4-PBA ameliorates cellular homeostasis in fibroblasts from osteogenesis imperfecta patients by enhancing autophagy and stimulating protein secretion

Abstract

The clinical phenotype in osteogenesis imperfecta (OI) is attributed to the dominant negative function of mutant type I collagen molecules in the extracellular matrix, by altering its structure and function. Intracellular retention of mutant collagen has also been reported, but its effect on cellular homeostasis is less characterized. Using OI patient fibroblasts carrying mutations in the α1(I) and α2(I) chains we demonstrate that retained collagen molecules are responsible for endoplasmic reticulum (ER) enlargement and activation of the unfolded protein response (UPR) mainly through the eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) branch. Cells carrying α1(I) mutations upregulate autophagy, while cells with α2(I) mutations only occasionally activate the autodegradative response. Despite the autophagy activation to face stress conditions, apoptosis occurs in all mutant fibroblasts. To reduce cellular stress, mutant fibroblasts were treated with the FDA-approved chemical chaperone 4-phenylbutyric acid. The drug rescues cell death by modulating UPR activation thanks to both its chaperone and histone deacetylase inhibitor abilities. As chaperone it increases general cellular protein secretion in all patients' cells as well as collagen secretion in cells with the most C-terminal mutation. As histone deacetylase inhibitor it enhances the expression of the autophagic gene Atg5 with a consequent stimulation of autophagy. These results demonstrate that the cellular response to ER stress can be a relevant target to ameliorate OI cell homeostasis.

Keywords: Autophagy; Chemical chaperone; Collagen; Endoplasmic reticulum stress; Osteogenesis imperfecta; Unfolded protein response.

Fig. 1

Mutations in type I collagen genes lead to collagen overmodifications and ER enlargement. (A) Electrophoretic analyses of type I collagen extracted from patients' fibroblasts with dominant OI. Fibroblasts were labeled with ³H-Proline for 18 h. Type I collagen was extracted from cell layer (upper panel) and medium (lower panel) fractions and analyzed by SDS-PAGE. Representative radiographs are shown. A typical broadening or doubling of the α(I) bands in both fractions is observed in all cells except the very N-terminal mutation α1-G226S and the α2-G859S. (B) Transmission electron microscopy analysis of OI fibroblasts revealed the presence of ER enlargement (*) and of cellular vacuolization (arrow). N = nucleus. Magnification 20,000×.

Fig. 2

Mutations in type I collagen genes activates the unfolded protein response in OI patients' fibroblasts. Western blot analyses of the collagen chaperone PDI and of proteins involved in the UPR (BIP, PERK, p-PERK, ATF4, ATF6, IRE1α and p-IRE1α) in cells with mutations in the α1 (A) and α2 chains (B). UPR resulted activated in all patients fibroblasts except in α2-G640C. (C) RT-PCR amplification of XBP1 mRNA from control and patients cells. The spliced XBP1-1s form of XBP1 transcript is found in patients cells with α1-G667RIII, α1-G994D, α2-G319V,α2-G697C, α2-G745C and α2-G859S mutations. Complete splicing is detected in control fibroblasts treated with thapsigargin (Thap). OI type II: lethal osteogenesis imperfecta. OI type III: non-lethal osteogenesis imperfecta. C-: negative control. * p < 0.05.

Fig. 3

OI cells react to cellular stress by activating autophagy. (A–B) Western blot analyses of proteins involved in the autophagic pathway (ATG5-12, ATG16L1, LC3-I/LC3-II) in OI fibroblasts with mutations in COL1A1 (A) and COL1A2 (B) genes. Protein components involved in the formation of the autophagosome are upregulated in patients with α1(I) mutations. The terminal autophagic marker LC3 is upregulated in the majority of cases. (C-D) LC3 Western blot analyses in the presence of chloroquine in OI fibroblasts with mutations in COL1A1 (C) and COL1A2 (D) genes. The terminal marker of autophagy evaluated in dynamic conditions is increased in all patients’ cells except in α2-G640C and α2-G745C. OI type II: lethal osteogenesis imperfecta. OI type III: non-lethal osteogenesis imperfecta. * p < 0.05.

Fig. 4

Autophagy activation in OI cells. Autophagy was evaluated in control and OI fibroblasts α1-G478S, α1-G667RIII, α1-G667RII, α1-G994D, α2-G640C, α2-G697C and α2-G859S by LC3 immunofluorescence in the presence of chloroquine. Representative confocal images of WT and mutant fibroblasts incubated with LC3 antibody are shown. Quantitation of the total area of punctate signal per cell confirms the activation of autophagy. DAPI (nuclei) in blue and LC3 in green. Magnification 40×, zoom 4×. OI type II: lethal osteogenesis imperfecta. OI type III: non-lethal osteogenesis imperfecta. * p < 0.05.

Fig. 5

Activation of apoptosis in OI patients cells. (A) Western blot analyses of the terminal marker of apoptosis cleaved caspase 3. (B) FACS analysis detection of apoptotic cells by concurrent staining with annexin V (FITC) and propidium iodide (PI). The fraction of apoptotic events in the cells is shown in representative plots and quantified in the histogram as mean ± SEM. Apoptosis is activated in all tested OI patients cells. OI type II: lethal osteogenesis imperfecta. OI type III: non-lethal osteogenesis imperfecta. * p < 0.05.

Fig. 6

4-PBA ameliorates OI fibroblasts homeostasis. To alleviate the stress and to ameliorate OI patients' cells homeostasis, fibroblasts with mutations in COL1A1 (A) and COL1A2 (B) genes were treated with 5 mM 4-PBA for 15 h. The effect of the drug was evaluated following by Western blotting the expression of p-PERK, as markers for UPR, and the expression of cleaved caspase 3, as signature for apoptosis. The levels of these proteins were compared in treated versus untreated cells and in treated OI cells versus untreated controls. 4-PBA treatment mainly restores control values for what concern the activation of the UPR sensor PERK and the activation of the terminal caspase 3 in cells with mutations in the α1 collagen chain. The treatment on α2 mutated cells was effective on α2-G697C and α2-G745C. * p < 0,05 mutant fibroblasts respect to control fibroblasts. # p < 0,05 treated fibroblasts respect to untreated mutant fibroblasts. § p < 0,05 treated fibroblasts respect to untreated control fibroblasts.

Fig. 7

4-PBA ameliorates OI fibroblasts homeostasis by stimulating protein secretion. (A) After 4-PBA treatment OI patients' fibroblasts were labeled with [35S] EXPRESS35S Protein Labeling Mix for 1 h and total proteins were extracted from medium and cell layer. Determination of the total counts incorporated in cell layer and medium revealed that 4-PBA increases protein secretion in all cells lines. (B) Collagen secretion was evaluated by incubating the cells for 4 h with 47.14 μCi of ³H-Pro/ml, and extracting the collagen from both medium and cell layer fractions at the indicated time points. Samples were run on SDS-PAGE and the ratio between the densitometric value of collagen α1(I) in the media and the total collagen α1(I) (collagen present in medium and in cell layer) was evaluated and shown in the plot. Collagen secretion is delayed in all mutant cells tested compared to controls. Collagen intracellular retention was significant in α1-G994D cells. 4-PBA increased collagen secretion in α1-G994D cells.

Fig. 8

4-PBA ameliorates OI fibroblasts homeostasis by stimulating autophagy. (A) Histone H3 acetylation was investigated by Western blot analyses. 4-PBA increased the level of the H3 modification in all cells with the exception of the α1-G478S. (B) The expression of the autophagic gene ATG5, evaluated by qPCR, was increased by the treatment in all cells with the exception of α1-G478S. (C) LC3 Western blot analyses in the presence of chloroquine revealed increased LC3-II levels in all α1 and α2 cells after the 4-PBA treatment, clearly indicating a stimulation of autophagy.