Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor

Abstract

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.

Fig 1. MSCs produce EBI3 in cells during chondrogenic differentiation.

(A-C) Human MSCs were cultured as aggregates in chondrogenic medium. (A) EBI3, IL-35 p35 and IL-27 p28 mRNA levels were evaluated by RT-qPCR at the indicated time points. All quantitative data are expressed as the mean ± SD (each n = 3). (B) EBI3, IL-35 p35, and IL-27 p28 protein levels in chondrogenic MSCs were detected at the indicated time points by Western blotting. β-actin was used as a loading control. Results are representative of 3 independent experiments with similar findings. (C) EBI3 protein levels in culture supernatants during chondrogenesis were detected by Western blotting. BSA was detected by CBB staining and used as loading control. Results are representative of 3 independent experiments with similar findings.

Fig 2. Endogenous EBI3 enhances chondrogenic differentiation of MSCs.

(A-G) MSCs were transfected with control siRNA or 2 different EBI3 siRNAs (si#1 and si#2). (A) EBI3 mRNA levels were evaluated by RT-qPCR at 48 hrs after transfection. (B) EBI3 protein levels were evaluated by western blotting at 48 hrs after transfection. β-actin was used as a loading control. (C-G) MSCs were cultured as pellets in chondrogenic medium for 21 days after transfection. (C) Photographs of the pellets. Scale bar, 1 mm. (D) The wet weight of the pellets. (E) The pellets cultured were stained with Safranin-O (S-O) or anti-type II collagen (Col II) antibody. Scale bar, 100 μm. (C and E) Results are representative of 4 independent experiments with similar findings. (F) Densitometric analysis of the staining in (E) was performed and shown by integral optical density (IOD). (G) The mRNA levels of the indicated genes in transfected pellets were determined by RT-qPCR. (A, D, F, and G) Quantified data are expressed as the mean ± SD (each n = 3 in A; n = 4 in D; n = 4 in F; n = 3 in G) with similar findings. * = P < 0.05; ** = P <0.01; *** = P <0.001 by Dunnett’s multiple comparison test.

Fig 3. EBI3 overexpression in MSCs inhibits chondrogenic differentiation.

(A-G) MSCs were transiently transfected with pEF6-EBI3-V5 plasmid DNA. (A) EBI3 mRNA levels were evaluated by RT-qPCR at 24 hrs after transfection. (B) EBI3 protein levels were evaluated by western blotting at 72 hrs after transfection. β-actin was used as a loading control. (C-G) MSCs were cultured as pellets in chondrogenic medium for 21 days after transfection with pEF6-EBI3-V5 or empty vector. (C) Photographs of the pellets. Scale bar, 1mm. (D) Wet weight of the pellets. (E) Aggregates were stained with Safranin O (S-O) or anti-type II collagen (Col II) antibody. Scale bar, 100 μm. Results are representative of 4 independent experiments with similar findings. (F) Densitometric analysis of the stained of (E) was performed and shown by integral optical density (IOD). (G) The mRNA levels of the indicated genes between transfected aggregates were determined by RT-qPCR on day 21. (A, D, F, and G) Quantified data are expressed as the mean ± SD with similar findings (each n = 3 in A, D, and G; n = 4 in F). * = P < 0.05; ** = P <0.01; *** = P <0.001 by Student’s unpaired 2-tailed t-test.

Fig 4. IL-1β up-regulates EBI3 and inhibits chondrogenic differentiation of MSCs.

(A-G) MSCs were cultured as cell pellets in chondrogenic medium. (A) The mRNA levels of the indicated genes were determined by RT-qPCR at the indicated time points. (B) EBI3 protein levels in chondrogenic MSCs treated with or without IL-1β were determined at the indicated time points by Western blotting. β-actin was used as a loading control. Results are representative of 3 independent experiments with similar findings. (C-G) MSCs were cultured with no stimulation (NS), IL-6/sIL-6R or IL-1β for 21 days. (C) Photographs of the pellets. Scale bar, 1mm. (D) Wet weight of the pellets. (E) Pellets were stained with Safranin O (S-O) or anti-type II collagen (Col II) antibody. Scale bar, 100 μm. Results are representative of 4 independent experiments with similar findings. (F) Densitometric analysis of (E) was performed to show integral optical density (IOD). (G) The mRNA levels of the indicated genes in cell pellets were determined by RT-qPCR on day 21. (A, D, F, G) Quantified data are expressed as the mean ± SD (each n = 2 in A; n = 3 in D; n = 4 in F; n = 4 in G). * = P<0.05; ** = P<0.01; *** = P<0.001 by Dunnett’s multiple comparison test.

Fig 5. EBI3 proteins localize in the ER of MSCs and modulate IRE1α under inflammatory conditions.

(A-C) MSCs were cultured in a monolayer in growth medium. (A) Human MSCs were stimulated with IL-6/sIL-6R (100 ng/ml), TNF-α (100 ng/ml) or IL-1β (3 ng/ml). The immunocytochemistry detected EBI3 and Calnexin. Nucleus was stained by DAPI (5 μg/ml). Images are representative of 3 independent experiments with similar findings. Scale bar, 50 μm. (B) Human MSCs were transfected with control siRNA or 2 different EBI3 siRNAs (si#1 and si#2) for 48 hrs. After that growth medium was refreshed, IL-1β and IL-6/sIL-6R were then added for 36 hrs. MSCs treated with Tunicamycin (Tm) and DTT were used as positive control of ER stress. Whole-cell lysates were analyzed for each ER stress sensors and EBI3 by Western blotting. (C) Densitometric analysis of the Western blotting in B was performed, and the data were normalized to β-actin. Quantified data are expressed as the mean± SD (each n = 3). * = P < 0.05; *** = P <0.001 by Dunnett’s multiple comparison test. (D) Synovial specimens from RA patients (n = 3) and OA patients (n = 3) were stained by immunofluorescence using specific antibodies against CD271, EBI3, and IRE1α. Results are representative of 3 independent experiments with similar findings. Scale bar, 100 μm.