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Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation.

Nistala H, Lee-Arteaga S, Smaldone S, Siciliano G, Carta L, Ono RN, Sengle G, Arteaga-Solis E, Levasseur R, Ducy P, Sakai LY, Karsenty G, Ramirez F - J. Cell Biol. (2010)

Bottom Line: This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization.Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition.Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10021, USA.

ABSTRACT
Extracellular regulation of signaling by transforming growth factor (TGF)-β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2- (Fbn2(-/-)) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1(-/-) mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

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Abnormally high TGF-β activity in differentiating Fbn1- osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1- (Fbn1−/−) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day −3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1−/− calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1- cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1- cOb cultured in low serum with or without 1 µg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2- cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
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fig6: Abnormally high TGF-β activity in differentiating Fbn1- osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1- (Fbn1−/−) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day −3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1−/− calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1- cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1- cOb cultured in low serum with or without 1 µg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2- cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).

Mentions: Although neonatal lethality of Fbn1−/− mice limits performing extensive analyses of bone formation (Carta et al., 2006), these mutant animals nonetheless enabled us to compare and contrast osteogenic differentiation on a matrix deficient for either fibrillin-1 or -2. Fbn1- cOb proliferated normally but, in contrast to Fbn2- cells, they yielded more mineral nodules than WT cultures (Fig. 6, A and B); they also displayed a modest increase in Osx expression, a significant up-regulation of Col1a2 and Bglap1, and normal Runx2 and Fbn2 activity (Fig. 6 C). In vivo levels of Col1a2 and Bglap1 (but not Osx) transcripts were appreciably higher than control, and collagen accumulation was slightly greater in mutant than WT bones (Fig. 6 D and Fig. S1 C). Moreover, AP-positive cells and mineral deposits appeared earlier and grew faster in Fbn1- than WT cOb cultures (Fig. S1 D). Collectively, these observations were consistent with the notion that loss of fibrillin-1 accelerates osteoblast maturation.


Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation.

Nistala H, Lee-Arteaga S, Smaldone S, Siciliano G, Carta L, Ono RN, Sengle G, Arteaga-Solis E, Levasseur R, Ducy P, Sakai LY, Karsenty G, Ramirez F - J. Cell Biol. (2010)

Abnormally high TGF-β activity in differentiating Fbn1- osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1- (Fbn1−/−) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day −3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1−/− calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1- cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1- cOb cultured in low serum with or without 1 µg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2- cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig6: Abnormally high TGF-β activity in differentiating Fbn1- osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1- (Fbn1−/−) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day −3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1−/− calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1- cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1- cOb cultured in low serum with or without 1 µg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2- cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
Mentions: Although neonatal lethality of Fbn1−/− mice limits performing extensive analyses of bone formation (Carta et al., 2006), these mutant animals nonetheless enabled us to compare and contrast osteogenic differentiation on a matrix deficient for either fibrillin-1 or -2. Fbn1- cOb proliferated normally but, in contrast to Fbn2- cells, they yielded more mineral nodules than WT cultures (Fig. 6, A and B); they also displayed a modest increase in Osx expression, a significant up-regulation of Col1a2 and Bglap1, and normal Runx2 and Fbn2 activity (Fig. 6 C). In vivo levels of Col1a2 and Bglap1 (but not Osx) transcripts were appreciably higher than control, and collagen accumulation was slightly greater in mutant than WT bones (Fig. 6 D and Fig. S1 C). Moreover, AP-positive cells and mineral deposits appeared earlier and grew faster in Fbn1- than WT cOb cultures (Fig. S1 D). Collectively, these observations were consistent with the notion that loss of fibrillin-1 accelerates osteoblast maturation.

Bottom Line: This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization.Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition.Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10021, USA.

ABSTRACT
Extracellular regulation of signaling by transforming growth factor (TGF)-β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2- (Fbn2(-/-)) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2(-/-) phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1(-/-) mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

Show MeSH
Related in: MedlinePlus