Transforming growth factor-beta1 modulates insulin-like growth factor binding protein-4 expression and proteolysis in cultured periosteal explants

Abstract

Objective: Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-beta) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-beta signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-beta1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants.

Design: Periosteal explants from rabbits were cultured with or without TGF-beta1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity.

Results: TGF-beta1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70% on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-beta1-treated explants. PAPP-A mRNA levels were increased 1.6-fold, PAPP-A protein levels were increased threefold, and IGFBP-4 protease activity was increased 8.5-fold between 7 and 10days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-beta1-treated explants.

Conclusions: This study demonstrates that TGF-beta1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants.

Fig. 1

A) IGFBP-4 and B) PAPP-A mRNA levels in cultured periosteal explants (12 explants/group). Periosteal explants were cultured for up to 42 days with (blue line) or without (black line) 10 ng/mL TGF-β1 (for the first two days). RNA was extracted from the explants (pooled 2 explants/group) after 3, 5, 7, 10, 14, 21, 28, 35 and 42 days of culture, reverse transcribed and analyzed using Real Time PCR with rabbit-specific primers and probes for IGFBP-4, PAPP-A and GAPDH. Standard curves from specific oligonucleotides containing primers and probe sequences were generated to obtain copy number data. All samples (n=6) were run in duplicate and quantitated by normalizing the target signal with the GAPDH signal. The data are presented as means ± standard error. Post-hoc testing using Duncan's Multiple Range test was performed to determine significance between specific times ± TGF-β1.

Fig. 2

A) Western ligand blot analysis of IGFBP-4 in conditioned medium from cultured periosteal explants. Periosteal explants were cultured in serum containing medium until day 7 (with or without 10 ng/mL TGF-β1 for the first 48h). The medium was then changed to serum free medium and conditioned medium was collected 48h later. The conditioned medium was then analyzed by Western ligand blot. B) PAPP-A secretion from cultured periosteal explants. Periosteal explants were cultured in serum containing medium until day 7 (with or without 10 ng/mL TGF-β1 for the first 48h). The medium was then changed to serum free medium and conditioned medium was collected 96h later. The conditioned medium was then analyzed by ELISA. The data are presented as means ± standard deviation. Two-tailed students t-test was used to determine significance.

Fig. 3

IGFBP-4 proteolysis in conditioned medium from cultured periosteal explants. Periosteal explants were cultured in serum containing medium until day 7 (with or without 10 ng/mL TGF-β1 for the first 48h). The medium was then changed to serum free medium and conditioned medium was collected 96h later. Fifty microliters of periosteal explant conditioned medium were incubated in a microcentrifuge tube containing ¹²⁵I-IGFBP-4 with or without 5 nM IGF-II at 37 °C for 24 hr, as previously described , . A) Reaction products from three different samples for control and TGF-β groups were separated by SDS-PAGE, 7.5-15% gradient and visualized by autoradiography. B) Densitometry analysis of the IGFBP-4 fragment bands. The results are presented as means ± standard error. Two-tailed students t-test was used to determine significance.

Fig. 4

Aggrecan mRNA levels in cultured periosteal explants (12 explants/group). Periosteal explants were cultured for up to 42 days with (blue line) or without (black line) 10 ng/mL TGF-β1 (for the first two days). RNA was extracted from the explants (pooled 2 explants/group) after 3, 5, 7, 10, 14, 21, 28, 35 and 42 days of culture, reverse transcribed and analyzed using Real Time PCR with rabbit-specific primers and probes for aggrecan and GAPDH. Standard curves from specific oligonucleotides containing primer and probe sequences were generated to obtain copy number data. All samples (n=6) were run in duplicate and quantitated by normalizing the target signal with the GAPDH signal. The data are presented as means ± standard error. Post-hoc testing using Duncan's Multiple Range test was performed to determine significance between specific times ± TGF-β1.