Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes Atsushi Fujimura1, Hiroyuki Michiue1, Tei-ichi Nishiki1, Iori Ohmori1, Fan-Yan Wei2, Hideki Matsui1, Kazuhito Tomizawa2,3* 1 Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 2 Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan, 3 PREST, Japan Science Technology Agency (JST), Kawaguchi, Japan Abstract Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca2+ and dephosphorylated NFATc2 under low Ca2+ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/ NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development. Citation: Fujimura A, Michiue H, Nishiki T-i, Ohmori I, Wei F-Y, et al. (2011) Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes. PLoS ONE 6(3): e17685. doi:10.1371/journal.pone.0017685 Editor: Colin Combs, University of North Dakota, United States of America Received October 27, 2010; Accepted February 7, 2011; Published March 14, 2011 Copyright: 2011 Fujimura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan and by a Grant-in-aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tomikt@kumamoto-u.ac.jp Introduction The hair follicle is a highly developed organ and the mechanisms that regulate the hair cycle have been extensively investigated [1,2]. Immunosuppressant drugs such as cyclosporin A (CsA) and FK506, which are indispensable for transplantation, cause hypertrichosis [1,3]; recent studies have shown that this phenomenon is associated with inhibition of the calcineurin (Cn)/ nuclear factor of activated T cells (NFAT) signaling pathway in follicular keratinocytes and follicular stem cells [4,5]. Cn, a Ca2+/calmodulin-dependent serine/threonine phospha- tase, plays important roles in cooperation with NFAT in various tissues such as the immune system, cardiac muscle and neurons [6– 8]. After activation via a rise in intracellular Ca2+ concentration, Cn dephosphorylates cytoplasmic NFAT (NFATc1-c4); this results in translocation of NFAT into the nucleus where it binds to DNA to activate gene transcription [9]. In follicular keratinocytes, NFATc2 regulates the expression of cell cycle-associated proteins, including p21waf1/cip1 and p27kip1, and apoptosis-associated proteins such as p53, and, consequently, controls the transition from the anagen to catagen stages [4]. Similarly, in bulge stem cells, NFATc1 regulates proliferation of follicular stem cells through Cdk4 repression [5]. Cn, which is ubiquitously expressed, is a heterodimer comprised of a catalytic subunit (CnA) binding with calmodulin (CaM) and a calcium-binding regulatory B subunit (CnB) [10]. CnA has four principal domains involved in enzymatic regulation: the N- terminus catalytic domain, the CnB-binding domain, the CaM- binding domain, and the C-terminus autoinhibitory domain. The latter conformationally blocks phosphatase activity and is released from the catalytic domain in response to Ca2+/CaM binding [10]. Three isoforms (alpha, beta, gamma) of the CnA subunit have been identified. The alpha and beta (ß) isoforms are expressed in a range of tissues whereas the gamma isoform is predominantly expressed in the testis [11–13]. Although eleven splicing variants of CnAß have been identified, only four of these variants appear to have functional phosphatase activity as they contain a conserved catalytic domain (ID No. GC10M074866 in GeneCards). In- creased levels of intracellular Ca2+ are believed to trigger Cn activation and to be a key regulator of a variety of physiological and pathological processes [10,14,15]. In general, follicular keratinocytes are cultured under low Ca2+ (,50 mM) conditions and an increase in intracellular/extracellular Ca2+ concentration readily induces differentiation [16,17]. Currently, little is known about Cn activation under such low Ca2+ conditions in follicular PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17685