The type II collagen N-propeptide, PIIBNP, inhibits cell survival and bone resorption of osteoclasts via integrin-mediated signaling

Abstract

Objective: Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH(2)-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins α(V)β(3) and α(V)β(5). As osteoclasts rely on α(V)β(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function.

Methods: We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast functions in the presence of recombinant PIIBNP. Adhesion of osteoclasts to PIIBNP was analyzed by staining of attached cells with crystal violet. PIIBNP-induced cell death was evaluated by counting Trypan Blue stained cells. The mechanism of cell death was evaluated by DNA fragmentation, TUNEL staining and western blotting to detect cleaved caspases. To determine the role of α(V)β(3) integrin, osteoclasts were pretreated with α(V) or β(3) integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed.

Results: Osteoclasts adhered to PIIBNP via an RGD-mediated mechanism. When osteoclasts were plated on extracellular matrix proteins, PIIBNP induced apoptosis of osteoclasts via caspase 3/8 activation. Osteoblasts and macrophages were not killed. Reduction of α(V) or β(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption.

Conclusion: We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the α(V)β(3) integrins. Because of this property and the cell specificity, we propose that PIIBNP may play a role in vivo in protecting cartilage from osteoclast invasion and also could be a new therapeutic strategy for decreasing bone loss.

Figure 1

(a) Adhesion of RAW-osteoclasts and MC3T3-E1 osteoblasts to GST, GST-PIIBNP and GST-mutant PIIBNP. *indicates p<0.05 versus cells treated with 3 μM GST. (b) Adhesion of primary osteoclasts and osteoblasts to GST, GST-PIIBNP and GST-mutant PIIBNP. *indicates p<0.05 versus cells treated with 3 μM GST. (c) Effect of blocking of RGD or RAD peptides on RAW-osteoclast adhesion to 0.5 μM PIIBNP. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05, and * * indicates p<0.01 versus cells treated with individual concentrations of RAD peptides. (d) Effect of PIIBNP on osteoclast adhesion to type I collagen (COL1), fibronectin (FN) and vitronectin (VN) by PIIBNP. GST or GST-PIIBNP was added to RAW 264.7 osteoclasts before the incubation. Each assay was performed in triplicate wells and three independent experiments. Columns represent mean ± S.D. *indicates p<0.05 versus cells treated with 3 μM GST.

Figure 2

(a) Percent cell death of differentiated RAW-osteoclasts and MC3T3-E1 osteoblasts on type I collagen (COL1), fibronectin (FN) and vitronectin (VN) without serum after treatment of GST, GST-PIIBNP and GST-mutant PIIBNP for 16 h. (b) Percent cell death of primary osteoclasts and osteoblasts. Columns represent mean ± S.D. of percent of trypan blue stained cells/ total cells in medium and on the plate. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05 versus cells treated with 3 μM GST.

Figure 3

(a) Percent of detached cells of differentiated RAW osteoclasts treated for 16 h without serum on type I collagen (COL1), fibronectin (FN) and vitronectin (VN) by GST, GST-PIIBNP and GST-mutant PIIBNP (mPIIBNP). Columns represent mean ± S.D. of percent detached cells. (b) A time course experiment showing the kinetics of osteoclast detachment by PIIBNP on vitronectin (VN) coated plates. Left panel shows percent detached RAW-osteoclasts. Right panel shows percent cell death of RAW osteoclasts. Columns represent mean ± S.D. of the percent detached cells (Left panel) and percent trypan blue stained cells (Right panel). (c) DNA fragmentation in RAW-osteoclasts after 16 h of treatment with 3 μM GST and GST-PIIBNP on type I collagen (COL1), fibronectin (FN) and vitronectin (VN) coated plates. (d) Effect of GST-PIIBNP for apoptosis of osteoclasts on type I collagen. Columns represent mean ± S.D. of TUNEL positive cells, at least 300 cells were counted by a blinded observer in the same experiment. The results shown are the average of three individual samples. (e) Effect of PIIBNP on caspases 8, 9 and 3 analyzed by Western blotting. RAW-osteoclasts were treated for 16 h with 3 μM GST and GST-PIIBNP on type I collagen (COL1) fibronectin (FN) and vitronectin (VN) coated plates. The results shown represent three independent experiments. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05 versus (a) (d) cells treated with 3 μM GST and (b) non (0h) treatment.

Figure 4

The dose-dependent effect of reduction of α_V integrin by siRNA on PIIBNP-induced cell death. (a) The expression levels of α_V integrin mRNA were analyzed by realtime PCR. Expression ratios of αv integrin (αv siRNA/ Control siRNA) are shown. (b) The panels show percent cell death of RAW-osteoclasts without serum on type I collagen (COL 1) and vitronectin (VN) after the treatment of GST, and GST-PIIBNP for 16 h. Three concentrations of siRNA were used to reduce integrin expression. Columns represent mean ± S.D. of the percent of Trypan Blue stained cells/ total cells. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05 versus cells treated with control siRNA.

Figure 5

The dose-dependent effect of reduction of β₃ integrin by siRNA on PIIBNP-induced cell death. (a) The expression levels of β₃ integrin mRNA were analyzed by realtime PCR. Expression ratios of αv integrin (β₃ siRNA/ Control siRNA) are shown. (b) The panels show percent cell death of RAW-osteoclasts without serum on type I collagen (COL 1) and vitronectin (VN) after the treatment of GST, and GST-PIIBNP for 16 h. Three concentrations of siRNA were used to reduce integrin expression. Columns represent mean ± S.D. of the percent of Trypan Blue stained cells/ total cells. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05 versus cells treated with control siRNA.

Figure 6

Inhibition of osteoclast bone resorptive activity by PIIBNP in vivo. The calvarial bones were harvested from mice treated with PBS, LPS, LPS + GST, and LPS + PIIBNP. (a) Upper panels showed that the histological sections stained for TRAP and counterstained with hematoxylin. Red cells are TRAP-stained osteoclasts. Lower panels showed that the histlogical staining for in situ TUNEL staining and counterstained with methyl green. Black arrow indicates TUNEL positive cells. The histological sections were fixed with 4% neutral buffered formalin for 10 min and apoptotic cells were determined using TUNEL assay kit (Wako) according to the manufacturer's protocol. Bar =100 μm. (b) Quantitation of osteoclast numbers. Columns represent mean ± S.D. of the osteoclasts number/ mm². (c) Percent of eroded surface occupied by osteoclasts. Columns represent mean ± S.D. of the percentage of eroded bone surface length covered by osteoclasts. For each animal, the sections with the largest diameter of bone marrow were used for calculations. (d) Quantitation of serum type I collagen fragment (C-telopeptide, Ctx) at the time of sacrifice. Columns represent mean ± S.D. of the Ctx concentration in serum. Each assay was performed in triplicate wells and three independent experiments. *indicates p<0.05 versus samples treated with LPS + GST.