Deficiency of Two-Pore Segment Channel 2 Contributes to Systemic Lupus Erythematosus via Regulation of Apoptosis and Cell Cycle
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of various autoantibodies and immune complexes that interact with multiple organs such as kidneys, joints, and blood. SLE affects people of all races, genders, and ages, with a higher prevalence in adult women, occurring approximately three to six times more often in women than in men. Despite extensive genetic research using genome-wide association studies (GWAS) and exome-wide association studies, the exact causes of SLE remain unclear. This study aims to explore the role of two-pore segment channel 2 (TPCN2) in SLE pathogenesis.
TPCN2 is a member of the two-pore channels (TPCs) family, which localize to acidic Ca2+ stores within the endolysosomal system and are widely expressed in various tissue types. TPCs have been linked to several diseases, including Parkinson’s disease, non-alcoholic fatty liver disease, Ebola virus disease, diabetes, and cancer. Previous studies have identified TPCN2 as a susceptibility gene for SLE, but its role in the pathogenesis of SLE remains unclear.
To investigate the functional role of TPCN2 in SLE, this study employed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the expression of TPCN2 in SLE patients. The results showed that the expression level of TPCN2 was significantly decreased in SLE peripheral blood mononuclear cells (PBMCs) compared to healthy controls. To further understand the role of TPCN2, the study performed a loss-of-function assay using lentiviral constructs in Jurkat and THP-1 cell lines. Knockdown of TPCN2 was confirmed at both the RNA level by qRT-PCR and the protein level by Western blotting.
The study found that TPCN2 knockdown significantly inhibited cell proliferation in both Jurkat and THP-1 cells, as measured by the Cell Count Kit-8 (CCK-8) assay. Additionally, flow cytometry analysis revealed that TPCN2 knockdown induced apoptosis and G2/M cell-cycle arrest in these cell lines. Specifically, the proportion of Annexin V-positive Jurkat cells indicating early- and late-stage apoptosis was three times higher in the TPCN2-knockdown group than in the normal control (NC) group. Similar results were observed in THP-1 cells, where TPCN2 knockdown induced 13.86% and 20.26% apoptosis in the shTPCN2#1 (sh#1) and shTPCN2#2 (sh#2) groups, respectively.
To understand the molecular mechanisms underlying the effects of TPCN2 knockdown, the study performed RNA sequencing (RNA-seq) analysis on TPCN2-deficient Jurkat cells. The RNA-seq results showed that 906 genes were upregulated and 312 were downregulated in the sh#1 group, whereas 362 genes were upregulated and 598 were downregulated in the sh#2 group compared to the NC group. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that TPCN2 knockdown significantly influenced cellular processes and biological regulation. The pathway functional enrichment results indicated that most differentially expressed genes (DEGs) were involved in multiple pathways, such as the forkhead box O (FoxO), thyroid hormone, and T cell receptor pathways.
Gene Set Enrichment Analysis (GSEA) further identified that TPCN2 deficiency was associated with the G2/M checkpoint, inflammatory response, IFN-g signaling, and the complement system. Additionally, the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) and interleukin-6-Janus kinase-signal transducer and activator of transcription signaling pathways were enriched in the TPCN2-knockdown groups.
The study also validated the expression of key DEGs at the mRNA level in TPCN2-deficient Jurkat cells. The results showed that NCOA3, S100A8, AHNAK, GRB10, PMEPA1, and ERO1A were significantly upregulated, while ARHGDIB and CX3CR1 were significantly downregulated. Similar results were observed in THP-1 cells. These genes play crucial roles in cell growth and apoptosis, highlighting the importance of TPCN2 in these processes.
In conclusion, this study demonstrates that TPCN2 knockdown inhibits cell proliferation, induces apoptosis, and causes G2/M cell-cycle arrest in Jurkat and THP-1 cells. RNA-seq analysis revealed that TPCN2 deficiency affects multiple pathways involved in cell cycle regulation, apoptosis, and inflammatory responses. These findings suggest that TPCN2 might be a potential protective factor against SLE, providing insight into novel therapeutic approaches for the disease. Further research is needed to elucidate the precise mechanisms underlying the role of TPCN2 in SLE pathogenesis.
doi.org/10.1097/CM9.0000000000001893
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