Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.
Leptin, a product of the obesity gene, has been shown to produce cardiac hypertrophy. Although leptin’s mechanism of action is poorly understood activation of the RhoA/ROCK pathway has been proposed as a contributing mechanism. The Ca(2+)-dependent phosphatase calcineurin plays a critical role in the hypertrophic program although it is not known whether leptin can activate this signaling pathway or whether there is a relationship between RhoA activation and calcineurin. Accordingly, we determined the effect of leptin on calcineurin activation and assessed the possible role of RhoA. Experiments were performed using cultured neonatal rat ventricular myocytes exposed to 50ng/ml leptin for 24h which resulted in a robust hypertrophic response. Moreover, leptin significantly increased intracellular Ca(2+) and Na(+) concentrations which was associated with significantly reduced activity of the 3Na(+)-2K(+)ATPase. The hypertrophic response to leptin were completely abrogated by both C3 exoenzyme (C3), a RhoA inhibitor as well as the reverse mode 3Na(+)-1Ca(2+) exchange inhibitor KB-R7943 ((2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl]isothiourea methanesulfonate), however only the effect of the latter was associated with attenuation of intracellular Ca(2+) concentrations whereas Ca(2+) concentrations were unaffected by C3. Similarly, C3 and KB-R7943 significantly attenuated early leptin-induced increase in calcineurin activity as well as the increase in nuclear translocation of the transcriptional factor nuclear factor of activated T cells. The hypertrophic response to leptin was also associated with increased p38 and ERK1/2 MAPK phosphorylation and increased p38, but not ERK1/2, translocation into nuclei. Both p38 responses as well as hypertrophy were abrogated by KB-R7943 as well as the calcineurin inhibitor FK-506 although ERK1/2 phosphorylation was unaffected. Our study therefore demonstrates a critical role for the calcineurin pathway in mediating leptin-induced hypertrophy. Moreover, we report a novel RhoA-dependent leptin-induced calcineurin activation which acts independently of changes in intracellular Ca(2+) concentrations.
Calcineurin is a ubiquitously expressed calcium-dependent phosphatase that is inhibited by the immunosuppressant drugs cyclosporine and tacrolimus. Measuring calcineurin activity in transplant patients has been complicated by a lack of consistent correlation between drug level and enzyme activity, particularly with chronic use. Data from mice lacking the CnAα or CnAβ isoform of the catalytic subunit of calcineurin demonstrate that loss of CnAβ results in immunosuppression, whereas loss of CnAα does not. As such, methods to examine activity of the CnAβ isoform may be more clinically relevant than nonspecific assays.
Since its introduction in photomedicine in 1983 ECP (extracorporeal photopheresis) has over the past decades been established as a safe and effective treatment approach for the palliative management of patients with cutaneous T-cell lymphoma, the Sezary syndrome variant in particular. Subsequently its effectiveness has been well documented in a number of additional T-cell-mediated diseases, particularly in the treatment and prevention of acute and chronic graft-vs. -host disease. More recently, ECP has been successfully used to treat acute heart allograft rejection and chronic allograft dysfunction after lung transplantation without increasing infectious complications. As recently documented ECP was also used as a part of CNI (calcineurin inhibitors) sparing or staggering protocols. For this group of patients it is proposed that its efficacy may be partly attributed through direct induction of lymphocyte apoptosis (Tambur et al., 2000)  and subsequent production of regulatory T cells (Treg) (Lamioni et al., 2007) [2,3] without causing general immunosuppression. However, the exact indications for use of ECP within this framework are not yet finalized.
Induction and Maintenance Immunosuppression Treatment of Proliferative Lupus Nephritis: A Network Meta-analysis of Randomized Trials
- American journal of kidney diseases : the official journal of the National Kidney Foundation
- Published 8 months ago
Intravenous (IV) cyclophosphamide has been first-line treatment for inducing disease remission in lupus nephritis. The comparative efficacy and toxicity of newer agents such as mycophenolate mofetil (MMF) and calcineurin inhibitors are uncertain.
Topical calcineurin inhibitors (TCIs), commercially available since 2000-2001, are the first and only topical medications approved for chronic treatment of atopic dermatitis (AD) in pediatric patients and remain a welcomed alternative to topical corticosteroids. In January 2006, the US Food and Drug Administration (FDA) issued a boxed warning requirement based on a theoretical risk of malignancy (including lymphoma) with TCI use. However, in the years since, analyses of epidemiologic and clinical data have failed to demonstrate a causal relationship between TCI use and malignancy or lymphoma risk, especially for pimecrolimus cream. In fact, the observed number of malignancies and lymphomas observed both in post-marketing surveillance and reported to the FDA using its adverse events reporting system is much lower among TCI-exposed patients than the expected number for the general population. Furthermore, among children enrolled in post-marketing pediatric registry studies for both tacrolimus and pimecrolimus followed for up to 5.5 years [10,724 patient-years (PY)] or 6.5 years (16,219 PY), respectively, the observed number of malignancies and lymphomas is very low and similar to the number expected for a sample of similar size in the general population. In addition to reporting these comparative malignancy and lymphoma data, this article provides a historical overview of the boxed warning requirement and critically evaluates the preclinical, clinical, and epidemiological evidence that has thus far failed to substantiate a relationship between TCI use and malignancy. The authors also provide practical clinical advice for optimizing AD management and patient care in the context of the boxed warning.
Topical immunomodulators (TI)-including corticosteroids, calcineurin inhibitors, and vitamin D analogues-are commonly prescribed in multiple specialties, but cost comparisons are lacking.
Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell-intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.
CSACI position statement: safety of topical calcineurin inhibitors in the management of atopic dermatitis in children and adults
- Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology
- Published over 4 years ago
Atopic dermatitis (AD) is a condition frequently encountered in medical practices across the country. Arming ourselves with appropriate and safe treatment modalities to provide relief for this chronic and relapsing inflammatory condition is of utmost importance to our patients and their families. Utilizing topical calcineurin inhibitors (TCIs) for the treatment of AD not responsive to high-potency corticosteroids, or low-potency corticosteroids and localized to the face, eyelids, and skin folds of patients >2 years, is reasonable to include in common practice. Despite the FDA¿s Black Box warning, to date no evidence has been published linking the TCIs to an increased incidence of malignancy in either children or adults that establishes causation. The Canadian Society of Allergy and Clinical Immunology (CSACI) therefore recognizes that the benefits of TCIs should be carefully weighed with the theoretical risks in advising patients, and acknowledges that long-term studies remain in progress. The safety and efficacy of topical tacrolimus and pimecrolimus should therefore be considered when treating children and adults with AD in Canadian allergy and immunology practices.
Lupus nephritis (LN) therapy has limited efficacy due to its toxicity, and LN patients suffer high risks of renal and cardiovascular morbidity and mortality. Calcineurin inhibitors (CNIs) have been used for over >30 years in LN treatment and are an established alternative therapy for Class V nephritis, but uncertainty remains about their role in proliferative disease or in the maintenance of remission. More recently, the combination of CNIs with mycophenolate mofetil (MMF) and glucocorticoid combination therapy, ‘multitarget’ therapy and the use of tacrolimus as opposed to ciclosporin has received attention. Is the evidence now sufficient to support the routine use of regimens including CNIs in LN? Although CNIs appear to have similar efficacy to MMF-based regimens as induction therapy, and are comparable with azathioprine as maintenance treatment, CNI toxicities, such as new-onset hypertension, hyperglycaemia and nephrotoxicity, have been problematic. Multitarget therapy improves the rate of complete remission in short-term studies, but whether this benefit is maintained over the longer term is uncertain. However, patient tolerability is lower and the frequency of serious events is higher in multitarget versus cyclophosphamide-based regimens, and there is a paucity of evidence from non-Asian ethnic groups. CNI-based therapy is also complicated by the absence of standardized dosing and the need for drug level monitoring, as well as by pharmacogenetic differences. Also, multitarget therapy increases the complexity and the cost of treatment. There is insufficient evidence to support the routine use of CNI-based or multitarget therapy for proliferative LN. Further data on long-term renal and cardiovascular outcomes and strategies to improve tolerability and safety are required.