TRE17/ubiquitin-specific protease 6 (USP6) oncogene translocated in aneurysmal bone cyst blocks osteoblastic maturation via an autocrine mechanism involving bone morphogenetic protein dysregulation

Abstract

Aneurysmal bone cyst (ABC) is a pediatric osseous tumor characterized by extensive destruction of the surrounding bone. The molecular mechanisms underlying its pathogenesis are completely unknown. Recent work showed that translocation of the TRE17/USP6 locus occurs in over 60% of ABC cases resulting in TRE17 overexpression. Immature osteoblasts are presumed to be the cell type harboring translocation of TRE17 in at least a subset of ABCs. However, the effects of TRE17 overexpression on transformation and osteoblast function are unknown. TRE17 encodes a ubiquitin-specific protease (USP) and a TBC (TRE2-Bub2-Cdc16) domain that promotes activation of the Arf6 GTPase. Here we report that TRE17 potently inhibits the maturation of MC3T3 pre-osteoblasts in a USP-dependent and Arf6-independent manner. Notably, we find that TRE17 function is mediated through an autocrine mechanism. Transcriptome analysis of TRE17-expressing cells reveals dysregulation of several pathways with established roles in osteoblast maturation. In particular, signaling through the bone morphogenetic protein (BMP) pathway, a key regulator of osteogenesis, is profoundly altered. TRE17 simultaneously inhibits the expression of BMP-4 while augmenting the BMP antagonist, Gremlin-1. Osteoblastic maturation is restored in TRE17-expressing cells by the addition of exogenous BMP-4, thus establishing a functional role for BMP-4 during TRE17-induced transformation. Because bone homeostasis involves a precise balance between the activities of osteoblasts and osteoclasts, our studies raise the possibility that attenuated osteoblast maturation caused by TRE17 overexpression may contribute to the bone loss/destruction observed in ABC.

FIGURE 1.

Identification of a TRE17 mutant deficient in Arf6 binding. A, domain structure of TRE17 isoforms and mutants. TRE17(long) and TRE17(short) are naturally occurring isoforms. TRE17(long)/USP− and TRE17(A6−) are point mutants that ablate USP activity and Arf6 binding, respectively. TBC, RabGAP homology domain; C and H, cysteine and histidine subdomains of the ubiquitin-specific protease domain. B, HeLa cells were co-transfected with HA-tagged Arf6 Q67L (QL) or T27N (TN) together with GST-tagged TRE17 WT or the Arf6 binding-deficient mutant (A6−). TRE17 was precipitated with glutathione-Sepharose beads, and associated Arf6 was detected by anti-HA blotting. vect, vector; Pdn, pulldown; WCL, whole cell lysate. C, HeLa cells were transfected with the indicated HA-tagged TRE17 allele. Cell extracts were subjected to pulldowns using calmodulin (CaM) agarose (+) or control (−) beads. Associated TRE17 was detected by anti-HA immunoblotting. D, HeLa cells were co-transfected with HA-Arf6 and control vector, TRE17 WT, or TRE17(A6−). Extracts were subjected to pulldowns with GST-GGA3; associated Arf6 (active Arf6) was detected by anti-HA immunoblotting and quantified using ImageJ software (NIH). Data represent the mean ± S.D. of five experiments; **, p < 0.01.

FIGURE 2.

Stable expression of TRE17 in MC3T3 mouse pre-osteoblasts induces morphological transformation. A, MC3T3 cells stably expressing the indicated HA-tagged TRE17 mutants were induced with dox for 24 h. TRE17 peptides were detected using anti-TRE17 (left) or anti-HA (right); extracts were blotted for p70S6k (left) or actin (right) as loading controls. Arrowheads mark migration of TRE17 peptides; asterisks denote nonspecific bands detected by the HA antibody. vect, vector; USP−, TRE17(long)/USP−; A6−, Arf6 binding-deficient mutant. B, MC3T3 cell lines expressing HA-tagged TRE17(long) or TRE17(A6−) were treated with dox to induce TRE17 expression. Lysates were subjected to anti-HA immunoprecipitation followed by anti-Arf6 immunoblotting (top panel). Alternatively, extracts were subjected to GST-GGA3 pulldowns to monitor levels of active Arf6 (second panel). Asterisk denotes a nonspecific band that derives from the GST-GGA3 affinity reagent. Whole cell lysates were immunoblotted for total levels of Arf6 and TRE17. C, MC3T3 cells stably expressing the indicated TRE17 mutants were treated with dox for 24 h and then photographed using a Nikon Eclipse TE2000 inverted microscope; 40× magnification.

FIGURE 3.

TRE17 blocks osteoblastic maturation in a USP-dependent manner. A, MC3T3 cells stably expressing the indicated TRE17 alleles were grown in differentiation-inducing medium in the presence of dox for 5 days. Cells were fixed, and ALP activity was monitored by staining with BCIP/NBT. vec, vector; USP−, TRE17(long)/USP−; A6−, Arf6 binding-deficient mutant. B, ALP activity was measured using a spectrophotometric-based assay with p-nitrophenyl phosphate substrate as described under “Experimental Procedures.” Data represent the mean ± S.D. of eight experiments; *, p < 0.05; **, p < 0.01. vect, vector. C, RNA was isolated from MC3T3 cells expressing the indicated TRE17 mutants, grown under differentiation-inducing conditions for 5 days. RT-PCR was performed using primers against ALP, BSP, and Col1A1. D, real time PCR was performed to quantify changes in gene expression, using the ABI Prism 7900 system. For each gene, two independent primer pairs were used (denoted ALP-1 and ALP-2, etc.), and expression was normalized against GAPDH levels. E, MC3T3 cells co-expressing TRE17(long) and TRE17(short) were blotted with anti-TRE17, and ALP activity was monitored by BCIP/NBT staining.

FIGURE 4.

Microarray analysis reveals broad dysregulation of pathways controlling maturation in TRE17(long)-expressing cells. A, control vector (vect)-expressing MC3T3 cells were incubated with CM from MC3T3 cells expressing the indicated TRE17 allele for 5 days under differentiation conditions, and ALP activity was measured. Data represent the mean ± S.D. of three experiments. Statistically significant differences relative to cells treated with control CM are indicated; *, p < 0.05; **, p < 0.01. USP−, TRE17(long)/USP−; A6−, Arf6 binding-deficient mutant. B, heat map showing signaling pathways with altered expression of multiple components in MC3T3 expressing TRE17(long), but not TRE17(long)/USP−. Decreases and increases in gene expression are indicated in green and red, respectively. -Fold changes in expression for individual genes are specified in the table. vec, vector; osteoblast diff., osteoblast differentiation; osteo, osteoblast. C, real time PCR was performed to confirm expression changes in select genes identified from the microarray. For each gene, two independent primer pairs were used (denoted −1 and −2 and), and expression was normalized against GAPDH levels.

FIGURE 5.

BMP pathway is dysregulated in TRE17(long)/MC3T3 and can be rescued by exogenous BMP-4. A, RNA was isolated from MC3T3 cells expressing the indicated TRE17 alleles under differentiation-inducing conditions, in the absence or presence of dox as indicated. RT-PCR was performed using primers against Grem1 and BMP-4 (left panel and right panel, respectively). Real time PCR was also performed to quantify changes in expression (graphs). For each gene, two independent primer pairs were used (denoted −1 and −2), and expression was normalized against GAPDH levels. vec, vector; USP−, TRE17(long)/USP−. B, MC3T3 cell lines were treated with dox under serum-free conditions. The conditioned medium were precipitated with trichloroacetic acid and then subjected to immunoblotting with anti-BMP-4. Purified recombinant BMP-4 (reBMP4), which has a slightly reduced electrophoretic mobility, was used as a positive control for blotting. A6−, Arf6 binding-deficient mutant. C and D, TRE17(long) or vector control MC3T3 were grown in differentiation medium, in the presence or absence of dox and the indicated concentration of recombinant BMP-4 for 5 days. In C, real time PCR was performed to quantify ALP expression levels, using two independent primer pairs and normalizing against GAPDH. In D, ALP activity was measured by BCIP/NBT staining.