The role of protein kinase C isoenzymes in the pathogenesis of human autoimmune diseases.

The physiological role of protein kinase C (PKC) enzymes in the immune system is presented briefly. From earlier publications of others data were collected how the defects of one/two isoenzymes of PKC system suggested their involvement in the pathogenesis of human autoimmune diseases. Our observations on the defects of seven PKC isoenzymes in the peripheral blood mononuclear cells (PBMC) demonstrate that these molecular impairments are not prerequisits of the pathogenesis of systemic lupus erythematosus (SLE), mixed connective tissue disease and Sjögren's syndrome. However, these defects can modulate the disease activity and symptoms especially in SLE by several pathways. The role of PKC system in other forms of autoimmune diseases is also very small. It was of note that we detected decreased expression of PKC isoenzymes in PBMC of a European white family with an X-linked genetic background showing seasonal undulations in the lupus patient and also in her healthy mother.

Glucose-based spiro-oxathiazoles as in vivo anti-hyperglycemic agents through glycogen phosphorylase inhibition.

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ∼36% at 30 mg kg-1 and ∼43% at 60 mg kg-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels.

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2(-/-) mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2(-/-) mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells.

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

3-Glucosylated 5-amino-1,2,4-oxadiazoles: synthesis and evaluation as glycogen phosphorylase inhibitors.

Glycogen phosporylase (GP) is a promising target for the control of glycaemia. The design of inhibitors binding at the catalytic site has been accomplished through various families of glucose-based derivatives such as oxadiazoles. Further elaboration of the oxadiazole aromatic aglycon moiety is now reported with 3-glucosyl-5-amino-1,2,4-oxadiazoles synthesized by condensation of a C-glucosyl amidoxime with N,N'-dialkylcarbodiimides or Vilsmeier salts. The 5-amino group introduced on the oxadiazole scaffold was expected to provide better inhibition of GP through potential additional interactions with the enzyme's catalytic site; however, no inhibition was observed at 625 µM.

3-Aminobenzamide protects primary human keratinocytes from UV-induced cell death by a poly(ADP-ribosyl)ation independent mechanism.

Poly(ADP-ribosyl)ation (PARylation) is a NAD(+)-dependent protein modification carried out by PARP [poly(ADP-ribose) polymerase] enzymes. Here we set out to investigate whether PARylation regulates UVB-induced cell death in primary human keratinocytes. We used the benchmark PARP inhibitor 3-aminobenzamide (3AB) and a more potent and specific inhibitor PJ34 and found that UVB (0.05-0.2J/cm(2)) induced a dose dependent loss of viability that was prevented by 3AB but not by PJ34. Similarly to PJ34, two other new generation PARP inhibitors also failed to protect keratinocytes from UVB-induced loss of viability. Moreover, silencing PARP-1 in HaCaT human keratinocytes sensitized cells to UVB toxicity but 3AB provided protection to both control HaCaT cells and to PARP-1 silenced cells indicating that the photoprotective effect of 3AB is independent of PARP inhibition. Lower UVB doses (0.0125-0.05J/cm(2)) caused inhibition of proliferation of keratinocytes which was prevented by 3AB but augmented by PJ34. UVB-induced keratinocyte death displayed the characteristics of both apoptosis (morphology, caspase activity, DNA fragmentation) and necrosis (morphology, LDH release) with all of these parameters being inhibited by 3AB and apoptotic parameters slightly enhanced by PJ34. UVA also caused apoptotic and necrotic cell death in keratinocytes with 3AB protecting and PJ34 sensitizing cells to UVA-induced toxicity. 3AB prevented UVB-induced mitochondrial membrane depolarization and generation of hydrogen peroxide. In summary, PARylation is a survival mechanism in UV-treated keratinocytes. Moreover, 3-aminobenzamide is photoprotective and acts by a PARP-independent mechanism at a premitochondrial step of phototoxicity.

Glucopyranosylidene-spiro-iminothiazolidinone, a new bicyclic ring system: synthesis, derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods.

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-ß-D-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-D-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO2Cl where R=tBu, Ph, 4-Me-C6H4, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N'-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (Ki=9µM) and the 2-naphthoylimino-thiazolidinones (Ki=10 µM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.

Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and ß-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused ß-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC.

RACK1 is involved in endothelial barrier regulation via its two novel interacting partners.

BACKGROUND: RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. TIMAP, TGF-ß inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function. The interaction of TIMAP with protein phosphatase 1 (PP1cδ) was characterized, yet little is known about its further partners. RESULTS: We identified two novel interacting partners of RACK1, namely, TGF-ß inhibited membrane-associated protein, TIMAP, and farnesyl transferase. TIMAP is most abundant in endothelial cells where it is involved in the regulation of the barrier function. WD1-4 repeats of RACK1 were identified as critical regions of the interaction both with TIMAP and farnesyl transferase. Phosphorylation of TIMAP by activation of the cAMP/PKA pathway reduced the amount of TIMAP-RACK1 complex and enhanced translocation of TIMAP to the cell membrane in vascular endothelial cells. However, both membrane localization of TIMAP and transendothelial resistance were attenuated after RACK1 depletion. Farnesyl transferase, the enzyme responsible for prenylation and consequent membrane localization of TIMAP, is present in the RACK1-TIMAP complex in control cells, but it does not co-immunoprecipitate with TIMAP after RACK1 depletion. CONCLUSIONS: Transient parallel linkage of TIMAP and farnesyl transferase to RACK1 could ensure prenylation and transport of TIMAP to the plasma membrane where it may attend in maintaining the endothelial barrier as a phosphatase regulator.

Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors.

Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had Ki values of 2.0µM against AMP (competitive) and 3.36µM against glucose-1-phosphate (non-competitive).

Synthesis, enzyme kinetics and computational evaluation of N-(ß-D-glucopyranosyl) oxadiazolecarboxamides as glycogen phosphorylase inhibitors.

All possible isomers of N-ß-D-glucopyranosyl aryl-substituted oxadiazolecarboxamides were synthesised. O-Peracetylated N-cyanocarbonyl-ß-D-glucopyranosylamine was transformed into the corresponding N-glucosyl tetrazole-5-carboxamide, which upon acylation gave N-glucosyl 5-aryl-1,3,4-oxadiazole-2-carboxamides. The nitrile group of the N-cyanocarbonyl derivative was converted to amidoxime which was ring closed by acylation to N-glucosyl 5-aryl-1,2,4-oxadiazole-3-carboxamides. A one-pot reaction of protected ß-D-glucopyranosylamine with oxalyl chloride and then with arenecarboxamidoximes furnished N-glucosyl 3-aryl-1,2,4-oxadiazole-5-carboxamides. Removal of the O-acetyl protecting groups by the Zemplén method produced test compounds which were evaluated as inhibitors of glycogen phosphorylase. Best inhibitors of these series were N-(ß-D-glucopyranosyl) 5-(naphth-1-yl)-1,2,4-oxadiazol-3-carboxamide (Ki = 30 µM), N-(ß-D-glucopyranosyl) 5-(naphth-2-yl)-1,3,4-oxadiazol-2-carboxamide (Ki =33 µM), and N-(ß-D-glucopyranosyl) 3-phenyl-1,2,4-oxadiazol-5-carboxamide (Ki = 104 µM). ADMET property predictions revealed these compounds to have promising oral drug-like properties without any toxicity.

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis.

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5'-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, ß-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

Poly(ADP-ribose) polymerase-2: emerging transcriptional roles of a DNA-repair protein.

Poly(ADP-ribose) polymerase (PARP)-2 is a nuclear enzyme that belongs to the PARP family and PARP-2 is responsible for 5-15 % of total cellular PARP activity. PARP-2 was originally described in connection to DNA repair and in physiological and pathophysiological processes associated with genome maintenance (e.g., centromere and telomere protection, spermiogenesis, thymopoiesis, azoospermia, and tumorigenesis). Recent reports have identified important rearrangements in gene expression upon the knockout of PARP-2. Such rearrangements heavily impact inflammation and metabolism. Metabolic effects are mediated through modifying PPARγ and SIRT1 function. Altered gene expression gives rise to a complex phenotype characterized primarily by enhanced mitochondrial activity that results both in beneficial (loss of fat, enhanced insulin sensitivity) and in disadvantageous (pancreatic beta cell hypofunction upon high fat feeding) consequences. Enhanced mitochondrial biogenesis provides protection in oxidative stress-related diseases. Hereby, we review the recent developments in PARP-2 research with special attention to the involvement of PARP-2 in transcriptional and metabolic regulation.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

Activation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP-ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide-induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis-inducing factor could not be observed, but cells could be saved by methyl pyruvate and alpha-ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis-necrosis switch in severely damaged cells.

Synthesis of 1-(D-glucopyranosyl)-1,2,3-triazoles and their evaluation as glycogen phosphorylase inhibitors.

1-(D-Glucopyranosyl)-1,2,3-triazoles were prepared from per-O-acetylated alpha- and beta-D-glucopyranosyl azides as well as per-O-benzoylated (beta-D-gluco-hept-2-ulopyranosylazide)onamide and onic acid methylester by using azide-alkyne cycloaddition catalysed by in situ generated Cu(I) under aqueous conditions. The O-acyl protecting groups were removed by the Zemplén protocol. The test compounds were assayed against rabbit muscle glycogen phosphorylase b to show that the beta-D-glucopyranosyl derivatives were superior inhibitors as compared to the two other series of triazoles.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.

Role of calcineurin in thrombin-mediated endothelial cell contraction.

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.

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

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.

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A.

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.