Ruxolitinib altered IFN-β induced necroptosis of human dental pulp stem cells during osteoblast differentiation

Objective: This study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-β) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). Design: hDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-β protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. Results: In the IFN-β-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-β-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. Conclusion: Ruxolitinib interfered with the IFN-β-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication