With its high-energy phosphate bonds, adenosine triphosphate (ATP) is the main intracellular energy carrier. It also functions in most signaling pathways, as a phosphate donor or a precursor for cyclic adenosine monophosphate. We show here that inositol pyrophosphates participate in the control of intracellular ATP concentration. Yeasts devoid of inositol pyrophosphates have dysfunctional mitochondria but, paradoxically, contain four times as much ATP because of increased glycolysis. We demonstrate that inositol pyrophosphates control the activity of the major glycolytic transcription factor GCR1. Thus, inositol pyrophosphates regulate ATP concentration by altering the glycolytic/mitochondrial metabolic ratio. Metabolic reprogramming through inositol pyrophosphates is an evolutionary conserved mechanism that is also preserved in mammalian systems.

Inorganic polyphosphate (poly-P) consists of just a chain of phosphate groups linked by high energy bonds. It is found in every organism and is implicated in a wide variety of cellular processes (e.g. phosphate storage, blood coagulation, and pathogenicity). Its metabolism has been studied mainly in bacteria while remaining largely uncharacterized in eukaryotes. It has recently been suggested that poly-P metabolism is connected to that of highly phosphorylated inositol species (inositol pyrophosphates). Inositol pyrophosphates are molecules in which phosphate groups outnumber carbon atoms. Like poly-P they contain high energy bonds and play important roles in cell signaling. Here, we show that budding yeast mutants unable to produce inositol pyrophosphates have undetectable levels of poly-P. Our results suggest a prominent metabolic parallel between these two highly phosphorylated molecules. More importantly, we demonstrate that DDP1, encoding diadenosine and diphosphoinositol phosphohydrolase, possesses a robust poly-P endopolyphosphohydrolase activity. In addition, we prove that this is an evolutionarily conserved feature because mammalian Nudix hydrolase family members, the three Ddp1 homologues in human cells (DIPP1, DIPP2, and DIPP3), are also capable of degrading poly-P.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds(1). Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)(2)-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution(2-4).The IP6 kinases (IP(6)Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules(5,6). Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP8 (7,8).Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion(9), telomere length(10,11), chemotaxis(12), vesicular trafficking(13), phosphate homeostasis(14) and HIV-1 gag release(15). Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT(16). Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins(17). The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus(18,19). These procedures use acidic conditions that might lead to inositol pyrophosphate degradation(20) and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates(20). Following IP(6)K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

The past ten years have seen a contained explosion of interest in inositol pyrophosphates. The early cloning of the IP6Ks and the more recent identification of the PP-IP5Ks have allowed the development of essential experimental tools to investigate the physiological role of inositol pyrophosphates. However, for this exciting field of research to gain momentum, simpler and more reliable research protocols need to be further developed. The ability to resolve and quantify inositol pyrophosphates using gel electrophoresis (Losito et al., 2009) has dramatically altered the way we are studying this class of molecules, opening new avenues for research. The use of this technology to resolve, detect and characterize inositol pyrophosphates extracted from cells certainly represents one desirable aim. The most crucial objective, however, is to obtain definite proof of the new mechanism of post-translational modification by identifying with biophysical methods the presence in vivo of pyrophosphorylated serines. This will hopefully precipitate the development of new ways to detect this modification, for example through the production of antibodies that specifically recognize pyrophosphorylated serines.

We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.

We aimed to elucidate the role of the Ca-independent PKC isoenzyme PKCdelta in the regulation of spontaneous in vitro chondrogenesis occurring in a 6-day-long culturing period in chicken limb bud-derived high density cell cultures (HDC). PKCdelta expression and activity were detectable throughout the entire culturing period with a peak on days 2 and 3, when most of the chondroblasts differentiate. To inhibit the activity of PKCdelta, either the natural compound rottlerin was transiently applied to the culture medium of HDC in 2.5, 5 or 10 μM concentrations, or gene silencing was performed by using PKCdelta shRNA. Rottlerin significantly reduced the overall PKC activity in enzyme activity assays of cell-free samples of untreated control HDC, probably via the inhibition of PKCdelta. On the contrary, we were unable to detect any consistent change of PKC enzyme activity assayed in samples of HDC treated with rottlerin during culturing. PKCdelta gene silencing resulted in a significantly lower PKC activity. Both rottlerin and PKCdelta shRNA caused a severe reduction in cartilage formation, furthermore protein and phospho-protein levels of Sox9, the key transcription factor of chondrogenesis, were also significantly decreased. Rottlerin lowered, while PKCdelta gene silencing elevated the phosphorylation status of ERK1/2. Our data suggest that PKCdelta stimulates chondrogenesis via influencing Sox9 and ERK1/2 phosphorylation, but the inhibition of cartilage formation in the rottlerin-treated HDC is probably PKCdelta independent and rottlerin might have different effects when applied to cells or to an in vitro enzyme activity assay.

Background: Inositol pyrophosphates are a recently characterized cell signalling molecules responsible for the pyrophosphorylation of protein substrates. Though likely involved in a wide range of cellular functions, the study of inositol pyrophosphates has suffered from a lack of readily available methods for their analysis.Principal Finding: We describe a novel, sensitive and rapid polyacrylamide gel electrophoresis (PAGE)-based method for the analysis of inositol pyrophosphates. Using 4',6-diamidino-2-phenylindole (DAPI) and Toluidine Blue we demonstrate the unequivocal detection of various inositol pyrophosphate species.Conclusion: The use of the PAGE-based method reveals the likely underestimation of inositol pyrophosphates and their signalling contribution in cells when measured via traditional HPLC-based techniques. PAGE-based analyses also reveals the existence of a number of additional, previously uncharacterised pyrophosphorylated inositol reaction products, defining a more complex metabolism associated with the catalytically flexible kinase class responsible for the production of these highly energetic cell signalling molecules.

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 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.

The 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.

Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.

We 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 140nM peak of cytosolic Ca2+ 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 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; 0.1mg/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 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.

It is known that PMA (phorbol-12-myristate-13-acetate) can activate the classical and novel protein kinase C isoenzymes (cPKC alpha, beta, gamma and nPKC delta, epsilon, eta, theta), while the calcium ion can induce only the activity of cPKC. Calcineurin binding protein (Cabin 1) belongs to the group of endogenous inhibitors of calcineurin. Cabin 1 becomes hyperphosphorylated in response to PKC activation and may play a negative role in calcineurin signalling. It was observed that both PMA treatment and the increase in intracellular Ca2+ contributed to the reduction of calcineurin activity in human peripheral blood mononuclear cells without modulating the mRNA and the protein levels of calcineurin. PMA and Ca-ionophore (A23187), the activating agents of PKC, applied alone or in combination, significantly increased the phosphorylation state of Cabin 1 as revealed by immunoprecipitation of Cabin 1 detecting its phospho-Ser content by specific antibodies. GF109203X, an inhibitor of the classic and the novel protein kinase C isoenzymes, and Gö6976, the selective inhibitor of the classical cPKC isoenzymes were able to abolish the effect of PMA or/and Ca-ionophore on the calcineurin activity with concomitant reversal of the hyperphosphorylation of Cabin 1. The calcineurin/Cabin 1 system was not influenced by Rottlerin, an inhibitor of PKC delta isoenzyme either in the absence or in the presence of Ca-ionophore and PMA. We presented evidence for the prominent role of cPKC alpha, beta, gamma isoenzymes in the inhibition of calcineurin as induced by PMA and Ca-ionophore. We demonstrated also that hyperphosphorylation of Cabin 1 by PMA/Ca2+-activated cPKC isoenzymes resulted in a simultaneous inhibition of calcineurin in peripheral blood mononuclear cells. These results suggest a negative regulatory role for Cabin 1 in calcineurin signalling and provide a possible mechanism of feedback inhibition through cross-talk between PKC and calcineurin.

Oxidative stress has been implicated in the pathogenesis of various diseases affecting chondrogenesis or the function of articular cartilage. DNA damage caused by oxidative stress may trigger the activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) which may contribute to tissue injury. We aimed at investigating the effects of peroxynitrite (100-600 microM) and hydrogen peroxide (0.1-4 mM) on PARP activation and extracellular matrix production of high density micromass cultures (HDC) prepared from chick limb bud mesenchymal cells. We found that both oxidative species strongly inhibited matrix formation of HDCs treated on day 2 but not on day 5. The PARP inhibitor 3-aminobenzamide (3-AB) stimulated matrix production in non-stressed cells and prevented suppressed matrix production in oxidatively stressed cells. Both hydrogen peroxide and peroxynitrite induced PARP activation and poly(ADP-ribose) accumulation. Decreased proliferation, viability and NAD+ content were not or only slightly improved by 3-AB, indicating that 3-AB directly affects matrix formation. In conclusion, oxidative stress stimulates poly(ADP-ribose) metabolism and inhibits extracellular matrix production of HDCs in a PARP-dependent manner. Our findings may have implications for potential therapeutic approaches aimed at restoring the matrix production capacity of chondrogenic cells.