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. 2020 Jul;2(7):407-414.
doi: 10.1002/acr2.11155. Epub 2020 Jun 12.

The Follistatin-like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage

Yury Chaly  1 , Bruce Hostager  1 , Sonja Smith  1 , Raphael Hirsch  1
Affiliations

The Follistatin-like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage

Yury Chaly et al. ACR Open Rheumatol. 2020 Jul.

Abstract

Objective: We sought to determine whether follistatin-like protein 1 (FSTL1), a protein produced by articular chondrocytes, promotes healthy articular cartilage and prevents chondrocytes from undergoing terminal differentiation to hypertrophic cells.

Methods: In vitro experiments were performed with immortalized human articular chondrocytes. The cells were transduced with a lentivirus encoding human FSTL1 small hairpin RNA or with an adenovirus encoding FSTL1. A quantitative polymerase chain reaction was used for gene expression analysis. Protein expression was assessed by Western blotting. Co-immunoprecipitation was used to identify interacting partners of FSTL1. FSTL1 expression in human articular cartilage was analyzed using confocal microscopy.

Results: Downregulation of FSTL1 expression in transforming growth factor β (TGFβ)-stimulated chondrocyte pellet cultures led to chondrocyte terminal differentiation characterized by poor production of cartilage extracellular matrix and altered expression of genes and proteins involved in cartilage homeostasis, including MMP13, COL10A1, RUNX2, COL2A1, ACAN, Sox9, and phospho-Smad3. We also showed that FSTL1 interacts with TGFβ receptor proteins, Alk1 and endoglin, suggesting a potential mechanism for its effects on chondrocytes. Transduction of chondrocytes with an FSTL1 transgene increased COL2A1 expression, whereas it did not affect MMP13 expression. FSTL1 protein expression was decreased in human osteoarthritic cartilage in situ.

Conclusion: Our data suggest that FSTL1 plays an important role in maintaining healthy articular cartilage and the FSTL1 pathway may represent a therapeutic target for degenerative diseases of cartilage.

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Figures

Figure 1
Figure 1
Suppression of follistatin‐like protein 1 (FSTL1) in human articular chondrocytes reduces cartilage proteoglycan content and volume. CH‐hTERT/E6/E7 chondrocytes were transduced with lentivirus encoding FSTL1 short hairpin RNA (shRNA) or scrambled shRNA (control). Chondrocyte differentiation was performed in the presence of transforming growth factor β. A, FSTL1 expression was assayed by real‐time PCR at day 0 of differentiation. The mean + SEM is shown (n = 3, *P = 0.004, two‐tailed t test). B, After 21 days, pellets were sectioned and stained with Safranin O/Fast green to demonstrate proteoglycan content (scale bars, 100 μm). The representative data shown are from one of three experiments. C, The plate was scanned and pellet diameters were measured with ImageJ software and used to calculate pellet volumes. The mean + SEM is shown (n = 3, *P = 0.0055, two‐tailed t test).
Figure 2
Figure 2
Follistatin‐like protein 1 (FSTL1) expression regulates chondrocyte phenotype. CH‐hTERT/E6/E7 chondrocytes were transduced with lentivirus encoding FSTL1 short hairpin RNA shRNA) or scrambled shRNA (control). Chondrocyte differentiation was performed in the presence of transforming growth factor β. A, After 21 days of culture, FSTL1, COL2A1, ACAN, MMP13, COL10A1, and RUNX2 were assayed by real‐time PCR. The mean + SEM is shown (n = 3; FSTL1 *P = 0.01, COL2A1 *P = 0.01, ACAN **P = 0.028, MMP13 *P = 0.01, COL10A1 ***P = 0.0015, RUNX2 ****P = 0.0039; two‐tailed t test). B, After 7, 14, or 21 days of culture, cell lysates were assayed by Western blotting for FSTL1, MMP13, or actin.
Figure 3
Figure 3
Follistatin‐like protein 1 (FSTL1) regulates transforming growth factor β (TGFβ) signaling. CH‐hTERT/E6/E7 chondrocytes were transduced with lentivirus encoding FSTL1 short hairpin RNA (shRNA) or scrambled shRNA (control). Chondrocyte differentiation was performed in the presence of TGFβ. After 3 days of culture, cell lysates were assayed by Western blotting for Sox9 (A) or phosphor‐(p) and total Smad3 (B). Duplicate blots were probed for actin to confirm equivalent lane loading. The results shown are from one representative experiment of three performed. C, HEK 293T cells were transfected with empty expression vector or plasmids encoding FLAG‐FSTL1 and BP‐epitope–tagged Alk1 or endoglin. FLAG‐FSTL1 was immunoprecipitated from lysates using anti‐FLAG antibody. FSTL1 was visualized with anti‐FSTL1 antibody. Alk1 and endoglin were visualized with anti‐BP antibody. The results shown are from one representative experiment of three performed.
Figure 4
Figure 4
Overexpression of Follistatin‐like protein 1 (FSTL1) enhances COL2A1 gene expression. CH‐hTERT/E6/E7 chondrocytes were infected with adenovirus encoding FSTL1 (FSTL1) or a control virus lacking a transgene (control). Chondrogenic differentiation was performed in a 21‐day pellet culture with transforming growth factor β (TGFβ). FSTL1 (A), COL2A1 (B), and MMP13 (C) expression was assessed by real‐time PCR. The mean + SEM is shown (n = 3; FSTL1 *P = 0.014, COL2A1 **P = 0.0098, MMP13 P = 0.55; two‐tailed t test).
Figure 5
Figure 5
Follistatin‐like protein 1 (FSTL1) expression is reduced in OA articular cartilage. Cryosections from healthy‐appearing, low Mankin (LM) score and diseased‐appearing, high Mankin (HM) score tissues were stained with rabbit anti‐human FSTL1 followed by Alexa Fluor 647‐anti‐rabbit IgG (red). Hoechst 33342 was used to counterstain nuclei (blue). Slides were imaged by confocal microscopy using a ×63 oil‐immersion objective. A, Images are representative of at least 15 microscope fields from one of four donors. B, The mean + SEM of the fold change in the average fluorescence signal (LM vs HM) is shown (*P < 0.01, Mann‐Whitney U test). C, COL2A1 and FSTL1 expression in tissues with LM and HM scores (LM, n = 3; HM, n = 3) were assayed by real‐time PCR and normalized to GAPDH. Each circle represents an individual data point. The Pearson's correlation coefficient (r) and P value are shown.
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