Cathepsin K is the principal protease in giant cell tumor of bone

Abstract

Giant cell tumor (GCT) of bone is a neoplasm of bone characterized by a localized osteolytic lesion. The nature of GCT is an enigma and the cell type(s) and protease(s) responsible for the extensive localized clinicoradiological osteolysis remain unresolved. We evaluated protease expression and cellular distribution of the proteolytic machinery responsible for the osteolysis. mRNA profiles showed that cathepsin K, cathepsin L, and matrix metalloproteinase (MMP)-9 were the preferentially expressed collagenases. Moderate expression was found for MMP-13, MMP-14, and cathepsin S. Specific protease activity assays revealed high cathepsin K activity but showed that MMP-9 was primarily present (98%) as inactive proenzyme. Activities of MMP-13 and MMP-14 were low. Immunohistochemistry revealed a clear spatial distribution: cathepsin K, its associated proton pump V-H(+)-ATPase, and MMP-9 were exclusively expressed in osteoclast-like giant cells, whereas cathepsin L expression was confined to mononuclear cells. To explore a possible role of cathepsin L in osteolysis, GCT-derived, cathepsin L-expressing, mononuclear cells were cultured on dentine disks. No evidence of osteolysis by these cells was found. These results implicate cathepsin K as the principal protease in GCT and suggest that osteoclast-like giant cells are responsible for the osteolysis. Inhibition of cathepsin K or its associated proton-pump may provide new therapeutic opportunities for GCT.

Figure 1

Specificity of the cathepsin K-specific monoclonal antibody (TNO B1446) (Western blot analysis: lane 1, cathepsin K; lane 2, negative control buffer; lane 3, pro-MMP-3; lane 4, cathepsin L; lane 5, cathepsin S; and lane 6, cathepsin V), showing minimal cross-reactivity (∼5%) with cathepsin V and no cross reactivity with cathepsins L and S. MMP-3 was included as a negative control.

Figure 2

Normalized protease mRNA expression in human GCTs (GAPDH = 1) assessed by real-time PCR cycles presented on a log scale. Mean (SD) of seven individual tumors. ND, not detectable (>40 PCR cycles).

Figure 3

Immunohistochemical staining of human GCT. A: control; B: cathepsin K; C: MMP-9; D: cathepsin L; E: cathepsin S; and F: V-H⁺-ATPase. Note that cathepsin K, MMP-9, and v-ATPase are exclusively expressed in the multinucleated giant cells whereas cathepsin L and S expression is confined to the mononuclear cells. Arrows in A indicate the multinucleated giant cells. Original magnifications, ×200.

Figure 4

Total (ie, latent and active) and active MMP-9, MMP-13, and cathepsin K activity in GCT [activity is expressed as recombinant enzyme equivalents (ng)/mg homogenate]. Mean (SD) of seven individual tumors. ND, not detectable.

Figure 5

Relative mRNA expression (GAPDH = 1) of cathepsin K, cathepsin L, cathepsin S, and MMP-9 in three separate GCT-derived mononuclear cell lines (mean SD). Values are based on the number of real-time PCR cycles. Cathepsin L expression was ∼42-fold higher than cathepsin K expression. MMP-9 expression was negligible.

Figure 6

Cathepsin L-expressing GCT-derived mononuclear cells do not induce osteolysis. Cathepsin L-expressing GCT-derived mononuclear cells were grown on devitalized dentine disks for 21 days. No evidence of bone resorption by these cells was found (A–C). D: A control dentine disk with cultured osteoclasts showing the typical pattern of resorption lines. Representative images were obtained in multiple experiments with three different cell lines (L980, L1050, L1180). Original magnifications, ×400.