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8 pages, 5 figures.-- PMID: 19189965 [PubMed].-- PMCID: PMC2666591.-- Supplementary information available at: http://www.jbc.org/content/suppl/2009/02/05/M805889200.DC1.html, Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity., This work was supported by grants from the Fundació La Marató de TV3 (Reference 030830), the Generalitat de Catalunya (Reference 2006 BE 00051), the Spanish Ministry of Education and Science (Grants SAF2006-04815, BIO2004-00998, BIO2007-60066, CTQ2005-00995/BQU), and the Fundación Mutua Madrileña 2007. The costs of publication of this article were defrayed in part by the payment of page charges.

The regulators of calcineurin (RCAN) proteins, previously known as calcipressins, have been considered to be a well conserved family from yeast to human based on the conservation of their FLISPP motif. Here, after performing a RCAN comparative genomic analysis we propose the existence of a novel functionally closely related RCAN subfamily restricted to vertebrates, the other RCAN proteins being considered only as distantly related members of the family. In addition, while three paralogous RCAN genes are found in vertebrates, there is only one in the other members of Eukarya. Moreover, besides the FLISPP motif, these paralogous genes have two others conserved motifs, the Cn-inhibitor RCAN (CIC) and the PxIxxT, which are restricted to vertebrates. In humans, RCAN1 and RCAN2 bind and inhibit Cn through their C-terminal region. Given the high amino acid identity in this region among human RCANs, authors in the field have hypothesized a role for RCAN3 in inhibiting Cn activity. Here, we demonstrate for the first time that human RCAN3, encoded by the RCAN3 (also known as DSCR1L2) gene, interacts physically and functionally with Cn. This interaction takes place only through the RCAN3 CIC motif. Overexpression of this sequence inhibits Cn activity towards the nuclear factor of activated T cells (NFAT) transcription factors and down-regulates NFAT-dependent cytokine gene expression in activated human Jurkat T cells.

Cooperation between calcineurin (CN)-NFATc and RAF-MEK-ERK signaling pathways is essential in thymocyte positive selection. It is known that the Regulators of Calcineurin (RCAN) proteins can act either facilitating or suppressing CN-dependent signaling events. Here, we show that RCAN genes are expressed in lymphoid tissues, and address the role of RCAN proteins in T cell development. Overexpression of human RCAN3 and RCAN1 can modulate T cell development by increasing positive selection-related surface markers, as well as the "Erk(hi) competence state" in double positive thymocytes, a characteristic molecular signature of positive selection, without affecting CN activity. We also found that RCAN1/3 interact with RAF kinases and CN in a non-exclusive manner. Our data suggests that the balance of RCAN interactions with CN and/or RAF kinases may influence T cell positive selection.

Inhibition of the calcineurin-NFAT signalling pathway is one of the main challenges for immunosuppression therapy to avoid the severe side effects of the current anticalcineurinic drugs, cyclosporin A and FK506. The members of the calcipressin family are endogenous inhibitors of calcineurin. We describe for the first time that two independent motifs within human calcipressin 1, the ELHA and the PxIxxT motifs, interact with calcineurin in an independent functional manner. However, the main finding here is that the ELHA-containing calcineurin-inhibitor CALP1 (CIC) motif is the responsible for the in vivo inhibition of calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells. We believe that the identification of the CIC motif could be used as a starting point for the development of new immunosuppressive drugs for use in transplantation and autoimmune diseases.

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27(kip1) and p21(waf1). The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation.