4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts

Abstract

Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography-mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients.

Keywords: endoplasmic reticulum (ER); extracellular matrix; fibril; glycosylation; osteoblast.

Figure 1

Intracellular localization of type I collagen.A, immunofluorescence staining of dermal fibroblasts. Type I collagen is in green. Endoplasmic reticulum marker protein, disulfide isomerase (PDI), is in red. Nuclei are in blue. Scale bar is 100 μm. B, calculated values of green area merged with red area divided by red area of immunofluorescence staining. Data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ∗p < 0.05 versus control, ∗∗p < 0.01 versus control.

Figure 2

Posttranslational modification of type I collagen.A, SDS-PAGE analysis of type I collagen in conditioned medium. B, percentage of posttranslational modifications at Lys⁸⁷, Lys⁹⁹, Lys¹⁷⁴, and Lys⁵⁶⁴ of α1 and Lys⁸⁷, Lys¹⁷⁴, and Lys²¹⁹ of the α2 chain of type I collagen analyzed by liquid chromatography–mass spectrometry. Control is the average of 2 cell lines. Differences in the ratio of GGHL to non-GGHL were tested by Pearson's Chi-square test. ∗p < 0.05 versus control, ∗∗p < 0.01 versus control. GGHL, glucosyl-galactosyl-hydroxylysine; GHL, galactosyl-hydroxylysine; Hyl, hydroxylysine.

Figure 3

Production of type I collagen.A, protein levels of type I collagen in conditioned medium and deposited on the dish measured by ELISA. B, real-time quantitative PCR of COL1A1 and COL1A2. C, calculated values of protein levels of type I collagen. The formula for calculation is as follows: protein levels deposited on the dish/(protein levels deposited on the dish + protein levels in conditioned medium). All data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ∗p < 0.05 versus control, ∗∗p < 0.01 versus control.

Figure 4

Detection of impaired type I collagen in extracellular matrix (ECM).A, fluorescence staining of deposited collagen. Misfolded triple-helical chains are stained in green by collagen hybridizing peptide (CHP). Type I collagen is stained in red. Scale bar is 200 μm. B, calculated values of fluorescence staining. The formula for calculation is as follows: (green area × green fluorescence density)/(red area × red fluorescence density). C, protein levels of type I collagen in conditioned medium. Control and OI #3 fibroblasts were cultured on plasma-treated polystyrene plates, control fibroblast-derived ECM, or OI #3 fibroblast-derived ECM. After a 24-h incubation period, the type I collagen level in conditioned medium was measured. All data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ∗∗p < 0.01 versus control, ##p < 0.01.

Figure 5

The effect of 4-phenylbutyric acid (4-PBA) on intracellular accumulation of type I collagen. Each cell line was treated with or without 5 mM 4-PBA. A, immunofluorescence staining of dermal fibroblasts. Type I collagen is in green. The endoplasmic reticulum marker protein, disulfide isomerase (PDI), is in red. Nuclei are in blue. Scale bar is 100 μm. B, calculated values of green area merged with red area divided by red area of immunofluorescence staining. Data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ##p < 0.01.

Figure 6

The effect of 4-phenylbutyric acid (4-PBA) on the production of type I collagen.A, protein levels of type I collagen in conditioned medium and deposited on the dish measured by ELISA. B, real-time quantitative PCR of COL1A1 and COL1A2. C, calculated values of protein levels of type I collagen. The formula for calculation is as follows: protein levels deposited on the dish/(protein levels deposited on the dish + protein levels in conditioned medium). All data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. #p < 0.05, ##p < 0.01.

Figure 7

The effect of 4-phenylbutyric acid (4-PBA) on type I collagen in the extracellular matrix.A, fluorescence staining of deposited collagen. Misfolded triple-helical chains are stained in green by collagen hybridizing peptide (CHP). Type I collagen is stained red. Scale bar is 200 μm. B, calculated values of fluorescence staining. The formula for calculation is as follows: (green area × green fluorescence density)/(red area × red fluorescence density). Data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ##p < 0.01.

Figure 8

Assessment of calcium deposition of osteoblasts differentiated from induced pluripotent stem cells.A, alizarin red S staining of deposited extracellular matrix derived from induced osteoblasts. Osteoblast differentiation from mesenchymal stromal cells was performed during the indicated day. B, the amount of calcium deposition of induced osteoblasts. Calcium deposition was quantified by a calcium assay kit at 7, 14, 21, and 28 days of osteoblast differentiation culture. C, calcium deposition amount, measured by a calcium assay kit, of induced osteoblasts at 28 days of osteoblast differentiation culture. All data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. ∗∗p < 0.01 versus control.

Figure 9

The effect of 4-phenylbutyric acid (4-PBA) on calcium deposition of osteoblasts differentiated from induced pluripotent stem cells.A and B, alizarin red S staining of deposited extracellular matrix derived from control (A) and OI #3 (B)-induced osteoblasts. Osteoblast differentiation culture from mesenchymal stromal cells was performed during the indicated day. C and D, calcium deposition amount, measured by a calcium assay kit, of induced osteoblasts of control (C) and OI #3 (D) at 16 days of osteoblast differentiation culture. Data are mean ± SEM. Differences were tested by ANOVA followed by Tukey's HSD post hoc test. #p < 0.05, ##p < 0.01.