Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation

Abstract

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation.

Introduction: Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures.

Materials and methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures.

Results: Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor.

Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.

FIG. 1

Smad3^−/− sternal chondrocytes spontaneous express maturation-associated genes. Sternal chondrocytes from wildtype and Smad3 ^−/− neonatal mice were harvested and cultured for 2, 4, or 8 days, and total RNA was extracted. (A, C, and E) Real-time RT-PCR and (B and D) Northern blot were performed to evaluate (A and B) col2, (C and D) colX, and (E) MMP9, MMP13, alkaline phosphatase, VEGF, and osteocalcin mRNA expression (2-day culture).

FIG. 2

Altered regulation of TGF-β and BMP signaling molecule expression in Smad3^−/− sternal chondrocytes. Sternal chondrocytes from wildtype and Smad3^−/− neonatal mice were harvested and cultured for 2 days. Total RNA for (A, C, and D) real-time RT-PCR or (B) total cellular lysates for Western blot were harvested and processed. Primers for the various RT-PCR reactions are shown in Table 1.

FIG. 3

Smad3^−/− chondrocytes have reduced TGF-β signaling and enhanced BMP signaling. Sternal chondrocytes from wild-type and Smad3^−/− neonatal mice were harvested, and after 12 h in culture, were transfected with either (A) 500 ng P3TP-Luc (TGF-β–responsive element), (B) 4× SBE-Luc (Smad3-specific reporter), or (C) 9× GCG-Luc (BMP-specific reporter) using Su-perfect reagent. SV-40 renilla luciferase construct (10 ng) was co-transfected with the above firefly reporters to standardize results for transfection efficiency. Luciferase activity in cell lysate was determined in a luminometer (Opticom 1; MGM Instruments, Hamden, CT, USA). Sternal chondrocytes were cultured for 48 h and treated with exogenous BMP (50 ng/ml) for the time specified. Total cellular protein was extracted, and Western blot for phosphoSmad 1/5/8 was performed. (D) β-actin was used to control for protein loading.

FIG. 4

Altered regulation of col2 by TGF-β and BMP in Smad3^−/− sternal chondrocytes. Sternal chondrocytes from wild-type and Smad3^−/− neonatal mice were harvested and cultured in the presence or absence of BMP-2 or TGF-β for 2, 4, or 8 days, and total RNA was extracted. Real-time RT-PCR or Northern blot was performed for col2 expression on cultures harvested after (A and B) 2, (C) 4, or (D) 8 days in culture.

FIG. 5

Altered regulation of colX expression and alkaline phosphatase activity by TGF-β and BMP signaling molecule expression in Smad3^−/− sternal chondrocytes. Sternal chondrocytes from wildtype and Smad3^−/− neonatal mice were harvested and cultured in the presence or absence of BMP-2 or TGF-β for 2, 4, or 8 days, and total RNA was extracted. Real-time RT-PCR was performed for colX expression on cultures harvested after (A) 2, (B) 4, or (C) 8 days in culture. (D) Cellular extracts of the 2-day cultures were harvested, and alkaline phosphatase activity was measured.

FIG. 6

Overexpression of Smad2 and Smad3 inhibit maturation in Smad3^−/− chondrocytes. Sternal chondrocytes from wild-type and Smad3^−/− neonatal mice were harvested, and after 24 h in culture, were exposed to concentrated retrovirus expressing either Smad2, Smad3, or GFP. After 48 h, 1 ml of complete media was added to each dish together with hexadimethrine bromide and incubated for another 48 h. (A and B) Expression of Smad2 and Smad3 in the cultures was determined by Western blot. Cultures infected with viral particles expressing GFP were used to determine the infection rate using (C) fluorescence microscopy and (D) brightfield analysis. (E) Total RNA was harvested, and real-time RT-PCR was performed to determine colX expression. (F) Cellular extracts were harvested, and alkaline phosphatase activity was measured.

FIG. 7

Recombinant noggin inhibits spontaneous maturation in Smad3^−/− chondrocytes. Sternal chondrocytes from wildtype and Smad3^−/− neonatal mice were harvested and were cultured for 48 h in the presence or absence of 500 ng/ml recombinant murine noggin. (A) Total RNA was harvested, and colX mRNA expression was evaluated by real-time RT-PCR (*p =0.0001 compared with WT). (B) Cellular extracts were harvested, and alkaline phosphatase activity was measured (*p =0.006 compared with WT).