and, moreover, converted this cytokine from the inhibitor of osteogenesis into its stimulator.Western-blot analysis of Inhibitory κBα (I-κBα) degradation was used to elucidate a mechanism of the anti-inflammatory effects.Les-3882 was more active, and it stimulated osteoblast differentiation at low dose (0.1 μM), presumably, via modulation of the NF-κB signaling pathway.K e y w o r d s: osteoblast differentiation, mouse mesenchymal precursor cells, inflammation, bone morphogenetic proteins, 4-thiazolidinone derivatives.
R heumatoid arthritis (RA) is a widespread inflammatory autoimmune disorder associa ted with a progressive disability and multiple systemic complications that can even lead to early death of patients [1].Synovial inflammation and joint swelling alone or combined with autoantibody production (rheumatoid factor and anti-citrullinated peptide antibodies), cartilage and bone dama ge, as well as multiple adverse systemic effects are common symptoms of RA [2].The actual etiology of the disease is still a mystery and many details of RA pathogenesis are poorly understood [3].
Different cytokines are known to control skele tal homeostasis as well as functions of the immune system that are misregulated in RA pathogenesis [4].The disturbed balance of proinflammatory versus antiinflammatory stimuli contributes to RA triggering.Multiple cytokines are involved in regulation of tissue and cellular functions during phases of RA pathophysiology via promoting autoimmuni ty, maintaining chronic inflammatory synovitis, and leading to a destruction of the adjacent joint tissues.Clinical studies performed with TNF blockers, predominantly with its soluble receptor, led to a successful use of these blockers in patients' treatment and confirmed a key role of this proinflammatory cytokine in RA pathogenesis, as well as in other inflammatory disorders [5,6].doi: https://doi.org/10.15407/ubj89.si01.111 The inflammation is considered to be a defensive reaction of organism characterized by the coordinated activation or inhibition of various signaling pathways that control expression of either pro and antiinflammatory mediators.TNFα, IL1β, T and Bcell receptors and pattern recognition Tolllike receptors are potent physiological inducers of key pro inflammatory signaling pathway called the NFκB (nuclear factor κlightchainenhancer of activated B cells).Activation of NFκB signaling is important for expression of products of multiple proinflammatory genes, including cytokines, chemoattractant chemokines, and adhesion molecules [7,8].
Several signaling pathways are misregulated in joints of the RA patients.The regulatory pathway of bone morphogenetic protein (BMP) and wingless (Wnt) are key signaling mechanisms involved in the induction and support of bone and cartilage formation and also important for skeleton maintenance.Although osteoblast differentiation is predominantly induced by the BMPs that belong to transforming growth factor β (TGFβ) superfamily, the properly coordinated activation of Wnt pathway is also essential for bone formation induction, maintenance, remodeling and reparation via regulation of the osteoblast and osteoclast proliferation and differentiation.A commitment of mesenchymal precursors to the osteo and chondrogenic lineages requires a precise coordination of BMP and Wnt signals.However, gene knockout studies and genetic analysis of bone diseases also identified the classical Wnt pathway as the master regulatory signaling in skeletal homeostasis [913].
Wnt/βcatenin pathway is crucially important in bone due to its multiple important functions.Wnts inhibit chondrocyte and adipocyte differentiation, thus, repressing the alternative directions for differentiation of mesenchymal stem cells, and therefore promoting osteoblast growth and differentiation, increasing their mineralization activity and simultaneously blocking their apoptosis.Wnt ligands via βcatenin stabilization increase the ratio between the antireceptor osteoprotegerin (OPG) and RANKL (receptor activating NFκB ligand), thus, repress the osteoclastogenesis.In a healthy skeleton, deposition and resorption of boned adjacent to joints are precisely balanced, while chronic inflammatory disorders such as RA lead to a disruption of such balance.TNFα produced by the inflammatory cells hampers osteoblast functions via the target genes of IL6 and DKK1.As a result repressed osteoblasts produce less of OPG, while bone resorption is enhanced by dumping of OPG/RANKL ratio that is a key factor controlling osteoclast activation and differentiation [1316].Therefore, proinflammatory cytokines massively block skeletogenic signaling pathways at the affected joints and also promote adjacent bone resorption, thus, causing bone erosions.
In spite of a high incidence of RA in the world (up to 1.8% of adult human population), yet there is no efficient cure of the RA.Several protocols, including a use of TNF and IL6 blockers, were develo ped and widely used.These drugs can slow down or even stop RA development, or at least keep joint destruction minimal.Unfortunately, none of the existing drugs can reverse the joint damage and regenerate destroyed tissues.Thus, it is important to develop new highly effective therapeutics for RA and other autoimmune diseases.
4Thiazolidinones are derivatives of thiazolidine with a carbonyl group at 4th position.This structure is a core for a series of synthetic pharmaceutical compounds displaying a wide spectrum of biological activities, such as antimicrobial, anticancer, anticonvulsant, antiinflammatory and analgesic, antidiabetic, antiparasitic, antiviral, including antiHIV, antihyperlipidemic, antihypertensive, and monoamine oxidase inhibitors.Such diversity in the biological responses of 4thiazolidinones has attracted attention of many researchers [17,18].
In this study, we addressed the antiinflammatory activity of novel 4thiazolidinonebased derivatives towards TNFα-induced inflammatory processes during osteoblast differentiation in mouse mesenchymal precursor (stem) cells (MP/SC).We optionally have focused at the 4thiazolidinone compounds that possess minimal cytotoxicity in order to prevent destructive side effects in the inflamed joint.

materials and methods
Materials.Synthesis and physicochemical data of the compounds used in this study were described previously: Les3882 and Les3288 [19], Les4368 and Les4370 [20,21].
Immediately before use, 4thiazolidinone derivatives were dissolved in the pure dimethyl sulfoxide (DMSO) at concentration 1 mM.The final concentration of the DMSO in the medium of cultured cells was not higher than 0.1%.Subsequent dilutions of these compounds were performed in the Dulbecco's modified Eagle's medium (DMEM, Biowest, France).
Cell culture and ligands.The studies were performed using the immortalized mouse mesenchymal precursor/stem cells of C2C12 line.The cells were cultured in the DMEM containing 10% fetal bovine serum (FBS, Biowest, France).The cells were grown in a 5% CO 2 containing atmosphere at 37 °C and 100% humidity.Culture medium was refreshed every 23 days.Before subcultivation, cells were washed with phosphatebuffered saline (PBS) that contained 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.4 mM KH 2 PO 4 with pH 7.4.During osteoblast differentiation assays, C2C12 cells in appropriate variants of the experiment were treated with 10 ng/ml of recombinant TNFα (R&D Systems, Minneapolis, MN, USA).
Cell viability assay.MTT (3(4,5dimethylthia zol2yl)2,5diphenyltetrazolium bromide) assay was used for assessing cell metabolic activity.C2C12 cells were grown for 24 h in 24well plates (500 μl) at 30,000 cells per well.After that, cells were incubated for 72 h with various additions of the synthesized compounds (final concentration of 0.02; 0.01; 0.5; 1.0 μM).Then, the MTT assay of viable cells was used in accordance with the manufacturer's recommendations (Sigma Aldrich, St. Louis, MS, USA).The reaction product was quantitatively determined by an Absorbance Reader BioTek EL*800 (BioTek Instruments, Inc., Winooski, VT, USA) at the wavelength of 620 nm.The viability of the untreated cells was counted as 100%.
Induction of osteoblast differentiation.C2C12 cells were induced to differentiate into osteoblasts by different BMPs, including BMP2 and BMP7.Cells were split at a density of 1.5×10 4 cells per cm 2 in 24 well plates.Next day, the cells were transduced with a combination of adenoviral constructs encoding the recombinant BMP2 and BMP7 at the multiplicity of infection (MOI) even to 500 for each one construct [22] to induce a production of the BMP2/BMP7 heterodimers along with the appropriate homodimers.During osteogenesis assay, C2C12 cells were cultured for 4 days in a differentiationsupporting medium supplemented with 50 µg/ml ascorbic acid.Plasmids encoding recombinant BMPs were purchased from Open Biosystems/GE Dharmacon, Lafayette, CO, USA.
Alkaline phosphatase assay.The alkaline phosphatase activity produced by C2C12 was analyzed spectrophotometrically using a πnitrophenylphosphate (πNPP) as a substrate [23].Four days after induction of osteogenesis, the cells were washed twice with 0.4 ml of 1X PBS per well.Afterwards, cells were lysed in 0.2 ml of alkaline phosphatase (ALP) lysis buffer (10 mM glycine, 100 µM MgCl 2 , 10 µM ZnCl 2 , 0.1% Triton X100) per well and agitated gently for 5 min.Then, 10 µl aliquot of cell lysate was placed into a 96well plate and ALP activity was revealed with 90 µl/well of ALP assay buffer (100 mM glycine, 1 mM MgCl 2 , 100 µM ZnCl 2 ) supplemented with 6 mM πNPP (PierceThermo Fisher Scientific, Grand Island, NY, USA.) [23].Plates were agitated gently and incuba ted at room temperature until color developed.The absorption was measured at 405 nm (OD405) in a 96well plate Absorbance Reader BioTek EL*800 (BioTek, Winooski, VT, USA).The level of expression of the enzyme is directly proportional to the intensity of mesenchymal precursor cells osteoblast differentiation.
Western-blot analysis.After cell treatment with the tested compound, cells were lysed and 20 μg of total cellular protein per well was subjected to electrophoresis on 12% SDS/PAGE gel.Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for immunoblot analysis, as described [24].The antiIκBα (Santa Cruz Biotechnolo gy, Santa Cruz, CA, USA) antibodies were used at a 1:1,000 dilution: Equal protein loading of each lane was evaluated by the immunoblotting of the same membrane with antiβactin monoclonal mouse AC 15 (1:5,000, Sigma Aldrich, St. Louis, MS, USA).Appropriate secondary peroxidaselabelled antibo dies (Cell Signaling Technology, Beverly, MA, USA) were used at a dilution of 1:10,000.
Statistical analysis.All experiments were repeated twice, using three parallel wells in each variant .Results of spectrophotometric measurements of ALP activity are expressed as mean ± standard deviation.Data were analyzed using GraphPad Prism 6 and Microsoft Office Excel programs.Statistical differences between experimental variants were assessed by Student's t-test.Appropriate P values were shown in graphs to demonstrate the significance of the results.Only differences with P-values lower than 0.05 were considered as significant.

results and discussion
As mentioned above, 4thiazolidinonebased compounds possess a broad spectrum of the biological activities [1821].In a current study, we focused at the compounds possessing a capability of modu-lating osteoblast differentiation in mouse mesenchymal precursor cells and demonstrating minimal cytotoxicity in order to avoid of any destructive side effects in the targeted tissue.
Cell viability study of novel 4-thiazolidinone derivatives.The molecular design of new compounds based on commonly used modifying chemical groups is one of the most successful approached in drug development.4Thiazolidinone derivatives (Les4368, Les4370, Les3882 and Les3288) differing by the presence of specific functional groups and the position of these groups in the molecule structure were synthesized (Fig. 1).
Cytotoxic action of Les4368, Les4370, Les 3882, and Les3288 towards MP/SC of C2C12 line used as an experimental model was studied.These compounds were added to the cultured medium at different concentrations (0.02, 0.1, 0.5 and 1.0 μM) for 72 h.DMSO vehicle dissolved in DMEM at the same concentrations as the 4thiazolidinone compounds was used as a negative control (Fig. 2).Les 4368, Les4370, Les3882, and Les3288 demonstrat- The anti-inflammatory activity of novel 4-thiazolidinone derivatives towards TNFα-induced inhibition of the osteoblast differentiation.The main goal of our study was to evaluate the antiinflammatory effects of Les4368, Les4370, Les3882, and Les3288 towards TNFα-induced proinflammatory effects during osteoblast differentiation.We used the immortalized mouse MP/SC of C2C12 line that are known as a useful tool to study differentiation of the myoblasts and osteoblasts.These cells can be induced by different BMPs, including BMP2 and BMP7, to differentiate efficiently into the early osteoblasts.
It was found that treatment of C2C12 cells with 10 ng/ml of TNFα completely inhibited their myoblast differentiation (data not shown), and it also strongly inhibited BMP2/7induced osteoblast differentiation (Fig. 3, control variant).We have tested which of the 4thiazolidinone derivatives will show the antiinflammatory effects.Les4368, Les4370, Les3882, or Les3288 were added to the cultured  medium in 1 μM concentration 56 h after BMP2/7 induction of C2C12 cells.DMSO vehicle dissolved in the DMEM at concentrations of 0.1% (v/v) was used as a negative control.TNFα was added 1 h after the addition of compounds.In case of using Les4368 and Les3882 for treatment of C2C12 cells, they rescued the osteoblast differentiation due to negative control of TNFα.Moreover, these compounds converted their action from the inhibition of osteoblast differentiation to its stimulation compared with a control.For confirmation of modulating osteoblast differentiation, spectrophotometric measurement of ALP activity that is a known marker of the osteoblast differentiation was used (Fig. 3).Les4370 and Les3288 were inefficient in rescuing the osteogenesis from the negative control of inflammation (Fig. 3).

Fig. 3. 4-thiazolidinone derivatives Les-4368 and Les-3882 (1 µM) rescue osteogenesis in C2C12 cells from negative control of the pro-inflammatory cytokine TNFα (10 ng/ml). Spectrophotometric measurement of the alkaline phosphatase activity in C2C12 cells induced to the osteoblast differentiation with the BMP2/
In next experiments, we have shown that Les 4368 and Les3882 possessed the antiinflammatory properties in a wide range of concentrations (0.02, 0.1 and 0.3 μM).The scheme of the experiment was similar to one described above.It was found that Les4368 used in 0.02 μM and 0.3 μM concentra-tions and Les3882 at all applied concentrations stimulated osteoblast differentiation comparing with a control.The best effect was found for Les3882 that stimulated osteoblast differentiation at a low 0.1 μM dose (Fig. 4).

The elucidation of the molecular mechanism of anti-inflammatory effects of Les-4368 and Les-3882.
A modulation of the NFκB activation by Les3882 and Les4368 compounds potentially could be a key mechanism mediating its antiinflammatory effects.The IκBα is a crucially important component in the control of activation of the NFκB pathway.Therefore, in next experiment we used the immunoblot assay with antiIκBα antibodies for validation of our hypothesis based on the determination of the level of NFκB signaling activation.
At the beginning we performed an experiment without use of 4thiazolidinone compounds: C2C12 cells were treated with TNFα for different time periods within a range of 5 min 2 h.At the first time term (520 min), an expected degradation of the IκBα was detected, and 30 min after TNFα treatment, de novo synthesized IκBα protein reappears  (Fig. 5, a).In the next experiment C2C12 cells were pretreated with Les4368 and Les3882 used in 0.1 and 0.3 μM concentrations.TNFα (10 ng/ml) was added 1 h after cell pretreatment with tested compounds for additional 1 h.βactin was used as an internal control of equal protein loading (Fig. 5B).We found that the pretreatment of C2C12 cells with Les3882 and Les4368 compounds with a subsequent 1 h stimulation with TNFα differently modulates the activation of the NFκB signaling pathway.The action of Les3882 led to a lower level of the IκBα protein after 1 h of the TNFα treatment, while Les4368 increased the level of the IκBα protein.It is known that the NFκB signaling is a major pathway that mediates the effects of proinflammatory cytokines, and TNFα is one of the most potent physiological inducers of this pathway [7,8,2528].The NFκB transcription factors consist of five components (p50/NFκB1, p52/NFκB2, p65/RelA, cRel and RelB) that function as dimers regulating the expression of specific target genes and participating in a regulation of a wide range of the biological pro-cesses.In resting cells, the NFκB dimers are inactivated via complex formation with the IκB proteins among which IκBα is most widely and robustly expressed.In the classical (canonical) NFκB signaling, binding of the TNFα or other ligands to their specific cell surface receptors activates an Inhibitory κB kinase (IKK) complex consisting of the IKKβ, IKKα, and IKKγ/NEMO (NFκB essential modulator) that phosphorylates the IκB proteins.Phosphorylation Fig. 6.Scheme of the canonical NF-κB signaling pathway [25][26][27][28] of the IκB proteins triggers their ubiquitination and leads to the proteasomal destruction, thus, releasing the NFκB complexes.Active NFκB complexes are further activated by the posttranslational modifications and then translocated to the nucleus where, either alone or in combination with other transcription factors, they induce target gene expression [2528] (Fig. 6).IκBα is a direct NFκB target gene, and its expression is immediately induced upon the NFκB activation, thus, representing a very efficient negative feedback loop limiting maximal NFκB activation.

Fig. 5. Representative Western-blot assay demonstrates the effect of TNFα on the IκBα level after 5 min to 2 h incubation (A) and the effect of Les-3882 or Les-4368 at indicated concentrations on the IκBα level after 1 h incubation of C2C12 cells with 10 ng/ml of TNFα (B and C). Densitometric quantification of the Western-blot bands is shown on panel C. The β-actin expression level was used as an internal control for protein loading
In this study, we found that two 4thiazolidinones (Les3882 and Les4368) demonstrated high efficiency in modulation of the antiosteogenic effects of TNFα and had an opposite effect on the IκBα level.The explanation of such difference and more detailed mechanism of such effects requires additional study of structurefunction interrelations of novel synthetic 4thiazolodinone derivatives.
Novel 4thiazolidinone derivatives, Les3882 and Les4368, rescue osteogenesis from the negative control of inflammation.The highest activity was demonstrated here by Les3882 compound that stimulated osteoblast differentiation at a low dose (0.1 μM), presumably, via modulation of the NFκB signaling pathway.