Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes

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

Selective killing of Burkitt's lymphoma cells by mBAFF-targeted delivery of PinX1

Increased expression of BAFF (B cell-activating factor belonging to the TNF family) and its receptors has been identified in numerous B-cell malignancies. A soluble human BAFF mutant (mBAFF), binding to BAFF receptors but failing to activate B-lymphocyte proliferation, may function as a competitive inhibitor of BAFF and may serve as a novel ligand for targeted therapy of BAFF receptor-positive malignancies. Pin2/TRF1-interacting protein X1 (PinX1), a nucleolar protein, potently inhibits telomerase activity and affects tumorigenicity. In this study, we generated novel recombinant proteins containing mBAFF, a polyarginine tract 9R and PinX1 (or its C/N terminal), to target lymphoma cells. The fusion proteins PinX1/C–G4S–9R–G4S–mBAFF and PinX1/C–9R–mBAFF specifically bind and internalize into BAFF receptor-positive cells, and subsequently induce growth inhibition and apoptosis. The selective cytotoxicity of the fusion proteins is a BAFF receptor-mediated process and depends on mBAFF, PinX1/C and 9R. Moreover, the fusion proteins specifically kill BAFF receptor-expressing Burkitt's lymphoma (BL) cells by inhibiting telomerase activity and the consequent shortening of telomeres. Therapeutic experiments using PinX1C–G4S–9R–G4S–mBAFF in severe combined immunodeficient (SCID) mice implanted with Raji cells showed significantly prolonged survival times, indicating the in vivo antitumor activity of the fusion protein. These results suggest the potential of PinX1/C–G4S–9R–G4S–mBAFF in targeted therapy of BL