Calcineurin-dependent Pathway Research Articles

Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans.

Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the C. albicans genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of C. albicans strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC.

Defective induction of the proteasome associated with T‐cell receptor signaling underlies T‐cell senescence

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T-cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4+ T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4+ T cells upon T-cell receptor (TCR) stimulation underlie T-cell senescence. Proteasome dysfunction promotes senescence-associated phenotypes, including defective proliferation, cytokine production and increased levels of PD-1+ CD44High CD4+ T cells. Proteasome induction by TCR signaling via MEK-, IKK- and calcineurin-dependent pathways is attenuated with age and decreased in PD-1+ CD44High CD4+ T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4+ T-cell senescence.

Canonical Transient Potential Receptor-3 Channels in Normal and Diseased Airway Smooth Muscle Cells.

All seven canonical transient potential receptor (TRPC1-7) channel members are expressed in mammalian airway smooth muscle cells (ASMCs). Among this family, TRPC3 channel plays an important role in the control of the resting [Ca2+]i and agonist-induced increase in [Ca2+]i. This channel is significantly upregulated in molecular expression and functional activity in airway diseases. The upregulated channel significantly augments the resting [Ca2+]i and agonist-induced increase in [Ca2+]i, thereby exerting a direct and essential effect in airway hyperresponsiveness. The increased TRPC3 channel-mediated Ca2+ signaling also results in the transcription factor nuclear factor-κB (NF-κB) activation via protein kinase C-α (PKCα)-dependent inhibitor of NFκB-α (IκBα) and calcineurin-dependent IκBβ signaling pathways, which upregulates cyclin-D1 expression and causes cell proliferation, leading to airway remodeling. TRPC3 channel may further interact with intracellular release Ca2+ channels, Orai channels and Ca2+-sensing stromal interaction molecules, mediating important cellular responses in ASMCs and the development of airway diseases.

Effects of cyclosporine and dexamethasone on canine T cell expression of interleukin-2 and interferon-gamma

Cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases in dogs. Cyclosporine inhibits calcineurin-dependent pathways of T cell activation and resultant T cell cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. Little work has been done comparing the effects of these agents on T cell cytokine production in dogs. Our study measured T cell interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production using flow cytometry and T cell IL-2 and IFN-γ gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in activated canine T cells incubated with cyclosporine and dexamethasone in vitro. For flow cytometric assays, diluted whole blood was cultured for 7 h in the presence of cyclosporine (10, 100, 500, and 1000 ng/mL) or dexamethasone (10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL). For qRT-PCR, whole blood was cultured for 5 h with the same drugs at the same concentrations, and RNA was then extracted from leukocytes. Flow cytometry and qRT-PCR both demonstrated inhibition of IL-2 and IFN-γ that was concentration-dependent in response to cyclosporine, and was more variable for dexamethasone. Quantitative RT-PCR but not flow cytometry documented significant reduction of IL-2 expression after dexamethasone treatment, while both methods showed concentration-dependent suppression of IFN-γ. Quantitative RT-PCR also revealed additional cytokine suppression at higher cyclosporine concentrations, an effect not found using flow cytometry, and may therefore be the preferred method for cytokine determination in dogs. Suppression of IL-2 and IFN-γ in activated T cells may have potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine T cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients.

TRPC6 expression in neurons is differentially regulated by NR2A- and NR2B-containing NMDA receptors.

The expression of transient receptor potential canonical 6 (TRPC6) in central nervous system (CNS) is important for neuronal functions and certain neural disorders. However, the regulatory mechanism of TRPC6 expression in neurons is still obscure. In this study, we show that TRPC6 expression in the primary cultured cortical neurons is bidirectionally regulated by glutamate. Activation of NR2A-containing NMDARs induces TRPC6 transcription through a calcineurin-dependent pathway. In contrast, activation of NR2B-containing NMDARs causes TRPC6 degradation through calpain. Thus, TRPC6 expression in neurons is regulated by glutamate in a bidirectional manner that is dependent on NR2A and NR2B.

The impact of calcineurin inhibitors on neutrophil gelatinase-associated lipocalin and fibroblast growth factor 23 in long-term kidney transplant patients.

Neutrophil gelatinase-associated lipocalin (NGAL), a protein with bacteriostatic functions rapidly excreted from stimulated or damaged epithelial cells, is elevated in acute and chronic kidney disease. A calcineurin dependent signaling pathway for fibroblast growth factor 23 (FGF23) has been revealed, but the effect of calcineurin inhibitors (CNIs) on the levels of NGAL and markers of mineral metabolism in long-term kidney transplant patients has not been explored. In a cross-sectional study, 39 patients who received a first kidney transplant more than 10years ago were split into two groups based on whether (n=28) or not (n=11) they used CNIs. Only patients with well-functioning grafts defined as an estimated glomerular filtration rate ≥45mL/min per 1.73m2 were included. The median levels of NGAL, intact parathyroid hormone (iPTH), and iFGF23 were significantly higher in CNI users vs CNI nonusers, 167.0 (134.0-235.0) ng/mL vs 105.0 (91.3-117.0) ng/mL, P<.001, 13.8 (10.0-17.3)pmol/L vs 8.4 (6.4-9.9) pmol/L, P=.003, and 81.6 (56.4-116.5) pg/mL vs 61.8 (43.3-72.1) pg/mL, P=.04 respectively. The median levels of iFGF23 were higher in CNI users compared to CNI nonusers giving support to the notion of a CNI induced FGF23 resistance in the parathyroid. The net result of CNIs side effects needs to be further explored.

Analysis of Taurine's Anti-Down Syndrome Potential in Caenorhabditis elegans.

Down syndrome (DS) patients overexpress human DS critical region gene 1 (hDSCR-1), whose translational product inhibits calcineurin-dependent signaling pathways of genetic transcription. Compared to hDSCR-1, C. elegans rcn-1 has 40% sequence similarity and its proteins share an analogous function with hDSCR-1 in regulating calcineurin. Taurine has had a positive effect on DS patients. According to animal research studies, taurine reduces the expression of MCIP1, a calcineurin inhibitory protein, on C2C12 myotubes and fibroblast in mouse. This study utilizes two C. elegans models for DS: rcn-1 overexpression model, displaying a calcineurin-deficient phenotype, and calcineurin loss-of function mutants. C. elegans larvae were treated with taurine to characterize its effect and mechanism in helping DS patients. RCN-1 expression and behavioral changes were examined in rcn-1 overexpression and calcineurin-deficient models at different concentrations of taurine. When treated with taurine, transgenic worms harboring an rcn-1 reporter (RCN-1::GFP) showed a reduced level of rcn-1 mRNA expression and improved behaviors that were comparable to those in the wild type. These results indicate that taurine exerts a down-regulating effect on the expression of rcn-1 and, consequently, a positive effect on the expression of calcineurins. In summary, taurine may improve the DS symptoms by prompting a positive interaction between RCN-1 and calcineurin. Furthermore, these results suggest that novel mechanisms may regulate interactions among taurine, RCN-1 and calcineurin.

Insulin Increases Expression of TRPC6 Channels in Podocytes by a Calcineurin-Dependent Pathway

Background/Aims: Insulin signaling to podocytes is relevant for the function of the glomerulus. Now, we tested the hypothesis that insulin increases the surface expression of canonical transient receptor potential canonical type 6 (TRPC6) channels in podocytes by a calcineurin-dependent pathway. Methods: We used quantitative RT-PCR, immunoblotting, immunofluorescence and fluorescence spectrophotometry in cultured podocytes. Activation of Nuclear Factor of Activated T-cells (NFATc1) was measured using a specific calorimetric assay. Results: Insulin increased the expression of TRPC6 transcripts and protein in podocytes. Insulin increased TRPC6 transcripts in a time and dose-dependent manner. The insulin-induced elevation of TRPC6 transcripts was blocked in the presence of tacrolimus, cyclosporine A, and NFAT-inhibitor (each p < 0.01 by ANOVA and Bonferroni's multiple comparison test). Transcripts of NOX4, another target gene of the calcineurin-NFAT pathway, were affected in a similar way. Immunoblotting showed that the administration of 100 nmol/L insulin increased TRPC6-proteins 2-fold within 48 hours. Insulin increased the activity of NFATc1 in nuclear extracts (p < 0.001) whereas tacrolimus, cyclosporine A, and NFAT-inhibitor blocked that insulin effect (p < 0.001; two way ANOVA). Immunofluorescence showed that insulin increased TRPC6-expression on the cell surface. Fluorescence-spectrophotometry and manganese quench experiments indicated that the increased TRPC6-expression after insulin administration was accompanied by an elevated transplasmamembrane cation influx. Insulin-stimulated surface expression of TRPC6 as well as transplasmamembrane cation influx could be reduced by pretreatment with tacrolimus. Conclusion: Insulin increases the expression of TRPC6 channels in podocytes by activation of the calcineurin-dependent pathway.

CONTROL OF GAP JUNCTION CONDUCTANCE BY CALCINEURIN-DEPENDENT CX-43 PHOSPHORYLATION: IMPLICATIONS OF ARRHYTHMOGENESIS

Slower action potential conduction, and consequent arrhythmias, follow decreased gap junction conductance, Gj. We investigated how the Ca2+-dependent phosphatase, calcineurin, regulates Gj through altering the phosphorylation of Cx43 protein, through...

Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin.

Glutaredoxin 6 (Grx6) of Saccharomyces cerevisiae is an integral thiol oxidoreductase protein of the endoplasmic reticulum/Golgi vesicles. Its absence alters the redox equilibrium of the reticulum lumen toward a more oxidized state, thus compensating the defects in protein folding/secretion and cell growth caused by low levels of the oxidase Ero1. In addition, null mutants in GRX6 display a more intense unfolded protein response than wild-type cells upon treatment with inducers of this pathway. These observations support a role of Grx6 in regulating the glutathionylation of thiols of endoplasmic reticulum/Golgi target proteins and consequently the equilibrium between reduced and oxidized glutathione in the lumen of these compartments. A specific function influenced by Grx6 activity is the homeostasis of intracellular calcium. Grx6-deficient mutants have reduced levels of calcium in the ER lumen, whereas accumulation occurs at the cytosol from extracellular sources. This results in permanent activation of the calcineurin-dependent pathway in these cells. Some but not all the phenotypes of the mutant are coincident with those of mutants deficient in intracellular calcium transporters, such as the Golgi Pmr1 protein. The results presented in this study provide evidence for redox regulation of calcium homeostasis in yeast cells.

Cyclosporin A enhances neural precursor cell survival in mice through a calcineurin-independent pathway.

Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neural precursor cells; NPCs) in the adult central nervous system. Administration of CsA in vitro or in vivo promotes the survival of NPCs and expands the pools of NPCs in mice. Moreover, CsA administration is effective in promoting NPC activation, tissue repair and functional recovery in a mouse model of cortical stroke. The mechanism(s) by which CsA mediates this cell survival effect remains unknown. Herein, we examined both calcineurin-dependent and calcineurin-independent pathways through which CsA might mediate NPC survival. To examine calcineurin-dependent pathways, we utilized FK506 (Tacrolimus), an immunosuppressive molecule that inhibits calcineurin, as well as drugs that inhibit cyclophilin A-mediated activation of calcineurin. To evaluate the calcineurin-independent pathway, we utilized NIM811, a non-immunosuppressive CsA analog that functions independently of calcineurin by blocking mitochondrial permeability transition pore formation. We found that only NIM811 can entirely account for the pro-survival effects of CsA on NPCs. Indeed, blocking signaling pathways downstream of calcineurin activation using nNOS mice did not inhibit CsA-mediated cell survival, which supports the proposal that the effects are calcinuerin-independent. In vivo studies revealed that NIM811 administration mimics the pro-survival effects of CsA on NPCs and promotes functional recovery in a model of cortical stroke, identical to the effects seen with CsA administration. We conclude that CsA mediates its effect on NPC survival through calcineurin-independent inhibition of mitochondrial permeability transition pore formation and suggest that this pathway has potential therapeutic benefits for developing NPC-mediated cell replacement strategies.

Cav3.2 T-type calcium channel is required for the NFAT-dependent Sox9 expression in tracheal cartilage.

Intracellular Ca(2+) transient is crucial in initiating the differentiation of mesenchymal cells into chondrocytes, but whether voltage-gated Ca(2+) channels are involved remains uncertain. Here, we show that the T-type voltage-gated Ca(2+) channel Cav3.2 is essential for tracheal chondrogenesis. Mice lacking this channel (Cav3.2(-/-)) show congenital tracheal stenosis because of incomplete formation of cartilaginous tracheal support. Conversely, Cav3.2 overexpression in ATDC5 cells enhances chondrogenesis, which could be blunted by both blocking T-type Ca(2+) channels and inhibiting calcineurin and suggests that Cav3.2 is responsible for Ca(2+) influx during chondrogenesis. Finally, the expression of sex determination region of Y chromosome (SRY)-related high-mobility group-Box gene 9 (Sox9), one of the earliest markers of committed chondrogenic cells, is reduced in Cav3.2(-/-) tracheas. Mechanistically, Ca(2+) influx via Cav3.2 activates the calcineurin/nuclear factor of the activated T-cell (NFAT) signaling pathway, and a previously unidentified NFAT binding site is identified within the mouse Sox9 promoter using a luciferase reporter assay and gel shift and ChIP studies. Our findings define a previously unidentified mechanism that Ca(2+) influx via the Cav3.2 T-type Ca(2+) channel regulates Sox9 expression through the calcineurin/NFAT signaling pathway during tracheal chondrogenesis.

α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization

Homozygosity for a common null polymorphism (R577X) in the ACTN3 gene results in the absence of the fast fibre-specific protein, α-actinin-3 in ∼16% of humans worldwide. α-Actinin-3 deficiency is detrimental to optimal sprint performance and benefits endurance performance in elite athletes. In the general population, α-actinin-3 deficiency is associated with reduced muscle mass, strength and fast muscle fibre area, and poorer muscle function with age. The Actn3 knock-out (KO) mouse model mimics the human phenotype, with fast fibres showing a shift towards slow/oxidative metabolism without a change in myosin heavy chain (MyHC) isoform. We have recently shown that these changes are attributable to increased activity of the calcineurin-dependent signalling pathway in α-actinin-3 deficient muscle, resulting in enhanced response to exercise training. This led us to hypothesize that the Actn3 genotype influences muscle adaptation to disuse, irrespective of neural innervation. Separate cohorts of KO and wild-type mice underwent 2 weeks immobilization and 2 and 8 weeks of denervation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse, as measured by a change in MyHC isoform. KO mice had a lower threshold to switch from the predominantly fast to a slower muscle phenotype (in response to immobilization) and a higher threshold to switch to a faster muscle phenotype (in response to denervation). We propose that this change is mediated through baseline alterations in the calcineurin signalling pathway of Actn3 KO muscle. Our findings have important implications for understanding individual responses to muscle disuse/disease and training in the general population.

Orm Proteins Integrate Multiple Signals to Maintain Sphingolipid Homeostasis

Sphingolipids are structural components of membranes, and sphingolipid metabolites serve as signaling molecules. The first and rate-limiting step in sphingolipid synthesis is catalyzed by serine palmitoyltransferase (SPT). The recently discovered SPT-associated proteins, Orm1 and Orm2, are critical regulators of sphingolipids. Orm protein phosphorylation mediating feedback regulation of SPT activity occurs in response to multiple sphingolipid intermediates, including long chain base and complex sphingolipids. Both branches of the TOR signaling network, TORC1 and TORC2, participate in regulating sphingolipid synthesis via Orm phosphorylation in response to sphingolipid intermediates as well as nutritional conditions. Moreover, sphingolipid synthesis is regulated in response to endoplasmic reticulum (ER) stress by activation of a calcium- and calcineurin-dependent pathway via transcriptional induction of ORM2. Conversely, the calcium- and calcineurin-dependent pathway signals ER stress response upon lipid dysregulation in the absence of the Orm proteins to restore ER homeostasis.

Translation of Myocyte Enhancer Factor-2 is induced by hypertrophic stimuli in cardiomyocytes through a Calcineurin-dependent pathway

The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates gene expression during cardiomyocyte differentiation and adaptation of the myocardium to stress. MEF2 activity is enhanced by increasing its transcription and by MAPK-dependent phosphorylation, and is reduced by binding to class-II Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here we show that MEF2 protein abundance is regulated at the translational level, determining myocyte size, during hypertrophy. In order to reduce MEF2 protein expression, its silencing through RNA interference required serum deprivation and, even in this condition, MEF2 protein abundance recovered to basal levels in presence of phenylephrine. Hypertrophic agonist stimulation of neonatal ventricular cardiomyocytes increased Mef2 expression by enhancing its translation, without changing its transcription or blocking degradation of the protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA levels. Calcineurin activity influenced expression of Polypyrimidine Tract Protein (PTB), contributing to MEF2 translation. Thus, our results show a previously unrecognized but relevant level of MEF2 activity regulation through the control of its translation that involves Calcineurin and PTB.

Angiotensin II signalling and calcineurin in cardiac fibroblasts: differential effects of calcineurin inhibitors FK506 and cyclosporine A

Cardiac remodelling is controlled by complex systems, including activation of the renin-angiotensin system (RAS) and signalling through MAP kinases and Ca2+-activated calcineurin. Whether Ang II, which increases [Ca2+]i and stimulates MAP kinases, mediates myocardial effects through calcineurin-dependent pathways remain unclear. We investigated effects of two calcineurin inhibitors, cyclosporine A (CsA) and tacrolimus (FK506) (10-10-10-6 mol/L, 20 mins) on activation of MAP kinases and on growth, pro-fibrotic and pro-inflammatory responses in Ang II-stimulated rat cardiac fibroblasts. Ang II increased phosphorylation of ERK1/2 and p38MAPK (1.5-1.8-fold, p<0.05) without effect on JNK. FK506, but not CsA, attenuated Ang II-stimulated MAP kinase activation. Molecular indices of cell growth (proliferating cell nuclear antigen (PCNA)), fibrosis (fibronectin, pro-collagen) and inflammation (iNOS), were upregulated by Ang II (12 hrs). FK506 and CsA inhibited PCNA effects. Ang II-induced pro-fibrotic and pro-inflammatory responses were inhibited by CsA. Ang II receptors, AT1R and AT2R, were not influenced by calcineurin inhibitors. Our data indicate differential calcineurin inhibitor sensitivity of MAP kinases and cellular responses in Ang II-stimulated fibroblasts. p38MAP kinase and ERK1/2 are regulated in a FK506-sensitive manner, whereas fibrosis and inflammation are CsA-sensitive. Cell proliferation is inhibited by both FKC506 and CsA. These are post-receptor phenomena, since AT1R and AT2R status was unaltered by treatment. Our findings identify an important role for calcineurin in MAP kinase/growth/pro-fibrotic/pro-inflammatory signalling by Ang II in cardiac fibroblasts. Although both FK506 and CsA inhibit calcineurin, they exert differential effects on molecular and cellular responses. Such differences may contribute to variable clinical responses of these agents.

Apoptosis inducing effect of tanshinone ⅡA on human nasopharyngeal carcinoma CNE cells

To investigate anticancer effect and potential mechanism of tanshinone II(A) (Tan II(A)) on human nasopharyngeal carcinoma cell line CNE cells. Antiproliferative effect of Tan II(A) on CNE cells was evaluated by morphological examination, cell growth curves, colonial assay and MTT assay. Apoptosis detection was carried out using Hoechest 33258 and PI double-dyeing method. Intracellular Ca2+ concentration and mitochondria membrane potential were detected by fluorospectrophotometer. Bad and MT-1A transcript analysis in CNE cells was analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Tan II(A) could inhibit CNE cells proliferation in dose- and time-dependent manner. 50% inhibiting concentration of Tan II(A) on CNE cells in 24, 48, 72 h was 45.7, 24.8, 3.3 mg x L(-2), respectively. Typical apoptotic morphology such as chromatin aggregation was observed in CNE cells with Tan II(A) treated for 24 h, and the apoptotic inducing effect was in a dose-dependent manner. After treated with Tan II(A), intracellular Ca2+ concentration of CNE cells was increased, mitochondria membrane potential of the cells was decreased, relative mRNA level of Bad and MT-1A was up-regulated. Tan II(A) had anticancer effect on CNE cells through apoptosis via calcineurin-dependent pathway and MT-1A downregulation.

Calcineurin Finds a New Partner in the L-Type Ca 2+ Channel

See related articles, pages 51–60 Calcineurin is a Ca2+/calmodulin-sensitive phosphatase that sits near the top of signaling pathways leading to pathological cardiac hypertrophy.1 Pathological stressors activate calcineurin, for which nuclear factor of activated T cells (NFAT) is a principal substrate. Dephosphorylation unmasks a nuclear localization signal on NFAT, which then translocates to the nucleus and, there, serves as part of a transcription factor complex able to initiate gene expression cascades that induce cardiac hypertrophy and subsequent heart failure. Determining how calcineurin is specifically activated in response to hypertrophic stressors by the same Ca2+ signal used for excitation–contraction coupling has been a major challenge. Triggered by Ca2+ influx through CaV1.2 voltage-gated Ca2+ channels at the sarcolemmal membrane, intracellular calcium briefly increases 10-fold with each heart beat to generate myocyte contraction. Almost as quickly, intracellular calcium falls to its baseline level, allowing myocyte relaxation and preparation for the next beat. There are 2 leading proposals for how Ca2+ could activate the calcineurin/NFAT pathway or function as a versatile regulator of multiple other signaling cascades in the face of the large Ca2+ oscillations driving each cycle of contraction and relaxation. One is that modulation of the amplitude and/or frequency of …

Role of placenta growth factor and its receptor flt‐1 in rheumatoid inflammation: A link between angiogenesis and inflammation

To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt-1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA). Expression of PlGF and flt-1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme-linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) in culture supernatants of either mononuclear cells or synoviocytes. The flt-1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti-CII antibody. PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast-like synoviocytes (FLS). When stimulated with IL-1beta, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFalpha and IL-6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin-dependent pathway. The response to PlGF was associated with increased expression of flt-1 on RA monocytes, which could be induced by IL-1beta and TNFalpha. A novel anti-flt-1 hexapeptide, GNQWFI, abrogated the PlGF-induced increase in TNFalpha and IL-6 production, and also suppressed CII-induced arthritis and serum IL-6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti-CII antibody-induced arthritis in mice. Our data suggest that enhanced expression of PlGF and flt-1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti-flt-1 peptide, GNQWFI, may be an effective strategy for the treatment of RA.

CCN2, the connective tissue growth factor

The role of extracellular matrix proteins in signaling control and regulation of response to injury, particularly in terms of scarring, is a topic of major importance. Blatant clinical examples of disturbed scarring are keloid production and systemic sclerosis, better known as scleroderma [1, 2]. Both these conditions involve the upregulation of a novel extracellular matrix-secreted protein termed connective tissue growth factor (CTGF). In this issue, Lang et al. implicate CTGF in the pathogenesis of cardiac fibrosis in a model of an infectious heart disease [3]. The investigators relied on a murine model of Coxsackie virus B3 (CVB3) myocarditis. They found that CTGF expression was induced in the course of CVB3 myocarditis. Particularly, fibroblasts expressed the cytokine. CTGF upregulation coincided with transforming growth factor-beta (TGF-β) expression and preceded procollagen type I mRNA expression. The investigators then used siRNAs. When they knocked down CVB3 replication, CTGF expression was diminished. However, suppression of CTGF had no effect on virus replication. Thus, CTGF is now implicated in the scarring after infectious heart disease. That CTGF is involved in cardiac fibrosis occurring with targetorgan damage has been described earlier. The same group has published that the serum and glucocorticoid-inducible kinase (SGK1) is important to cardiac fibrosis occurring in the course of desoxycorticosterone acetate (DOCA salt)induced hypertension and that CTGF is induced as a downstream target of the kinase [4]. We investigated earlier whether CTGF mediates the profibrotic effects of angiotensin II (Ang II) in the heart and kidney and the role of calcineurin-dependent pathways in CTGF gene regulation. We used transgenic rats harboring human renin and angiotensinogen genes. Ang II induced an age-dependent increase in myocardial CTGF expression, which was 3.5-fold greater than in normotensive control rats [5]. CTGF overexpression correlated closely with the Ang II-induced rise in blood pressure. CTGF mRNA and protein were located predominantly in areas with leukocyte infiltration, myocardial, and vascular lesions, and colocalized with TGF-β, collagen I, and collagen III mRNA expressions. Blockade of calcineurin activity with cyclosporine A completely normalized Ang II-induced CTGF overexpression in the heart and kidney, suppressed the inflammatory response, and mitigated Ang II-induced cell proliferation and apoptosis. In contrast, blockade of mTOR (target of rapamycin) pathway with everolimus increased CTGF expression. Apparently, CTGF is an important mediator of fibrosis, and therefore, we need to know about the protein in greater detail. CTGF belongs to the CCN family of matrix signaling modulators [6]. The family is named after three prototypical members, cysteine-rich protein 61 (Cyr61), also termed CCN1; CTGF, termed CCN2; and the nephroblastoma overexpressed protein (Nov), also known as CCN3. The italicized letters show the derivation of the designation “CCN”. However, because their description was almost two decades ago, the CCN family has grown. The molecules comprise up to four modules, an insulin-like growth factor binding protein (IGFBP) domain (module I), a Von Willebrand factor domain (module II), a thrombospondinhomology domain (module III), and a cysteine knot, heparin-binding domain (module IV). A schematic view of CTGF and its modules is shown in Fig. 1a. What do the CCN proteins do? They play key roles in angiogenesis, chondrogenesis, and wound healing. The CCN proteins work by participating in cell proliferation, J Mol Med (2008) 86:1–3 DOI 10.1007/s00109-007-0287-x