BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes

Abstract

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.

Fig. 1

Synthesis of type IIA procollagen, like type I procollagen, is a sign of dedifferentiation in mouse chondrocytes expanded in monolayer. Embryonic rib cage chondrocytes were serially cultured in monolayer in culture medium supplemented with 10% FBS. At each passage, cells were fixed 72 h after their seeding. On the panel shown on the left, cells were double-stained for the triple helix part of collagen II with Cy3-conjugated secondary antibodies (red) and for type IIA procollagen with Cy2-conjugated secondary antibodies (green). Note that with passaging, cells spread and synthesis of type II procollagen progressively switches from the IIB to the type IIA form, with cells synthesizing both forms (merged double-staining in yellow). On the panel shown in the middle, cells were stained for type I procollagen with Cy2-conjugated secondary antibodies (green). On the panel shown on the right, cells were stained for aggrecan with Cy2-conjugated secondary antibody (red). Nuclei were stained with Hoechst dye (blue) (×16).

Fig. 2

Both BMP-2 and TGF-β1 stimulate type II procollagen synthesis in dedifferentiated chondrocytes but only TGF-β1 favors synthesis of the type IIA form. P1 chondrocytes were cultured for 3 days in the presence of 1% FBS alone or supplemented with 50 ng/ml BMP-2 or 5 ng/ml TGF-β1 as indicated. Cells were stained for the triple helix part of collagen II with Cy2-conjugated secondary antibodies (red) and for type IIA procollagen with Cy3-conjugated secondary antibodies (green). Nuclei are stained with Hoechst dye (blue). While a significant amount of cells cultured with 1% FBS are visualized by the presence of their nuclei only, most cells cultured in the presence of BMP-2 or TGF-β1 are stained for total type II procollagen, as observed on the left. In parallel cultures, cells treated with TGF-β1 show intense staining for type IIA procollagen compared with cells cultured with 1% FBS or 1% FBS + 50 ng/ml BMP-2, as observed on the right (×16).

Fig. 3

The differential effect of BMP-2 and TGF-β1 on type II procollagen isoforms synthesis is dependent on the duration of treatment with the growth factors. P1 chondrocytes were cultured for 18 h in the presence of 10% FBS (day 0), then cultured for 1, 2 or 3 days in the presence of 1% FBS alone or supplemented with 50 ng/ml BMP-2 or 5 ng/ml TGF-β1. Western-blotting analysis of synthesis of global type II or type IIA procollagen in chondrocytes cultured for 1, 2, or 3 days reveals that both BMP-2 and TGF-β1 stimulate total type II procollagen synthesis in dedifferentiated chondrocytes. BMP-2 favors the IIB form whereas TGF-β1 stimulates the type IIA form of procollagen II.

Fig. 4

RT-PCR analysis of expression of selected genes in mouse chondrocytes in response to treatment with BMP-2 and TGF-β1. RT-PCR analysis was performed with chondrocytes freshly extracted from rib cages (Extr. Ch.), or cultured as P0 chondrocytes for 48 h in the presence of 10% FBS, cultured as P1 chondrocytes for 18 h in the presence of 10% FBS (D0) then for 3 days in the presence of 1% FBS alone or supplemented with BMP-2 or TGF-β1 (D3). The corresponding gene products are indicated on the left.

Fig. 5

Western-blot analysis of integrin and type I procollagen expression of mouse chondrocytes in response to BMP-2 or TGF-β1 treatment. (A) Western-blot analysis of chondrocytes cultured as P0 chondrocytes for 48 h in the presence of 10% FBS, cultured as P1 chondrocytes for 18 h in the presence of 10% FBS (D0) then for 3 days in the presence of 1% FBS alone or supplemented with BMP-2 or TGF-β1 (D3). As shown here, protein levels of type I procollagen and α11 integrin subunit are highly correlated in chondrocytes. (B) Immunolocalization of the α10 and α11 integrins subunits in chondrocytes adhered to glass coverslips. The arrows indicate clustering of α10 and α11 subunits likely corresponding to focal contacts. Note that α10 is detected in differentiated (P0) chondrocytes and α11 in dedifferentiated (P1) chondrocytes (×100).

Fig. 6

Adenoviral overexpression of Smad6 does not influence expression of type II procollagen and the ratio of type IIA and type IIB forms in mouse chondrocytes exposed to BMP-2. P1 chondrocytes were infected with adenoviruses carrying a β-galactosidase-cDNA as a control or a Smad6-coding cDNA (m.o.i. of 300 or/and 500). (A) Twenty-four h following infection, Western-blotting using anti-FLAG antibody confirms the synthesis of Smad6. (B– D) Infected P1 chondrocytes were cultured in the presence of 1% FBS alone or supplemented with 50 ng/ml BMP-2. (B) After 1 h, Western-blotting shows that the basal and BMP-2-stimulated level of phosphorylation of Smad1/5/8 is inhibited by Smad6. (C) After 3 days of culture, Western-blotting shows that overexpression of Smad6 (m.o.i. 500) does not modulate the type II procollagen synthesis stimulated by BMP-2. (D) In parallel, expression of the type IIA and type IIB procollagen spliced forms was analyzed by RT-PCR. The type IIA/IIB ratio was quantified on the scans of photographs of agarose gels using Image Quant software. Results obtained from four independent experiments were expressed as means ± SEM. Statistical analyses were performed using Student’s t-test (p < 0.05 was considered significant; NS: not significant). The BMP-2 effect on the IIA/IIB ratio is not affected by overexpression of Smad6.