STIM2β-mediated SOCE in the regulation of cell cycle and PPARG2 transcription during adipogenesis and Pro-viral role of NFAT5 during CoV infection

Abstract

In the first chapter, I investigated the role of store-operated Ca2+ entry (SOCE) in adipogenesis. Adipogenesis is a complicated process involving various signaling cascades that regulate the cell cycle and transcription of adipogenic genes. As a secondary messenger, intracellular Ca2+ also plays a role in the regulation of adipogenesis. However, the mechanism by which the intracellular Ca2+ levels are regulated during adipogenesis is poorly understood. Therefore, I examined the changes in intracellular Ca2+ levels and SOCE activity during the differentiation of 3T3-L1 preadipocytes and primary stromal vascular fraction (SVF) cells. During adipogenesis, the intracellular Ca2+ levels and SOCE activity exhibited dynamic changes, with the basal cytosolic Ca2+ levels decreasing and SOCE activity increasing as the differentiation progressed. I identified that these changes in intracellular Ca2+ levels were regulated by stromal interacting molecule 2 beta (STIM2β), an alternatively spliced isoform of STIM2. Knock-out (KO) of STIM2β altered the changes in intracellular Ca2+ levels during adipogenesis, resulting in increased basal cytosolic Ca2+ levels and elevated SOCE activity. Additionally, the alteration of STIM2β led to a reduction in mitotic clonal expansion (MCE) of 3T3-L1 cells, which is known as the prerequisite process for terminal adipogenic differentiation. Paradoxically, STIM2β KO preadipocytes exhibited enhanced adipogenesis, which was associated with increased transcription of peroxisome proliferator-activated receptor gamma 2 (PPARG2). This phenotype was also observed in STIM2β-deficient primary SVF cells, confirming that increased SOCE by STIM2β KO promotes PPARG2 transcription. A PPARG2 promoter assay revealed that PPARG2 transcription is mediated by nuclear factor of activated T cell (NFAT) and cyclic AMP response element binding protein (CREB), which are downstream transcription factors of SOCE. Furthermore, analysis of white adipose tissue development in STIM2β KO mice revealed hypertrophic adipose tissue, indicating that while STIM2β deficiency reduces MCE, it enhances adipogenic differentiation. Taken together, these findings reveal a previously unrecognized role of STIM2β-mediated SOCE in regulating MCE and PPARG2 transcription during adipogenesis, and provide evidence that MCE is not a prerequisite stage for adipogenesis. In the second chapter, I explored the physiological role of NFAT5 under coronavirus (CoV) infection conditions. NFAT5 is known to participate in inflammatory and immune responses. However, the physiological role of NFAT5 during CoV infection remains elusive. I discovered that full-length NFAT5 protein levels are reduced and a cleaved form appears upon HCoV-OC43 infection. This cleavage is mediated by the CoV protease NSP5. To investigate the role of cleaved NFAT5, I established stable cell lines expressing cleaved NFAT5 and performed a focus-forming assay (FFA), which revealed increased viral replication in cleaved NFAT5 expressing stable cell lines. In contrast, stable cell lines expressing the cleavage-resistant mutant of NFAT5 did not show increased viral replication, confirming that cleaved NFAT5 functions as a proviral factor. Considering that cleaved NFAT5 retains its DNA binding domain, we analyzed the effect of cleaved NFAT5 on the Type I interferon (IFN) response, which is critical for the antiviral response. These results demonstrate a functional switch of NFAT5 from an antiviral to a proviral factor following CoV-mediated cleavage, revealing a previously unrecognized mechanism of immune evasion.