János Fodor

Publications of János Fodor

• Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts.

  Authors: Tamás Oláh, János Fodor, Sarah Oddoux, Olga Ruzsnavszky, Isabelle Marty, László Csernoch

  Pflügers Archiv : European journal of physiology. 08/2011; 462(4):599-610.

  To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation-contraction coupling has been studied in detail, the role of theTo date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation-contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP(3) receptors (IP(3)R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP(3)-mediated Ca(2+) release were assessed by measuring changes in [Ca(2+)](i) following the stimulation by bradykinin or vasopressin. The amplitude of the Ca(2+) transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca(2+). Similar observations were made when 0.1 μM vasopressin was used to initiate Ca(2+) release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca(2+) entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca(2+) transients; rather, they were due to the enhanced activity of IP(3)R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca(2+) release via IP(3)R.

• Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells.

  Authors: Tamás Oláh, János Fodor, Olga Ruzsnavszky, János Vincze, Celine Berbey, Bruno Allard, László Csernoch

  Cell calcium. 06/2011; 49(6):415-25.

  When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins asWhen the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.

• Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture.

  Authors: Lívia Kosztka, Zoltán Rusznák, Dénes Nagy, Zsuzsanna Nagy, János Fodor, Géza Szucs, Andrea Telek, Mónika Gönczi, Olga Ruzsnavszky, Norbert Szentandrássy, László Csernoch

  Melanoma research. 04/2011; 21(4):308-22.

  TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it hasTASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.

• Switch of voltage-gated K+ channel expression in the plasma membrane of chondrogenic cells affects cytosolic Ca2+-oscillations and cartilage formation.

  Authors: Zoltan Varga, Tamás Juhász, Csaba Matta, János Fodor, Éva Katona, Adam Bartok, Tamás Oláh, Attila Sebe, László Csernoch, Gyorgy Panyi, Róza Zákány

  PloS one. 01/2011; 6(11):e27957.

  Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stemUnderstanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca(2+)-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation. Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated Na(V)1.4, K(V)1.1, K(V)1.3 and K(V)4.1 channels, although K(V)1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of Na(V)1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K(+) channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced K(V) currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca(2+)-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive K(V)1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive K(V)4.1 did not affect cartilage formation. These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca(2+)-oscillations, which are modulated by K(V)-driven membrane potential changes. K(V)1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.

• Inhibition of calcineurin by cyclosporine A exerts multiple effects on human melanoma cell lines HT168 and WM35.

  Authors: Tamás Juhász, Csaba Matta, Gábor Veress, Georgina Nagy, Zsolt Szíjgyártó, Zsanett Molnár, János Fodor, Róza Zákány, Pál Gergely

  International journal of oncology. 05/2009; 34(4):995-1003.

  The immunosuppressant cyclosporine A (CsA) is a specific pharmacological inhibitor of calcineurin, the Ca2+-calmodulin activated phospho-Ser/Thr-specific protein phosphatase. AlthoughThe immunosuppressant cyclosporine A (CsA) is a specific pharmacological inhibitor of calcineurin, the Ca2+-calmodulin activated phospho-Ser/Thr-specific protein phosphatase. Although calcineurin-inhibiting compounds are applied for local treatment of psoriasis or atopic dermatitis in dermatological practice, little is known about the functions of calcineurin in epidermis-derived malignancies. We investigated the effects of CsA on two human melanoma cell lines, the metastasis forming HT168 and WM35 established from an RGP primary lesion. CsA of 2 microM lowered the enzyme activity by 50% and caused elevation in both mRNA and protein expression of calcineurin. Cell proliferation was diminished, as well as the cellular morphology and the actin organization were altered in both cell lines. CsA increased cell death moderately in both cell lines and reduced the metabolic activity of HT168 cells, but not that of WM35 cells. CsA also elevated the expressions of both Bcl-2 and ERK1/2. Fibronectin guided migration of HT168 cells was stimulated under the effect of CsA, while that of WM35 cells was reduced, moreover, HT168 cells switched from the expression of beta3 to beta1 integrin, but WM35 cells continued to express beta3. Based on our results we propose a multiple, partly malignancy-dependent role of calcineurin in these melanoma cell lines.

• Ionotropic purinergic receptor P2X(4) is involved in the regulation of chondrogenesis in chicken micromass cell cultures.

  Authors: János Fodor, Csaba Matta, Tamás Juhász, Tamás Oláh, Mónika Gönczi, Zsolt Szíjgyártó, Pál Gergely, László Csernoch, Róza Zákány

  Cell calcium. 04/2009;

  We have previously demonstrated that elevation of free cytosolic Ca(2+) concentration at the time of differentiation of chondroblasts was mainly due to a Ca(2+) influx and it was indispensable toWe have previously demonstrated that elevation of free cytosolic Ca(2+) concentration at the time of differentiation of chondroblasts was mainly due to a Ca(2+) influx and it was indispensable to cartilage formation in chicken high density mesenchymal cell cultures (HDC) [C. Matta, J. Fodor, Z. Szijgyarto, T. Juhasz, P. Gergely, L. Csernoch, R. Zakany, Cytosolic free Ca(2+) concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium 44 (2008) 310-323]. Here, we report that chondrogenic cells secreted ATP and administration of ATP to the culture medium evoked Ca(2+) transients exclusively in the presence of extracellular Ca(2+) and only on day 3 of culturing, when the final commitment of chondroblasts occurs. Moreover, ATP caused elevated protein expression of the chondrogenic transcription factor Sox9 and stimulated cartilage matrix production. Expression pattern of different types of both ionotropic and metabotropic purinergic receptors was detected. Agonists of metabotropic receptors, ADP and UDP did not evoke any Ca(2+) transients and had no influence on cartilage formation, while UTP caused transient elevation of cytosolic Ca(2+) concentration in 3-day-old HDC without stimulating matrix production. Suramin, which blocks all P2X receptors but not P2X(4) did not impede the effects of ATP, furthermore, P2X(4) appeared in the plasma membrane fraction and gave signals with immunocytochemistry only from day 3. In summary, we suggest a role of ionotropic purinergic signalling of P2X(4) in the generation of ATP-dependent Ca(2+) transients of differentiating chondroblasts.

• Mitochondrial expression of the two-pore domain TASK-3 channels in malignantly transformed and non-malignant human cells.

  Authors: Zoltán Rusznák, Gábor Bakondi, Lívia Kosztka, Krisztina Pocsai, Beatrix Dienes, János Fodor, Andrea Telek, Mónika Gönczi, Géza Szucs, László Csernoch

  Virchows Archiv : an international journal of pathology. 05/2008; 452(4):415-26.

  The presence of TASK-3 channels has been described in a number of healthy and malignantly transformed cells, showing mainly intracellular distribution with relatively insignificant labelling of theThe presence of TASK-3 channels has been described in a number of healthy and malignantly transformed cells, showing mainly intracellular distribution with relatively insignificant labelling of the cell surface membrane. In this work, immunochemical and molecular biology methods were utilised to establish the intracellular organelle whose TASK-3 expression accounts for this strong intracellular labelling using cultured melanoma and HaCaT cells. Before the immunocytochemical experiments, the presence of TASK-3 mRNA was also confirmed in melanoma cells. Comparison of the results of the TASK-3- and mitochondrion-specific labelling indicated that the TASK-3 channel subunits were strongly expressed by mitochondria in both investigated cell types. Moreover, prominent TASK-3 expression of keratinocytes could also be demonstrated in histological sections excised from the human skin. These results indicate that TASK-3 channels are present in the mitochondria in both malignantly transformed and healthy cells, suggesting that they might have roles in ensuring mitochondrial functions.

• Cytosolic free Ca(2+) concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells.

  Authors: Csaba Matta, János Fodor, Zsolt Szíjgyártó, Tamás Juhász, Pál Gergely, László Csernoch, Róza Zákány

  Cell calcium. 03/2008;

  We measured changes of cytosolic Ca(2+) concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevationWe measured changes of cytosolic Ca(2+) concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevation was detected in Fura-2-loaded cells on day 3 of culturing, when majority of chondrogenic cells of HDC become differentiated. This 140nM peak of cytosolic Ca(2+) concentration is a result of increased Ca-influx and is indispensable to proper chondrogenesis, because addition of 0.8mM EGTA to culture medium on day 2 or 3 significantly decreased the intracellular Ca(2+) concentration abolishing the Ca(2+)-peak of day 3 and inhibited cartilage formation. Uncontrolled Ca(2+) influx evoked by a Ca(2+) ionophore exerted dual effects on chondrogenesis in a concentration-dependent manner; 0.1mg/L A23187 increased, whereas 5mg/L A23187 almost totally blocked cartilage formation. Intracellular Ca-stores seemed not to have any significant participation in the regulation of changes of cytosolic Ca(2+) concentration of chondrifying cells. Activity of Ca-calmodulin-dependent protein phosphatase, calcineurin responded to changes of intracellular Ca(2+) concentration induced by EGTA or A23187 in a differentiation stage-dependent manner. Since inhibition of calcineurin with cyclosporine A eliminated the peak in the cytosolic Ca(2+) concentration, an active regulatory role of calcineurin on Ca(2+) influx of chondrifying cells can be supposed.

• Functional genomics of calcium channels in human melanoma cells.

  Authors: Tamás Deli, Norbert Varga, Attila Adám, István Kenessey, Erzsébet Rásó, László G Puskás, József Tóvári, János Fodor, Mónika Fehér, Gyula P Szigeti, László Csernoch, József Tímár

  International journal of cancer. Journal international du cancer. 08/2007; 121(1):55-65.

  Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to theCa(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.

• Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells

  Authors: Csaba Matta, János Fodor, Zsolt Szíjgyártó, Tamás Juhász, Pál Gergely, László Csernoch, Róza Zákány

  Cell Calcium.

  We measured changes of cytosolic Ca2+ concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevationWe measured changes of cytosolic Ca2+ concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevation was detected in Fura-2-loaded cells on day 3 of culturing, when majority of chondrogenic cells of HDC become differentiated. This 140 nM peak of cytosolic Ca2+ concentration is a result of increased Ca-influx and is indispensable to proper chondrogenesis, because addition of 0.8 mM EGTA to culture medium on day 2 or 3 significantly decreased the intracellular Ca2+ concentration abolishing the Ca2+-peak of day 3 and inhibited cartilage formation. Uncontrolled Ca2+ influx evoked by a Ca2+ ionophore exerted dual effects on chondrogenesis in a concentration-dependent manner; 0.1 mg/L A23187 increased, whereas 5 mg/L A23187 almost totally blocked cartilage formation. Intracellular Ca-stores seemed not to have any significant participation in the regulation of changes of cytosolic Ca2+ concentration of chondrifying cells. Activity of Ca–calmodulin-dependent protein phosphatase, calcineurin responded to changes of intracellular Ca2+ concentration induced by EGTA or A23187 in a differentiation stage-dependent manner. Since inhibition of calcineurin with cyclosporine A eliminated the peak in the cytosolic Ca2+ concentration, an active regulatory role of calcineurin on Ca2+ influx of chondrifying cells can be supposed.

• Ionotropic purinergic receptor P2X4 is involved in the regulation of chondrogenesis in chicken micromass cell cultures

  Authors: János Fodor, Csaba Matta, Tamás Juhász, Tamás Oláh, Mónika Gönczi, Zsolt Szíjgyártó, Pál Gergely, László Csernoch, Róza Zákány

  Cell Calcium.

  We have previously demonstrated that elevation of free cytosolic Ca2+ concentration at the time of differentiation of chondroblasts was mainly due to a Ca2+ influx and it was indispensable toWe have previously demonstrated that elevation of free cytosolic Ca2+ concentration at the time of differentiation of chondroblasts was mainly due to a Ca2+ influx and it was indispensable to cartilage formation in chicken high density mesenchymal cell cultures (HDC) [C. Matta, J. Fodor, Z. Szijgyarto, T. Juhasz, P. Gergely, L. Csernoch, R. Zakany, Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium 44 (2008) 310–323]. Here, we report that chondrogenic cells secreted ATP and administration of ATP to the culture medium evoked Ca2+ transients exclusively in the presence of extracellular Ca2+ and only on day 3 of culturing, when the final commitment of chondroblasts occurs. Moreover, ATP caused elevated protein expression of the chondrogenic transcription factor Sox9 and stimulated cartilage matrix production. Expression pattern of different types of both ionotropic and metabotropic purinergic receptors was detected. Agonists of metabotropic receptors, ADP and UDP did not evoke any Ca2+ transients and had no influence on cartilage formation, while UTP caused transient elevation of cytosolic Ca2+ concentration in 3-day-old HDC without stimulating matrix production. Suramin, which blocks all P2X receptors but not P2X4 did not impede the effects of ATP, furthermore, P2X4 appeared in the plasma membrane fraction and gave signals with immunocytochemistry only from day 3. In summary, we suggest a role of ionotropic purinergic signalling of P2X4 in the generation of ATP-dependent Ca2+ transients of differentiating chondroblasts.

• Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture.

  Authors: János Fodor, Monika Gönczi, Monika Sztretye, Beatrix Dienes, Tamás Oláh, László Szabó, Eszter Csoma, Péter Szentesi, Gyula Szigeti, Isabelle Marty, László Csernoch

  The Journal of Physiology.

  The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in theThe 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.