Juvenile idiopathic arthritis fibroblast-like synoviocytes influence chondrocytes to alter BMP antagonist expression demonstrating an interaction between the two prominent cell types involved in endochondral bone formation

Abstract

Background: To examine critical interactions between juvenile idiopathic arthritis synovial fibroblasts (JFLS) and chondrocytes (Ch), and their role in bony overgrowth seen in patients with juvenile idiopathic arthritis (JIA).

Methods: Control (CFLS) and JFLS were cultured in synoviocyte media containing recombinant BMP4. Ch were cultured in either CFLS or JFLS conditioned-media without stimulation. Media supernatants were analyzed by ELISA. RNA from conditioned media experiment was analyzed by ClariomS microarray.

Results: As expected, genes expressed in untreated JFLS and CFLS cultured in synoviocyte media were similar to each other and this expression differed from untreated Ch cultured in chondrocyte media. JFLS favor BMP ligand gene expression while downregulating TGFβ receptors' expression. Noggin and chordin, antagonists with high affinity for BMP4, are JFLS- but not Ch-preferred regulators of BMP signaling. Compared to Ch, JFLS overexpress collagen X (COLX), a marker of chondrocyte hypertrophy. Exogenous BMP4 causes JFLS to significantly decrease expression of noggin and collagen II (COL2), a marker of chondrocyte proliferation, and causes overexpression of COLX and alkaline-phosphatase (ALP). Chondrocytes cultured in JFLS-conditioned media (Ch-JFLS) express BMP genes and favor chordin protein expression over other antagonists. Ch-JFLS have significantly increased expression of COL2 and significantly decreased expression of COLX.

Conclusions: These data suggest JFLS, in the presence of BMP4, undergo hypertrophy and that JFLS-conditioned media influence chondrocytes to become highly proliferative. To the authors' knowledge, no prior study has shown that JFLS and chondrocytes play a direct role in the bony overgrowth in joints of patients with JIA and that BMPs or regulation of these growth factors influence the interaction between two prominent synovial cell types.

Keywords: BMP antagonists; BMP4; Chondrocyte; Endochondral bone; Fibroblast; Hypertrophy; Juvenile idiopathic arthritis; Proliferation; Synoviocyte; TGFβ.

Fig. 1

Unsupervised hierarchal clustering comparing chondrocytes (Ch) and fibroblast-like synoviocytes (FLS) after 6 h in culture and differentially expressed genes specific to TGFβ and BMP signaling in these cells. a, Unsupervised hierarchical clustering of 104 differentially expressed genes (estimated percentage false prediction (pfp < 0.01, Rank Product) JFLS have similar gene expression patterns to CFLS and differ from Ch. b, Using a manually curated list of 27 genes specific to these signaling pathways, we examine changes in gene expression over 24 h using linear expression values. CFLS have significantly higher levels of BMP signaling via the activation of SMAD1/5 while JFLS overexpress BMP ligand, BMP7 compared to Ch (p < 0.05, t-test). Concurrently, there is a reduction in the gene expression of TGFβ receptors in CFLS and JFLS (TGFBR2/TGFBR3 and TGFBR1/TGFBR3, respectively) when compared to Ch (p < 0.05)

Fig. 2

Protein expression of BMP antagonists: chordin, noggin, follistatin, and gremlin. JFLS significantly overexpress chordin (a) and noggin (b) when compared to Ch (p < 0.05, t-test) as determined by ELISA on cell media supernatants. CFLS exhibit a significant decrease in follistatin when compared to untreated Ch (c) while gremlin remained unchanged in FLS when compared to Ch (d)

Fig. 3

Protein expression of collagen II (COL2), a marker of proliferating chondrocytes and collagen X (COLX), a marker of chondrocyte hypertrophy as determined by ELISA on cell media supernatants from FLS and Ch. There were no significant changes in COL2 expression between FLS and Ch (a). COLX was significantly overexpressed in JFLS compared to Ch (p < 0.05, t-test) (b)

Fig. 4

Using ELISA to study the effects of exogenous BMP4 on protein expression of BMP antagonists in cell media supernatants over 24 h. JFLS, in the presence of BMP4, significantly decrease expression of noggin and follistatin (a and b) (p < 0.05) while gremlin and chordin remained unchanged in FLS exposed to exogenous BMP4 (c and d)

Fig. 5

Using ELISA to study the effects of exogenous BMP4 on protein expression of markers of chondrocyte differentiation. Exogenous BMP4 causes a significant decrease in COL2 in JFLS (a) (p < 0.05) and an increase in COLX in both CFLS and JFLS (B) (p < 0.05). CFLS and JFLS, in the presence of BMP4, overexpress bone-derived alkaline phosphatase (ALP), a marker secreted by bone cells and hypertrophic chondrocytes (c) (p < 0.05)

Fig. 6

Unsupervised hierarchal clustering comparing chondrocytes cultured in FLS-conditioned media and untreated chondrocytes and differentially expressed genes specific to TGFβ and BMP signaling in these chondrocytes. LIMMA revealed 11 differentially expressed genes with a 5% FDR when comparing Ch and Ch-CFLS (Fig. 4a, teal bar) and 13 differentially expressed genes between Ch and Ch-JFLS (Fig. 4a, purple bar) after 6 h of exposure to conditioned media. Overlapping genes revealed those regulated by TGFβ/BMP (ID1, ID3, and PMEPA1). Analyzed changes in gene expression between untreated Ch compared to Ch-CFLS and Ch-JFLS over 24 h using the curated list of genes involved in TGFβ/BMP signaling (b). Ch-CFLS and Ch-JFLS had significantly lower expression levels of TGFβ-related genes when compared to untreated Ch (Ch-CFLS: SMAD4, SMURF2, TGFBR2, TGFBI and Ch-JFLS: SMAD2, SMAD7, TGFBR2) (p < 0.05). Ch-JFLS had significantly increased expression of BMP2 and BMP7 compared to untreated Ch (p < 0.05)

Fig. 7

Protein expression of BMP antagonists: chordin, noggin, follistatin, and gremlin. Using ELISA to measure protein expression, both Ch-CFLS and Ch-JFLS significantly overexpress chordin when compared to untreated Ch with the greater difference seen in Ch cultured in JFLS-conditioned media (a) (p < 0.05) while noggin, follistatin, and gremlin remained unchanged (b, c, and d)

Fig. 8

Protein expression of collagen II (COL2), a marker of proliferating chondrocytes and collagen X (COLX), a marker of chondrocyte hypertrophy as determined by ELISA on cell media supernatants. While both Ch-CFLS and Ch-JFLS decrease expression of proliferation marker, COL2, the difference was only significant in Ch cultured in CFLS-conditioned media (a). Hypertrophic marker, COLX is significantly downregulated in both Ch-CFLS and Ch-JFLS (b) (p < 0.05, t-test)

Fig. 9

Possible paradigms for the role of FLS in growth disturbances in JIA. JFLS in culture favor expression of genes related to BMP as opposed to TGFβ (a). In the presence of exogenous BMP4, JFLS show a decrease in BMP antagonists that have a high affinity for BMP4 which allows for these cells to differentiate further along chondrocyte lineage as demonstrated by an increase in COLX and ALP, late-stage markers, and a decrease in COL2, an early-stage marker (a). Cell hypertrophy is required for endochondral bone formation. Similar to JFLS in culture, chondrocytes cultured in JFLS-conditioned media favor expression of genes related to BMP as opposed to TGFβ (b). Unlike JFLS in culture, Ch-JFLS decrease protein expression of hypertrophic marker, COLX and have increased protein expression of BMP antagonist, chordin which can contribute to the inhibition of endochondral bone formation (b)