Abstract
Transforming growth factor β (TGFβ) receptor interacting protein-1 (TRIP-1) is an intracellular protein expressed in osteoblasts with high affinity for type 5b tartrate resistant acid phosphatase (TRAP). It is suggested that through this interaction, TRIP-1 serves as a positive regulator of TGFβ signaling and osteoblast differentiation during bone remodeling. We show here that TRIP-1 is abundant in osteoblasts in vivo and in vitro. TRIP-1 mRNA and protein expression were increased at early stages and decreased at later stages during osteoblast differentiation, suggesting a predominant role during early maturation. To investigate a role during bone remodeling, primary osteoblasts were treated with different hormones and factors that are known to affect remodeling. TRIP-1 levels were decreased with dexamethasone and increased with vitamin D(3) , dihydrotestosterone (DHT), TGFβ1, and bone morphogenic protein 2 (BMP-2). Treatment with parathyroid hormone (PTH) and β-estradiol did not affect TRIP-1 levels. Transfected small interfering RNA (siRNA) against TRIP-1 inhibited osteoblast differentiation as characterized by a decrease in alkaline phosphatase staining and enzyme activity, and decrease in the expression of collagen I, alkaline phosphatase, Runx2, osteopontin, and osteocalcin. The proliferation of osteoblasts was also affected by TRIP-1 siRNA. This particular effect was defined by decreased cell number, marked reduction of cyclin D1, a 38% decrease of cells in S phase (p < 0.001) and a 97% increase of cells in the G2/M phase (p < 0.01) of the cell cycle. However, TRIP-1 siRNA did not induce an effect in apoptosis. Using a TGFβ luciferase reporter we found that knocking down TRIP-1 decreased the activation of TGFβ signaling by 40% percent (p < 0.001). In conclusion, our characterization of TRIP-1 in osteoblasts provides the first evidence of its key role as a positive regulator of osteoblast function.