Expression, regulation and potential function of NFAT transcription factors in human mesenchymal stem cells

Introduction: Human mesenchymal stem cells (hMSCs) display immunomodulatory effects partially mediated by soluble factors like prostaglandin E2 (PGE2), and have been successfully used in the treatment of graft-versus-host-disease. Information about transcriptional mechanisms regulating hMSC function is scarce. Nuclear Factor of Activated T cells (NFAT) is a transcription factor known to control T-cell-dependent immune responses. In resting cells, NFAT is phosphorylated, cytoplasmic and inactive, while upon stimulation NFAT is dephosphorylated by the phosphatase calcineurin, leading to nuclear translocation and activation of the protein. Inactivation of NFAT is mediated by several kinases, e.g. glycogen synthase kinase 3b (GSK3b). Little is known about expression, regulation or function of NFAT in hMSCs. Methods: Bone marrow-derived primary hMSCs were used in all experiments. NFAT and cyclooxygenase 2 (COX2) expression was determined by real time PCR and western blotting. Subcellular localization of NFAT was analyzed by confocal microscopy. NFAT and COX2 promotor activity was measured by luciferase assays. PGE2 concentration was analyzed by ELISA. Results: hMSCs express considerable levels of all NFAT family members (predominantly NFATc3 and NFATc4), with the exception of NFATc2. In untreated hMSCs, NFAT proteins are partially constitutively active, as judged by their phosphorylation and subcellular localization status. Dephosphorylation, nuclear translocation and transcriptional activity of NFAT are further induced by treatment with ionomycin and reversed by cyclosporin A, indicating that the regulation of NFAT in hMSCs occurs in a calcineurin-dependent manner. Inhibition of GSK3b leads to cyclosporin A-dependent nuclear accumulation of NFAT, emphasizing the presence of a constitutive active calcineurin pathway in hMSCs. Treatment of hMSCs with ionomycin and phorbol ester induces, while CsA attenuates, the expression of COX2 and PGE2, suggesting that these genes are transcriptional targets of NFAT in hMSCs. Conclusion: hMSCs show an NFAT expression profile which is clearly distinct from that found in Jurkat T cells or CD34+ cells. Constitutive activation of NFAT in hMSCs suggests it may be involved in mediating biological properties of these cells, e.g. the modulation of lymphocyte function through the expression of PGE2. Dissection of NFAT-regulatory pathways and target genes in hMSCs will allow to identify and specifically manipulate hMSC functions.