The Lysosomal Trafficking Regulator (lyst) Gene Is Regulated Through Fgf-2 Via Fgf Receptor Mediated Signaling
Gabriel Mirhaidari, PhD1,2, Jacob Zbinden, PhD1,3, Kevin Blum, PhD1,3, Christopher Breuer, MD1, Jenny Barker, MD, PhD1,4.
1The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA, 2The Ohio State University, College of Medicine, Columbus, OH, USA, 3The Ohio State University, College of Biomedical Engineering, Columbus, OH, USA, 4The Ohio State University, Department of Plastic and Reconstructive Surgery, Columbus, OH, USA.
PURPOSE: The lysosomal trafficking regulator (LYST) gene encodes a large 430kDa protein known for its involvement in the size and trafficking of lysosomes. Mutations in the LYST gene impact global biological processes including paracrine signaling, cellular degranulation events, and cell membrane repair across diverse cell types. Recently, we have described that LYST is necessary for normal wound healing in vivo utilizing a mouse model with mutated LYST, the Beige (Bg) mouse. Translational therapeutic interventions aimed at LYST remain elusive, however, due to a limited understanding of its mechanisms of action, including regulatory factors. In this study, we explore factors that regulate LYST gene expression to inform future therapeutic interventions.
METHODS: Primary fibroblast cultures were derived from wild-type (WT), LYST-mutant Beige (Bg), and novel LYST Exon 52 KO mice (52 KO). Cells were subjected to varying cell culture media and drug treatment conditions with lysosomal size and LYST gene expression quantified as primary outcomes. RNA-sequencing with differential gene expression and pathway analysis was utilized to determine effects of LYST mutation on cellular processes. LYST protein localization through immunofluorescent staining and cell cycle assignment was performed to determine its relation to cell cycle progression.
RESULTS: Serum starvation (0.1% FBS) significantly upregulated LYST gene expression compared to a normal 10% FBS cell culture condition (mean 2.56 fold change, p <0.0001). Rescue with FGF-2 significantly reversed the upregulation of LYST gene expression (mean 1.122 fold change vs 2.512, p <0.0001). Selective blockade of the FGF receptor with infigratinib in the presence of FGF-2 and serum starvation blocked the downregulation by FGF-2 (mean 2.863 fold change vs 1.122, p < 0.0001). LYST gene expression was able to be selectively downregulated with addition of drugs blocking specific downstream FGF receptor mediators: fludarabine targeting STAT1 (mean 1.537 fold change vs 1.002, p < 0.0001), S31-201 targeting STAT3 (mean 1.775 fold change vs 1.009, p < 0.0001), wortmannin targeting PI3K/AKT (mean 1.652 fold change vs 1.002, p < 0.0001), and YK-4-279 targeting ETS (mean 1.458 fold change vs 1.002, p = 0.004). RNA-sequencing and pathway analysis demonstrated that in serum-starved Bg fibroblasts, there was significant disruption of pathways involved in DNA repair, cell division, and cell cycle progression compared to WT fibroblasts. LYST protein had significantly increased nuclear localization compared to cytoplasmic localization during G2 of the cell cycle indicating a previously undescribed relationship between LYST localization and cell cycle progression.
CONCLUSION: The present study reveals a role for FGF-2 in transcriptional downregulation of the LYST gene. We selectively modified LYST gene expression by targeting downstream effectors of the FGF receptor signaling pathway utilizing drugs that have current clinical use. Our results indicate a role for LYST in normal DNA repair and cell cycle progression. With expanding evidence for LYST as a critical underlying factor in biological functions such as wound healing and cancer, these findings provide a foundation for selective modulation of gene expression with pharmacological agents. Further work aimed at modulating LYST can provide novel therapies for diseases of dysregulated wound repair and tissue healing.
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