Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids

Abstract

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also in MPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease in MTOR transcript level was observed at these conditions.

Figure 1. Determination of count and viability of HDFa and MPS II cells respectively with 100 μM of genistein, 100 μM of kaempferol, 100 μM of daidzein, mix of genistein and kaempferol or genistein and daidzein (30 µM each), and with 0.05% DMSO (Ctrl) for different periods of time (24, 48 and 72 h).

Data are represented as mean and bars show SD values of experiments run in triplicate.

Figure 2. Distribution of up- (red) and down-regulated (blue) transcripts of whole genome of HDFa cells after 24 and 48 h treatment with (A) genistein (100 µM), (B) kaempferol (100 µM), (C) mix of genistein and keampferol (30 µM each), (D) daidzein (100 µM), and (E) mix of genistein and daidzein (30 µM each), at 0.5 ≥ FC ≥ 2 for n ≥ 3, with the p-value < 0.05.

DMSO-treated cells were used in control experiments.

Figure 3. Genes identified as changed under studied conditions with corresponding overlap between the datasets (0.5 ≥ FC ≥ 2, n ≥ 3, with the p-value < 0.05).

Up- and down-regulated genes of whole genome of HDFa cells after 24 and 48 h treatment with 100 µM genistein, 100 µM kaempferol, genistein-kaempferol of 30 µM each, 100 µM daidzein and genistein-daidzein of 30 µM each.

Figure 4. Number of genes whose expression was altered as a function of the FC in response to various flavonoids' treatment type (100 µM genistein, 100 µM kaempferol, 100 µM daidzein, and mixtures of them of 30 µM each), identified in the microarray analysis of whole genome sequences and transcripts of HDFa cells (n ≥ 3, with the p-value < 0.05).

Figure 5. Genes with significantly (i.e. more than 10-fold change) regulated expression upon various flavonoids’ treatment (100 µM genistein - GEN, 100 µM kaempferol - KAEM, 100 µM daidzein - DAIDZ, and mixtures of them of 30 µM each, GEN + KAEM or GEN + DAIDZ) identified in the microarray analysis of whole genome sequences and transcripts of HDFa cells (n ≥ 3, with the p-value < 0.05).

Figure 6. TFEB and MTOR expression analysis in HDFa and MPS cells via real-time qRT-PCR.

Data represent averaged values ± SD from n = 3, and mean significant differences for samples treated for 24 h with various flavonoids (100 µM genistein, 100 µM kaempferol, and mixtures of them of 30 µM each) against non-treated, with respect to endogenous reference gene GAPDH (in HDFa) and RPLPO (in MPS II), with p < 0.005 as determined by ANOVA with Tukey's HSD Post Hoc.

Figure 7. GO analysis by ‘Cellular Compartment’ (A) and ‘Biological Processes’ (B) category of the genes with up-regulated expression upon various flavonoids' treatment (100 µM genistein, 100 µM kaempferol, and mixtures of them of 30 µM each, for 24 and 48 hours) of HDFa cells, with false discovery rate (FDR) < 0.1, fold change ≥ 1.3 and below 0.7, and p < 0.001.

Figure 8. Graphs showing the enrichment plots generated by GSEA analysis of ranked gene expression data (UP: up-regulated, red; DOWN: down-regulated, blue).

Sizes of normalized enrichment score (NES), q-value of false discovery rate (FDR q-val), and nominal p-value (NOM p-val) for lysosome-associated gene sets regulated by tested flavonoids (100 µM genistein, 100 µM kaempferol, and mixtures of them of 30 µM each, for 24 and 48 hours) in fibroblasts are depicted. The enrichment score is shown as a scattered green line, and the horizontal black bars next to the plot indicate the position of lysosome-associated genes, which are mostly grouped in the fraction of up-regulated genes.