Calcineurin A-α suppression drives nuclear factor-κB-mediated NADPH oxidase-2 upregulation

A long-term consequence of calcineurin inhibitors (CNIs) is oxidative damage and nephrotoxicity. This study indicates that NF-κB is a novel calcineurin-regulated transcription factor that is activated with calcineurin inhibition, thereby driving oxidative damage in CNI nephropathy. These findings provide additional evidence of divergent calcineurin signaling pathways and suggest that selective CNIs could improve the long-term outcomes of patients by mitigating renal side effects.

Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration

Planarian flatworms regenerate their heads and tails from anterior or posterior wounds and this regenerative blastema polarity is controlled by Wnt/β-catenin signaling. It is well known that a regeneration blastema of appendages of vertebrates such as fish and amphibians grows distally. However, it remains unclear whether a regeneration blastema in vertebrate appendages can grow proximally. Here, we show that a regeneration blastema in zebrafish fins can grow proximally along the proximodistal axis by calcineurin inhibition. We used fin excavation in adult zebrafish to observe unidirectional regeneration from the anterior cut edge (ACE) to the posterior cut edge (PCE) of the cavity and this unidirectional regeneration polarity occurs as the PCE fails to build blastemas. Furthermore, we found that calcineurin activities in the ACE were greater than in the PCE. Calcineurin inhibition induced PCE blastemas, and calcineurin hyperactivation suppressed fin regeneration. Collectively, these findings identify calcineurin as a molecular switch to specify the PCE blastema of the proximodistal axis and regeneration polarity in zebrafish fin.

FK506 Induces the TGF-β1/Smad 3 Pathway Independently of Calcineurin Inhibition to Prevent Intervertebral Disk Degeneration

BackgroundIntervertebral disk (IVD) degeneration is the most common cause of lower back pain. Inhibiting inflammation is a key strategy for delaying IVD degeneration. Tacrolimus (FK506) is a potent immunosuppressive agent that is also beneficial to chondrocytes via alleviating inflammation. However, the potential function of FK506 in IVD and the underlying mechanisms remain unknown. The current study is aim at exploring the underlying mechanism of FK506 in preventing IVD degeneration.MethodsCell morphology was imaged using an optical microscope. mRNA levels of nucleus pulposus (NP) matrix components were determined by qRT-PCR, and protein expression NP matrix components was assessed by western blotting. A rat caudal IVD degeneration model was established to test for FK506 in vivo.ResultsFK506 improved the morphology of NP cells and the cell function at both the mRNA and protein level. FK506 could attenuate NP degeneration induced by IL-1β. Furthermore, FK506 exerted its function via TGFβ/Smad3 activation instead of through calcineurin inhibition. Inhibition of the TGF-β pathway prevented the protective effect of FK506 on IVD degeneration. In an in vivo study, FK506 injection reversed the development of rat caudal IVD degeneration influenced by Smad3.ConclusionOur current study demonstrates the positive effect of FK506 on delaying the degeneration of IVD via the TGFβ/Smad3 pathway.

Taurine Decreases Cellular Cholesterol Level in HepG2 Cells Partly through Upregulating Calcineurin

Objective: Taurine exerts cholesterol-lowering effect through inducing CYP7A1 and promoting the biotransformation of cholesterol into bile acids in livers, but its molecular mechanism remains unclear. Taurine also suppresses the expression of MCIP1, a calcineurin inhibitory protein. Here we aimed to explore whether calcineurin involves in the cholesterol-lowering effect and upregulation of CYP7A1 by taurine. Methods: High cellular cholesterol conditions were obtained by incubating with 0.2mM cholesterol contained DMEM in HepG2 cells. FK506, a calcineurin inhibitor, was used to depress cellular calcineurin. CnAb-/- cells are the HepG2 cells of which calcineurin was knocked down. Taurine was cultured in wild type, high-cholesterol conditions, calcineurin inhibition or deficiency HepG2 cells respectively for 24h or 48h. The levels of intracellular total cholesterol were determined by an enzymatic method and the expressions of CYP7A1, calcineurin, MEK1/2, c-Jun/p-c-Jun and SHP-1 were detected by western blotting. Results: High cellular cholesterol conditions in HepG2 cells were established and resulted in increased CYP7A1 and calcineurin expression. Taurine exhibited the decreasing-cholesterol effects on HepG2 cells regardless of whether cells with high cholesterol conditions or inhibited / deleted intracellular calcineurin. However, the extent of decreasing cholesterol after calcineurin repression or deficiency was much less than that of controls. Taurine could induce the expression of CYP7A1 but this induction was abolished when the cellular calcineurin was inhibited or deleted. Taurine was able to suppress MEK1/2, p-c-Jun and SHP-1, which are several key molecules in one inhibitory pathway of CYP7A1 transcription, whereas this suppression on MEK1/2 but not p-c-Jun or SHP-1 was reversed after completely knocking down calcineurin. Conclusions: Calcineurin was found to be required partly in taurine-decreasing cholesterol effect through inhibiting MEK1/2 which resulted in CYP7A1 upregulation.

Designing Selective and Non-Immunosuppressive Antifungal FK506 Analogs: Structures, Biophysics and Dynamics of Fungal and Human Calcineurin-Inhibitor Complexes

Calcineurin is an attractive antifungal target due to its central role in fungal pathogenesis. The development of specific antifungals targeting calcineurin is complex, as calcineurin inhibitors, such as FK506, are immunosuppressive. Using fungal calcineurin-inhibitor crystal structures we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection. To better understand the interaction of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition at a molecular level, and guide the design of fungal-selective and non-immunosuppressive FK506 analogs, here we report the high-resolution structures of the M. circinelloides FKBP12 bound to FK506 and the human, A. fumigatus and M. circinelloides FKBP12 proteins bound to the FK506 analog, APX879. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12:ligand complexes to enhance fungal-selectivity.

Targeting chronic NFAT activation with calcineurin inhibitors in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) represents the most common adult lymphoma and can be divided into 2 major molecular subtypes: the germinal center B-cell-like and the aggressive activated B-cell-like (ABC) DLBCL. Previous studies suggested that chronic B-cell receptor signaling and increased NF-κB activation contribute to ABC DLBCL survival. Here we show that the activity of the transcription factor NFAT is chronically elevated in both DLBCL subtypes. Surprisingly, NFAT activation is independent of B-cell receptor signaling, but mediated by an increased calcium flux and calcineurin-mediated dephosphorylation of NFAT. Intriguingly, although NFAT is activated in both DLBCL subtypes, long-term calcineurin inhibition with cyclosporin A or FK506, both clinically approved drugs, triggers potent cytotoxicity specifically in ABC DLBCL cells. The antitumor effects of calcineurin inhibitors are associated with the reduced expression of c-Jun, interleukin-6, and interleukin-10, which were identified as NFAT target genes that are particularly important for the survival of ABC DLBCL. Furthermore, calcineurin blockade synergized with BCL-2 and MCL-1 inhibitors in killing ABC DLBCL cells. Collectively, these findings identify constitutive NFAT signaling as a crucial functional driver of ABC DLBCL and highlight calcineurin inhibition as a novel strategy for the treatment of this aggressive lymphoma subtype.