Phenylbutyric Acid Modulates Apoptosis and ER Stress-Related Gene Expression in Glycogen Storage Disease Type Ib In Vitro Model

Abstract

Introduction: Chronic endoplasmic reticulum (ER) stress and increased apoptosis are involved in the pathogenesis of glycogen storage disease Ib (GSD Ib), whereas small molecule phenylbutyrate (4-PBA) showed the capability of reducing ER stress-induced apoptosis. The objective was to generate an in vitro system in which capability of small molecules (SMs) to influence ER stress and apoptosis could be screened at the expression level.

Methods: G6PT-deficient FlpInHEK293 cell line was created and validated using the CRISPR/Cas9 knockout method. Molecular markers of unfolded protein response (ATF4, DDIT3, HSPA5, XBP1s), and apoptosis (BCL2/BAX, CASP3, CASP7) in G6PT-deficient cells were analyzed using RT-qPCR method before and upon the treatment with 4-PBA.

Results: Treatment with the most effective dose of 1 mM 4-PBA reduced the expression of UPR markers and executioner caspases, while increased BCL2/BAX ratio in G6PT-deficient cells. Our results proved the concept that 4-PBA could alleviate markers of ER stress detected in the GSD Ib in vitro model system and prevent cell death.

Conclusion: This cost-effective in vitro model screens the therapeutic potential of SMs affecting ER stress and apoptosis in G6PT-deficient kidney cells, offering a first-line screening assay for promising compounds. 4-PBA's potential repurposing for GSD Ib patients opens new research directions.

Keywords: 4‐PBA; CRISPR/Cas9; ER stress; GSD Ib in vitro model system; apoptosis.

FIGURE 1

Establishment of SLC37A4 ^−/− FlpInHEK293 cell line. The scheme is illustrating human SLC37A4 gene locus along with the corresponding sgRNAs (colored in orange). The PAM sequences are shown in green, while the scissors are pointing to the double‐strand break region.

FIGURE 2

The establishment and characterization of SLC37A4 ^−/− FlpInHEK293 cell line. (A) PCR analysis was conducted on representative clonal cell lines carrying homozygous (SLC37A4 ^+/+, SLC37A4 ^−/−) or heterozygous (SLC37A4 ^+/−) alleles. Lower bands on the agarose gel denote edited fragments containing the c.14_100del variant in the SLC37A4 gene. The last lane of the agarose gel indicates a genetic marker corresponding to the amplified fragments. Molecular sizes in base pairs are provided on the right. (B) The sequencing chromatogram of the amplified fragment of the SLC37A4 locus of the wild‐type clone, where knockout has occurred, is shown in the upper part of the chromatogram, and the corresponding sequence of the SLC37A4 ^−/− clone at the bottom. PAM regions are indicated with a green frame, while the deleted region of the SLC37A4 gene is marked by arrowheads. (C) The relative expression of the SLC37A4 mRNA in an edited cell line is compared to the wild‐type control, which serves as the calibrator. To normalize the gained results, the GAPDH gene was used as an endogenous control. RT‐qPCR analysis was conducted with biological and technical duplicates. A t‐test was used to estimate the statistical significance between the groups. The error bars represent the means ± standard deviation (SD) obtained from biological and technical duplicates in two independent experiments. The Western blot analysis represents the G6PT protein expression in the knockout clone compared to the control. The β‐Actin protein was used as a loading control. (D) The sequencing chromatogram of amplified fragments of potential off‐target sites. The asterisks indicate the position of mismatches.

FIGURE 3

The treatment with 4‐PBA improves the viability, and proliferation of the SLC37A4 ^−/− cells. SLC37A4 ^−/− cells were exposed to varying concentrations of 4‐PBA (0.75, 1, 2.5, and 5 mM) for 48 h. Cell viability was assessed using the MTT assay. The absolute values obtained for each sample were expressed as a percentage relative to the absolute value gained for SLC37A4 ^+/+ cells, which were set as 100%. The statistical significance between the groups was estimated by a t‐test. The error bars represent the means ± standard deviation (SD) obtained from biological duplicates and technical triplicates in two independent experimental repeats of the MTT assays. The asterisks indicate a significant difference between groups with ** representing p < 0.001.

FIGURE 4

The treatment with 4‐PBA decreases apoptosis and ER stress in SLC37A4 ^−/− cells. (A) The image depicts the expression level of BCL2/BAX mRNAs ratio in untreated and treated SLC37A4 ^−/− clones with range concentrations of 4‐PBA from 0.75–5 mM for 48 h compared to the BCL2/BAX ratio of the SLC37A4 ^+/+ clone. Values below 1 indicate the increased level of apoptosis in the SLC37A4 ^−/− cell line. (B) The relative expression of BCL2 and BAX mRNA of the SLC37A4 ^−/− cell line before and after the treatment with selected doses of 4‐PBA for 48 h compared to the SLC37A4 ^+/+ used as a calibrator. The statistical significance was determined using the t‐test. (C) The relative expression of CASP3 and CASP7 mRNA of the knockout cells before and after the treatment with selected doses of 4‐PBA for 48 h compared to the SLC37A4 ^+/+ used as a calibrator. (D) Quantitative gene expression analysis demonstrates the relative expression level of ER stress markers (ATF4, DDIT3, HSPA5, and XBP1s) in knockout clones and treated clones with 1 mM 4‐PBA for 48 h, in comparison to the wild‐type clone used as the calibrator. Error bars in the figure represent means ± SD obtained from two individual experiments, each including two biological and technical replicates. The statistical significance between groups was determined by one‐way ANOVA with the post hoc t‐test. The asterisks denote a significant difference between groups: * represents p < 0.05, and ** represents p < 0.001, and NS denotes no significant difference.