Inhibition Of Calcineurin Activity Research Articles

FK506 alleviates proteinuria in rats with adriamycin-induced nephropathy by down-regulating TRPC6 and CaN expression

To investigate the roles of transient receptor potential cation channel 6 (TRPC6) and calcineurin (CaN) in proteinuria pathogenesis and the mechanism of action of the calcineurin inhibitor tacrolimus (FK506) in adriamycin-induced nephropathy. The adriamycin-induced nephropathy rats were established and randomly divided into adriamycin nephropathy (ADR), low-dose FK506 treated (ADR + FK0.5), high-dose FK506 treated (ADR + FK1.0) and Control groups. Twenty-four hour urinary protein and blood biochemistry were measured on weeks 2, 3, 5 and 7, and the distributions and expressions of TRPC6 and CaN in the renal tissue were detected by immunohistochemistry, real-time PCR and Western blot. The study showed the 24-hour urinary protein increased significantly in ADR rats compared with Controls whilst for ADR rats hypoalbuminemia, hypercholesterolemia, renal functional lesion and renal pathologic changes appeared. The areas and intensities of TRPC6 and CaN expressed in the glomerulus and tubulointerstitium of ADR rats increased significantly. The expression of TRPC6 mRNA in ADR rats began to increase on the 3rd week and persisted to the 7th week, and CaN mRNA increased throughout the period. Protein expressions of TRPC6 and CaN in ADR rats were significantly higher than those of Controls. FK506 can inhibit CaN activity in the renal tissues and further decrease TRPC6 expression and therefore reduce renal damage and proteinuria in a dose-dependent manner. These findings suggested that TRPC6 and CaN were up-regulated on mRNA and protein levels in adriamycin-induced rats. FK506 had a therapeutic effect on the progression of proteinuria and renal damage by down-regulating of TRPC6 and CaN in the renal tissues.

Midterm clinico-radiologic findings of an open label observation study of add-on tacrolimus with biologics or non-biologic DMARDs

Tacrolimus (TAC) suppresses immune-inflammation by an intermediary inhibition of calcineurin activation in the treatment of rheumatoid arthritis (RA). Various combination therapies for RA have been reported to be superior to monotherapies. The aim was therefore to study add-on TAC in a combination with biologics (BIO) and/or non-BIO disease-modifying anti-rheumatic drugs (DMARDs) in treatment-resistant patients. In eight RA patients, TAC was added on to BIO (TAC/BIO group) and in forty-one to non-BIO DMARDs (TAC/non-BIO group). The mean C-reactive protein (CRP) decreased from 33 mg/l at the baseline to 16 mg/l at first year in the TAC/BIO group (P < 0.05), from 41 to 14 mg/l in the TAC/non-BIO group (P < 0.05); the mean DAS28-CRP (28 joint count) disease activity score decreased from 5.3 to 4.4 in the TAC/BIO group (P < 0.05) and from 5.0 to 3.9 in the TAC/non-BIO group (P < 0.05). The median of Δ modified total Sharp score decreased from 43 during the year preceding the baseline to 3 during the first year of the follow-up in the TAC/BIO group (P < 0.05) and from 22 to 0 during the second year in the TAC/non-BIO group (P < 0.05). Twenty-six adverse events occurred in this study in 26 patients (53% in all); however, the only severe adverse event was one case of an atypical mycobacterial disease (2%). The combination therapy of TAC with BIO or non-BIO DMARDs represents an effective and relatively safe mode of therapy in treatment-resistant RA.

The Regulator of Calcineurin 1 (RCAN1/DSCR1) Activates the cAMP Response Element-binding Protein (CREB) Pathway

cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling.

Sildenafil inhibits calcineurin/NFATc2-mediated cyclin A expression in pulmonary artery smooth muscle cells

AimsTo examine whether calcineurin/NFAT signaling pathway leads to proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating cell cycle proteins and whether the phosphodiesterase-5 (PDE5) inhibitor sildenafil affects calcineurin/NFAT-induced cell proliferation. Main methodsA [3H]thymidine incorporation assay was used to examine DNA synthesis (cell proliferation); cyclin A and NFATc2 expressions were determined by Western blot. Cyclin-dependent kinase 2 (CDK2) activity was measured with an in vitro kinase activity assay, and calcineurin and NFAT activity were evaluated using a calcineurin assay kit and a luciferase activity assay, respectively. A chemical inhibitor or siRNA transfection was used to inhibit calcineurin/NFAT signaling pathway. Key findingsSerotonin dose-dependently stimulated cyclin A expression in PASMCs. This effect was accompanied by dose-dependent increases in CDK2 activity and the rate of DNA synthesis. At the same time, PASMCs treated with serotonin showed dose-dependent activation of calcineurin/NFAT signaling pathway. Inhibition of calcineurin activity by cyclosporine A or loss of NFATc2 protein by siRNA transfection abolished serotonin-induced cyclin A expression and consequent CDK2 activation and DNA synthesis. We further found that pretreatment of cells with sildenafil suppressed serotonin-triggered activation of calcineurin/NFATc2 signaling pathway and resultant cyclin A expression, CDK2 activation and cell proliferation, while the presence of DT-3 [a specific protein kinase G (PKG) peptide inhibitor] reversed the effects of sildenafil on PASMCs. SignificanceOur study suggests that enhanced PKG activity suppresses calcineurin/NFATc2 cascade-mediated cyclin A expression, CDK2 activation and PASMC proliferation to contribute to the overall effects of sildenafil in the treatment of pulmonary hypertension.

Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

Voclosporin: a potentially promising therapeutic agent for noninfectious uveitis

Voclosporin is a novel calcineurin inhibitor that functions by binding cyclophilin, consequently inhibiting calcineurin activity and preventing the transcription of many genes involved in lymphocyte proliferation and cytokine release. Voclosporin has been shown to be effective in a variety of autoimmune diseases, such as rheumatoid arthritis, psoriasis and renal transplant rejection. Noninfectious uveitis is also an immune-mediated disease which, if left untreated, can cause severe loss of vision. Presently, corticosteroids are the mainstay of therapy in noninfectious uveitis. However, there are several metabolic and ophthalmic adverse effects associated with the long-term use of corticosteroids. Voclosporin has recently been shown in the LX-211 Uveitis Multicenter Investigation of a New Approach to Treatment (LUMINATE) clinical trial program to be effective in the treatment of noninfectious uveitis. With an acceptable side-effects profile, voclosporin is an exciting addition to the therapeutic options available in the management of noninfectious uveitis.

Tacrolimus-Induced Hypertension

See related article, pp 1167–1175 Hypertension associated with solid-organ transplantation is of major clinical importance, because the degree of posttransplant hypertension has been shown to correlate with reductions in graft viability and survival.1,–,3 Effective blood pressure control with antihypertensives has been shown to enhance graft survival and reduce the risk of future cardiovascular disease and events in transplant recipients.3 The etiology of posttransplant hypertension is varied; however, hypertension produced by immunosuppressant therapy is an important cause of posttransplant-related hypertension. Tacrolimus (FK506) and cyclosporine A are 2 common immunosuppressants that have been shown to affect blood pressure. Thus, elucidation of mechanisms that contribute to posttransplant hypertension has major clinical implications. In general, the type of immune response evoked by a particular set of antigens, such as that associated with organ transplantation, depends on the expansion of specific T-lymphocyte (T-cell) subsets, as well as the duration of antigen exposure, which, in the case of organ transplantation, is life long. Tacrolimus and cyclosporine A limit the immune response via inhibition of calcineurin activity, through similar but slightly different mechanisms. With tacrolimus, the mechanism of calcineurin inhibition involves tacrolimus binding to its cytosolic binding partner, the ubiquitously expressed immunophilin FKBP12. Tacrolimus/FKBP12, along with calcium and calmodulin, forms a complex with calcineurin, thereby inhibiting calcineurin's phosphatase activity. Calcineurin signaling has been shown to regulate expression of a number of cytokines, including interleukin (IL)-2, IL-3, IL-4, and interferon-γ, as well as expression of the IL-2 receptor, which have been implicated in T-cell activation.4 For example, reducing IL-2 signaling with tacrolimus is important in limiting the immune response, because IL-2 promotes the expansion of cytotoxic T cells and has been shown to play an important role in immunologic memory. In this issue of Hypertension , Chiasson et al5 report …

Calcineurin inhibition by polaprezinc in rats with experimentally-induced colitis

Aims We investigated the therapeutic effect of polaprezinc (PZ), N-(3-aminopropionyl)- l-histidinato zinc, in rats with experimentally-induced colitis by focusing on calcineurin (CN) inhibition. CN plays a crucial role in T-cell activation and cytokine gene expression and is targeted by immunosuppressants such as cyclosporine and FK506. Main methods Colitis was induced into male Wistar rats by trinitrobenzene sulfonic acid and was treated with intrarectally administered PZ. The inflammation was assessed by the macroscopic damage score, colon wet weight, and proinflammatory mediator expression by RT-PCR analysis. Protein expression of calcineurin and the activation of its substrate, the nuclear factor of activated T cells (NFAT) transcription factor, were also studied. Calcineurin inhibition by PZ was investigated in in vitro experiments using colonic mucosa, purified calcineurin enzyme, and Jurkat T cells. Key findings CN was activated in the colitic mucosa; PZ treatment inhibited CN activation, the expression of proinflammatory cytokines in the mucosa, and thereby ameliorated the experimental colitis in rats. In in vitro experiments, PZ inhibited CN activity, NFAT activation, interleukin-2 expression, and the growth of Jurkat T cells. In the effective concentrations, PZ did not affect cell viability. Significance Our results suggest that PZ can be used as an immunosuppressive agent for the treatment of colitis through its inhibitory effect on CN activity.

BNP controls early load-dependent regulation of SERCA through calcineurin

Heart failure is characterised by reduced expression of sarcoplasmic reticulum calcium-ATPase (SERCA) and increased expression of B-type natriuretic peptide (BNP). The present study was performed to investigate causality of this inverse relationship under in vivo conditions in the transversal aortic constriction mouse model (TAC). Left ventricular SERCA-mRNA expression was significantly upregulated in TAC by 32% after 6 h, but not different from sham after 24 h. Serum proANP and BNP levels were increased in TAC after 24 h (BNP +274%, p < 0.01; proANP +60%, p < 0.05), but only proANP levels were increased after 6 h (+182%, p < 0.01). cGMP levels were only increased 24 h after TAC (+307%, p < 0.01), but not 6 h after TAC. BNP infusion inhibited the increase in SERCA expression 6 h after TAC. In BNP-receptor-knockout animals (GC-A), the expression of SERCA was still significantly increased 24 h after TAC at the mRNA level by 35% (p < 0.05), as well as at the protein level by 25% (p < 0.05). MCIP expression as an indicator of calcineurin activity was regulated in parallel to SERCA after 6 and 24 h. MCIP-mRNA was increased by 333% 6 h after TAC, but not significantly different from sham after 24 h. In the GC-A-KO mice, MCIP-mRNA was significantly increased in TAC compared to WT after 24 h. In mice with BNP infusion, MCIP was significantly lower 6 h after TAC compared to control animals. In conclusion, mechanical load leads to an upregulation of SERCA expression. This is followed by upregulation of natriuretic peptides with subsequent suppression of SERCA upregulation. Elevated natriuretic peptides may suppress SERCA expression by inhibition of calcineurin activity via activation of GC-A.

Stimulation of β 2-adrenergic receptors inhibits calcineurin activity in CD4 + T cells via PKA–AKAP interaction

The sympathetic nervous system (SNS) is able to modulate immune functions via adrenoceptor-dependent mechanisms. Activation of β 2-adrenergic receptors (AR) on CD4 + T lymphocytes has been shown to inhibit Th1-cytokine production and cell proliferation. Here, we investigated the role of the calcium/calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway, in β 2-AR-mediated suppression of T cell function. Purified rat splenic CD4 + T cells were stimulated with anti-CD3/anti-CD28 in presence or absence of the β 2-AR agonist terbutaline (TERB). Treatment with TERB induced a dose-dependent inhibition of cellular CaN activity, along with a reduction in IL-2 and IFN-γ production, and T cell proliferation. Co-administration of the β-AR antagonist nadolol abolished these effects. Blockade of the cAMP-dependent protein kinase A (PKA) with the inhibitor H-89 completely prevented TERB-induced CaN inhibition. However, a receptor-independent rise in the second messenger cAMP was not sufficient to suppress CaN activity. Disruption of the interaction between PKA and A-kinase anchoring protein (AKAP) by the inhibitor peptide St-Ht31 fully blocked TERB-induced CaN inhibition, demonstrating that PKA–AKAP interaction is essential for the β 2-AR-mediated CaN inhibition. Taken together, this study provides evidence for a link between the β 2-AR and TCR signaling pathways since expression of IL-2 and IFN-γ in activated T cells largely depends on dephosphorylation of the transcription factor NFAT by CaN, and identifies a novel intracellular mechanism that can lead to downregulation of T cell function after SNS activation.

Immunosuppressive activity on the murine immune responses of glycyrol from Glycyrrhiza uralensis via inhibition of calcineurin activity

Context: Calcineurin (CN), a unique protein phosphatase, plays an important role in immune regulation. Our laboratory has established an effective molecular drug-screening model based on CN activity.Objective: Our aim is to search for an effective immunosuppressant from Glycyrrhiza uralensis (Leguminosae).Materials and methods: As guided by CN inhibitory test, an active compound was purified and identified as glycyrol. Immunosuppressive activity of glycyrol in vitro was assayed by T lymphocytes proliferation and mixed lymphocyte reaction (MLR). In addition, delayed-type hypersensitivity reaction (DTH) and skin allograft test in vivo were also carried out. Further, we have investigated the effect of glycyrol on phorbol 12-myristate 13-acetate (PMA)/ionomycin (Io)-stimulated IL-2 expression in Jurkat cells.Results: The enzymatic assay showed glycyrol (IC50 = 84.6 μM) inhibited calcineurin activity in a dose-dependent manner. Glycyrol, at the non-cytotoxic concentration, significantly inhibited proliferation of murine spleen T lymphocytes induced by Concanavalin A (Con A) and mixed lymphocyte reaction (MLR) in vitro. In addition, mice treated with glycyrol had shown the dose-dependent decrease in delayed type hypersensitivity (DTH) and prolonged the graft survival by 59% compared to the control group (*p < 0.05). RT-PCR showed glycyrol suppressed IL-2 production in a concentration-dependent manner.Discussion and conclusion: Our results show the immunosuppressive activity of glycyrol and this activity should be due to its inhibitory effect on CN activity, thereby suppressing IL-2 production and regulating T lymphocytes. Thus, glycyrol could be a candidate for development as a novel immunomodulatory drug.

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Individualized Monitoring of Nuclear Factor of Activated T Cells-Regulated Gene Expression in FK506-Treated Kidney Transplant Recipients

The suggested key mechanism of both cyclosporine A (CsA) and FK506 is the inhibition of calcineurin phosphatase activity, preventing nuclear factor of activated T cells (NFAT)-translocation into the nucleus of T cells, with a subsequent transcriptional block of crucial cytokine genes. However, the two drugs exert different clinical activities as exemplified by the ability of FK506 to treat acute rejections. Inhibition of calcineurin activity by FK506 occurs in vitro at the same or even higher dose as for CsA; however, the magnitude of clinical and experimental immunosuppression is higher, indicating that FK506 may act in a calcineurin-independent way. To test this hypothesis, we measured the inhibition of NFAT-regulated gene expression in 262 stable kidney transplanted patients after FK506 intake. Previously, we showed that the optimal degree of NFAT inhibition in patients treated with CsA is between 15% and 30% residual gene expression. A considerable number of patients treated with FK506 do not achieve this level of immunosuppression despite therapeutic drug concentrations. Importantly, FK506 does inhibit protein translation. This insufficient degree of NFAT inhibition was associated with a higher rate of biopsy-proven acute rejection but also with a lower incidence of recurrent infections. Conversion of CsA to FK506 causes immediately reduced inhibition of NFAT-regulated gene expression. We could demonstrate that a considerable number of FK506-treated patients benefit from the drug, irrespective of the potency of NFAT inhibition in T cells by a yet unknown mechanism. Nevertheless, residual expression of NFAT-regulated genes seems to be a useful pharmacodynamic method to monitor FK506 therapy in renal transplant patients.

Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

Calcineurin activity augments cAMP/PKA-dependent activation of V-ATPase in blowfly salivary glands

We have examined the role of the Ca(2+)-dependent protein phosphatase 2B (calcineurin) in the regulation of the vacuolar H(+)-ATPase (V-ATPase) in blowfly salivary glands. In response to the neurohormone serotonin [5-hydroxytryptamine (5-HT)] and under the mediation of the cAMP/PKA signaling pathway, the secretory cells assemble and activate V-ATPase molecules at the apical membrane. We demonstrate that the inhibition of calcineurin activity by cyclosporin A, by FK-506, or by prevention of the elevation of Ca(2+) diminishes the 5-HT-induced assembly and activation of V-ATPase. The effect of calcineurin on V-ATPase is mediated by the cAMP/PKA signaling pathway, with calcineurin acting upstream of PKA, because 1) cyclosporin A does not influence the 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP)-induced activation of V-ATPase, and 2) the 5-HT-induced rise in cAMP is highly reduced in the presence of cyclosporin A. Moreover, a Ca(2+) rise evoked by the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor cyclopiazonic acid leads to an increase in intracellular cAMP concentration and a calcineurin-mediated PKA-dependent activation of V-ATPase. We propose that calcineurin activity mediates cross talk between the inositol 1,4,5-trisphosphate/Ca(2+) and the cAMP/PKA signaling pathways, thereby augmenting the 5-HT-induced rise in cAMP and thus the cAMP/PKA-mediated activation of V-ATPase.

The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis

BackgroundGlucocorticoid hormones (GCs) induce apoptosis of leukemic T-cells by transcriptional regulation via the GC receptor, GR. In the human leukemic CEM cell culture model, RCAN1 has been identified as one of the genes that is specifically upregulated only in the GC-sensitive CEM C7-14 cells, but not in the GC-resistant CEM-C1-15 sister cells in correlation with GC-evoked apoptosis. RCAN1 gene encodes two major protein isoforms of the regulator of calcineurin (RCAN1), RCAN1-1 and RCAN1-4 via alternative splicing of exons 1 and 4 respectively, to exons 5-7. Studies reported here evaluated the differential regulation and function of the two transcripts and protein products of RCAN1 by the synthetic GC dexamethasone (Dex), and by modulators of calcium signaling.ResultsDex selectively upregulates transcript specific for RCAN 1-1 in glucocorticoid (GC)-susceptible human leukemic CEM-C7-14 cells but not in GC-refractory CEM-C1-15 sister cells. Expression of the second major transcript, RCAN1-4, is upregulated by [Ca2+]i inducers, thapsigargin and A23187, but not by Dex, suggesting a mutually exclusive regulatory pathway for both RCAN1 transcripts. GC-mediated upregulation of RCAN1-1 transcript and RCAN1-1 protein was kinase dependent, and was blocked by staurosporine and the p38 MAP kinase inhibitor SB 202190. RCAN1-1 coimmunoprecipitates with calcineurin PP3C and Dex-mediated RCAN1-1 upregulation correlated with reduction in calcineurin PP3C activity.ConclusionData presented here suggest that GCs specifically upregulate RCAN1-1 transcript and protein while inducers of [Ca2+]i selectively upregulate RCAN1-4. GC-mediated increase in RCAN1-1 abundance and binding possibly inhibits calcineurin activity and modulates apoptosis in CEM-C7-14 cells.

Blockade of Protein Phosphatase 2B Activity in the Amygdala Increases Anxiety- and Depression-Like Behaviors in Mice

Organ transplant patients receive chronic administration of the calcineurin inhibitor cyclosporin-A (CsA) and demonstrate increased incidence of mood disorders. Significant calcineurin expression can be observed with immunohistochemistry in the amygdala, a brain area important for behaviors related to mood disorders and anxiety. It is therefore important to determine whether chronic blockade of calcineurin might contribute to symptoms of anxiety and depression in these patients. Pharmacological CsA and viral-mediated gene transfer (adeno-associated viral expression of short hairpin RNA [AAV-shRNA]) approaches were used to inhibit calcineurin activity globally and selectively in the amygdala of the mouse brain to determine the role of calcineurin in behaviors related to depression and anxiety. Systemic inhibition of calcineurin activity with CsA or local downregulation of calcineurin levels in the amygdala with AAV-delivered shRNAs targeting calcineurin A increased behavioral measures of anxiety in both the elevated plus maze and light/dark tests with no changes in locomotor activity. In the forced swim and tail suspension models of depression-like behavior, calcineurin blockade in the amygdala increased immobility similarly to manipulations that lead to a depression-like phenotype. Taken together, these data demonstrate that decreasing calcineurin activity in the amygdala increases anxiety- and depression-like behaviors. These studies suggest that chronic administration of CsA to organ transplant patients could have significant effects on anxiety and mood and that this should be recognized as a clinical consequence of treatment to prevent transplant rejection.

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15-21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni(2+) was found to be the strongest stimulator, while Zn(2+) was found to inhibit CaN phosphatase activity. Mn(2+) was a relatively less effective stimulator compared to Ni(2+). Fe(2+) was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe(2+) slightly inhibited CaN activity from bovine brain. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.

The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1−/− myopathic mice

We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.