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. 1999 Nov;10(11):3801-13.
doi: 10.1091/mbc.10.11.3801.

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

M Fujii  1 , K Takeda, T Imamura, H Aoki, T K Sampath, S Enomoto, M Kawabata, M Kato, H Ichijo, K Miyazono
Affiliations
Free PMC article

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

M Fujii et al. Mol Biol Cell. 1999 Nov.
Free PMC article

Abstract

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

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Figures

Figure 1
Figure 1
Histochemical analysis of alkaline phosphatase activity and phosphorylation of Smad1 and Smad5 by constitutively active forms of type I receptors in the adenovirus vector. (A) Alkaline phosphatase production in C2C12 cells by constitutively active forms of type I receptors is shown. C2C12 cells were infected with adenoviruses carrying constitutively active forms of type I receptor cDNA at an m.o.i. of 300 (ii–vii). Four days after infection, the cells were observed by phase-contrast microscopy. (B) Quantification of alkaline phosphatase activity in C2C12 cells by the constitutively active form of ALK-2 [ALK-2(QD)] is shown. Top, alkaline phosphatase activity was determined as described in MATERIALS AND METHODS; m.o.i. is shown in parentheses. Bottom, expression of ALK-2(QD) was determined by anti-HA immunoblotting. (C) Phosphorylation of Smad1 and Smad5 by the constitutively active forms of type I receptors is shown. C2C12 cells were infected with adenoviruses with HA-tagged type I receptor (m.o.i. of 200) and FLAG-tagged Smad1 or Smad5 cDNAs (ALK-HA; m.o.i. of 200). Top, cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-phosphoserine antibody. Middle, expression of Smad1 and Smad5 was confirmed by reblotting the membrane with anti-FLAG antibody. Bottom, expression of type I receptors was detected by immunoblotting using anti-HA antibody of the cell lysates. ALP, alkaline phosphatase; IP, immunoprecipitation; p-NP, p-nitrophenyl phosphate.
Figure 2
Figure 2
Induction of alkaline phosphatase activity by Smads in the adenovirus vector. (A and B) Effects of Smad1 and Smad5 in the presence and absence of Smad4 are shown. C2C12 cells were infected with adenoviruses carrying Smad1, Smad5, and Smad4 at an m.o.i. of 200–600 (shown in parentheses in B). Adenovirus carrying β-galactosidase (m.o.i. of 800) was used as a control (i). (A) Expression of Smads was confirmed by anti-FLAG immunoblotting. (B) C2C12 cells were stained for alkaline phosphatase activity. (C and D) Effects of Smad2, Smad3, and Smad4 on C2C12 cells are shown. C2C12 cells were infected with adenoviruses with Smad2, Smad3, or Smad4 alone (m.o.i. of 600; ii–iv) or with combinations of Smad2 or Smad3 and Smad4 (m.o.i. of 300 for each; v and vi). (C) Expression of Smads was confirmed by anti-FLAG immunoblotting. (D) C2C12 cells were stained for alkaline phosphatase activity.
Figure 3
Figure 3
Increase in alkaline phosphatase activity by Smad1 and Smad5 in the presence of BMP-6. (A) Effects of Smads in the presence of BMP-6. C2C12 cells were infected with adenoviruses carrying Smad1, Smad2, and Smad5 at an m.o.i of 300 and treated with 200 ng/ml BMP-6 (iii–v). Adenovirus carrying β-galactosidase (m.o.i. of 800; ii) was used as a control. Cells were stained for alkaline phosphatase activity 4 d after infection. (B) Nuclear translocation of Smad5 by BMP-6. The cells infected with Smad5 (m.o.i. of 100) and Smad4 (m.o.i. of 50) and treated with BMP-6 (200 ng/ml) were stained by indirect immunofluorescence using an anti-Smad5 antibody.
Figure 4
Figure 4
Smad6 and Smad7 inhibit the osteoblast differentiation of C2C12 cells. (A) C2C12 cells infected with adenoviruses carrying Smad5, Smad6, and Smad7 (m.o.i. of 150 for each) were treated with 200 ng/ml BMP-6 and stained for alkaline phosphatase activity 4 d after infection. (B and C) C2C12 cells infected with adenoviruses with the constitutively active forms of BMPR-IA/ALK-3 [ALK-3(QD); B] or BMPR-IB/ALK-6 [ALK-6(QD); C] (m.o.i. of 300) were coinfected with Smad6 (ii) or Smad7 (iii) (m.o.i. of 100). Cells were stained for alkaline phosphatase activity 4 d after infection. (D and E) Subcellular localization of Smad5 coinfected with the adenoviruses carrying BMPR-IA/ALK-3(QD) (D) or BMPR-IB/ALK-6(QD) (E) and Smad6 (ii) or Smad7 (iii) is shown. Each adenovirus was infected at an m.o.i. of 100. Cells were stained by indirect immunofluorescence using an anti-Smad5 antibody 4 d after infection.
Figure 5
Figure 5
Chondrogenic differentiation of ATDC5 cells by constitutively active forms of type I receptors. (A and B) ATDC5 cells were infected with adenoviruses carrying the constitutively active forms of type I receptors at an m.o.i. of 300 (iii–viii). (A) Expression of each receptor was determined by immunoblotting using anti-HA antibody. (B) Cells were stained by Alcian Blue 8 d after infection. Adenovirus carrying β-galactosidase (m.o.i. of 300) was used as a control (ii). (C) Incorporation of [35S]sulfate into ATDC5 cells by the constitutively active forms of type I receptors was determined 10 d after infection.
Figure 6
Figure 6
Prevention of chondrogenic differentiation of ATDC5 cells by kinase-inactive forms of type I receptors. ATDC5 cells were infected with adenoviruses carrying the kinase-inactive forms of type I receptors at an m.o.i. of 300 (iii–viii). (A) Expression of each receptor was determined by immunoblotting using anti-HA antibody. (B) Cells were stained by Alcian Blue 8 d after infection. Adenovirus carrying β-galactosidase (m.o.i. of 300) was used as a control (ii).
Figure 7
Figure 7
Chondrogenic differentiation of ATDC5 cells by Smads. (A) ATDC5 cells were infected with adenoviruses carrying Smad1–Smad7 at an m.o.i. of 300 (iii–ix). Cells were stained by Alcian Blue 8 d after infection. Adenovirus with β-galactosidase (m.o.i. of 300) was used as a control (ii). (B) Incorporation of [35S]sulfate into ATDC5 cells by Smad1–Smad7 was determined 10 d after infection.
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