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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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Elevated TGF-β signaling limits Fbn2- osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-, and Fbn2- cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 µm. (B) Maturation of WT and Fbn2- cOb cultures treated with 1 µM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 µM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
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fig5: Elevated TGF-β signaling limits Fbn2- osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-, and Fbn2- cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 µm. (B) Maturation of WT and Fbn2- cOb cultures treated with 1 µM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 µM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).

Mentions: Because LTBPs target TGF-β to microfibrils (Isogai et al., 2003), we examined LTBP1 incorporation in the matrix laid down by overconfluent cOb cultures and found less immunoreactive material in Fbn2- than WT cultures (Fig. 5 A). TMLC bioassays correlated this visual finding with an ∼47% decrease in the amount of TGF-β extracted from the matrix (relative to the amount extracted from cells) of mutant compared with WT samples (n = 3; P < 0.0001). Furthermore, qPCR analyses showed that Ltbp1 mRNA accumulation in differentiating mutant cOb (time points at days 0, 3, and 7 of osteoinduction) is less than control (∼14%) only 3 d after OS treatment (n = 3; P < 0.046). Taken at face value, these results strongly suggested that loss of fibrillin-2 promotes improper TGF-β activation mostly by impairing LLC sequestration in the osteoblast matrix. Decreased matrix incorporation of LTBP1 in the presence of robust Fbn1 expression further suggested that fibrillin-1 could not compensate for the loss of fibrillin-2 deposition in differentiating cOb cultures. This last observation is analogous to the previous finding that the BMP-dependent syndactyly of Fbn2−/− mice is not seen in Fbn1−/− mice even though both proteins are abundantly deposited in the ECM of the forming autopods (Arteaga-Solis et al., 2001; Carta et al., 2006).


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)

Elevated TGF-β signaling limits Fbn2- osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-, and Fbn2- cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 µm. (B) Maturation of WT and Fbn2- cOb cultures treated with 1 µM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 µM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101602&req=5

fig5: Elevated TGF-β signaling limits Fbn2- osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-, and Fbn2- cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 µm. (B) Maturation of WT and Fbn2- cOb cultures treated with 1 µM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 µM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < 0.05).
Mentions: Because LTBPs target TGF-β to microfibrils (Isogai et al., 2003), we examined LTBP1 incorporation in the matrix laid down by overconfluent cOb cultures and found less immunoreactive material in Fbn2- than WT cultures (Fig. 5 A). TMLC bioassays correlated this visual finding with an ∼47% decrease in the amount of TGF-β extracted from the matrix (relative to the amount extracted from cells) of mutant compared with WT samples (n = 3; P < 0.0001). Furthermore, qPCR analyses showed that Ltbp1 mRNA accumulation in differentiating mutant cOb (time points at days 0, 3, and 7 of osteoinduction) is less than control (∼14%) only 3 d after OS treatment (n = 3; P < 0.046). Taken at face value, these results strongly suggested that loss of fibrillin-2 promotes improper TGF-β activation mostly by impairing LLC sequestration in the osteoblast matrix. Decreased matrix incorporation of LTBP1 in the presence of robust Fbn1 expression further suggested that fibrillin-1 could not compensate for the loss of fibrillin-2 deposition in differentiating cOb cultures. This last observation is analogous to the previous finding that the BMP-dependent syndactyly of Fbn2−/− mice is not seen in Fbn1−/− mice even though both proteins are abundantly deposited in the ECM of the forming autopods (Arteaga-Solis et al., 2001; Carta et al., 2006).

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