Bukva, M. | Search bioRxiv

bioRxiv2025

JPT2/HN1L functions as an NAADP-binding protein in a cell type-specific manner

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger evoking Ca2+ release from intracellular Ca2+ stores by targeting several Ca2+ channels, including two-pore channels (TPC1/2), transient receptor potential mucolipin-1 (TRPML1), or ryanodine receptor type 1 (RYR1). For activation of Ca2+ channels, NAADP requires binding proteins, such as JPT2/HN1L and LSM12. So far, their function has been analyzed in several cell lines and only a very limited number of primary cells; however, their physiological relevance in cell types known to utilize NAADP signaling remains unclear. Here, we generated Jpt2/Hn1l-/- mice to evaluate the contribution of JPT2/HN1L proteins to platelet aggregation and Ca2+ signaling in cardiomyocytes, mast cells, and T cells. NAADP is known to contribute to collagen-related peptide (CRP-XL)- evoked platelet aggregation, but this was not altered by JPT2/HN1L deletion. Functional Ca2+ imaging revealed that JPT2/HN1L plays a strikingly cell-type specific role in NAADP-mediated Ca2+ release. In electrically paced ventricular cardiomyocytes, {beta}-adrenergic stimulation is known to evoke arrhythmogenic spontaneous diastolic Ca2+ transients, which were not altered in their frequency in Jpt2/Hn1l-/- myocytes. Further, antigen-evoked Ca2+ transients in peritoneal mast cells (PMCs) are not changed in Jpt2/Hn1l-/- PMCs. However, CD4+ T cells displayed a pronounced requirement for JPT2/HN1L. Following T cell receptor/CD3 stimulation, global Ca2+ elevations and early NAADP-driven Ca2+ microdomains, which occur within tens of milliseconds of TCR/CD3 engagement and serve as initiating signals for downstream immune activation, were significantly decreased in Jpt2/Hn1l-/- CD4+ cells. We conclude that JPT2/HN1L is indispensable for NAADP-mediated Ca2+ release in T cells, but dispensable in cardiomyocytes, platelets, and mast cells, at least for the agonists employed. Accordingly, LSM12 might compensate for the loss of JPT2/HN1L. Together, JPT2/HN1L is not universally required as an NAADP-binding protein but exhibits cell-type specificity, with an essential function in T cell Ca2+ signaling.

pharmacology and toxicology↗

bioRxiv2025

Adenosine-specific transcriptional programs in murine connective tissue type mast cells

Mast cells are tissue-resident immune cells that are critical for the pathogenesis of allergic and inflammatory disorders. In addition to classic host defense against parasites, food quality control through antigen avoidance has been recently elucidated as additional physiological mast cell function. The purine nucleoside adenosine (ADO), like other mast cell activators, such as antigens or Mrgprb2 agonists, increases intracellular Ca2+ concentration; however, it fails to induce degranulation of preformed mediators when applied to isolated mast cells alone, and there is limited knowledge of whether ADO evokes the de novo synthesis and release of inflammatory mediators such as cytokines in tissue mast cells. An unbiased genome-wide analysis of gene expression triggered by various mast cell activators should enable identification of the gene program specifically activated by adenosine in mast cells and thereby reveal new components of the associated inflammatory responses. To this end, we performed bulk RNA sequencing (RNA-Seq) in primary murine peritoneal mast cells (PMCs) as connective tissue mast cells. By comparing responses evoked by ADO stimulation with those of the Mrgprb2 compound 48/80 and antigens that activate Fc{varepsilon}RI receptors, we identified 393 genes whose expression was uniquely regulated by ADO, including upregulation of genes encoding de novo synthesized mediators such as Tgfa and Il7. Transcription factor activity inference, protein classification, functional enrichment analysis, protein-protein interaction network, and topology analysis revealed that these genes play roles in phosphoinositide signaling, vesicle trafficking, glycolysis, mitochondrial activity, and cell cycle arrest. In summary, our work elucidates a distinct ADO-triggered response and identifies a set of proteins including de novo synthesized mediators whose functional relevance in adenosine-evoked mast cell activation and inflammatory reactions can be evaluated in future studies.

pharmacology and toxicology↗