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By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility, invasion and lung colonization.

Interstitial fluid flow and associated shear stress are relevant mechanical signals in cartilage and bone (patho)physiology. However, their effects on chondrosarcoma cell motility, invasion and metastasis have yet to be delineated. Using human SW1353, HS.819.T and CH2879 chondrosarcoma cell lines as model systems, we found that fluid shear stress induces the accumulation of cyclic AMP (cAMP) and interleukin-1β (IL-1β), which in turn markedly enhance chondrosarcoma cell motility and invasion via the induction of matrix metalloproteinase-7 (MMP-7). Specifically, shear-induced cAMP and IL-1β activate PI3-K, ERK1/2 and p38 signaling pathways, which lead to the synthesis of MMP-7 via transactivating NF-κB and c-Jun in human chondrosarcoma cells. Importantly, MMP-7 upregulation in response to shear stress exposure has the ability to promote lung colonization of chondrosarcomas in vivo. These findings offer a better understanding of the mechanisms underlying MMP-7 activation in shear-stimulated chondrosarcoma cells, and provide insights on designing new therapeutic strategies to interfere with chondrosarcoma invasion and metastasis.


INTRODUCTION
Matrix metalloproteinase-7 (MMP-7) has a broad proteolytic activity against a number of extracellular matrix (ECM) substrates, such as casein, collagens, fibronectin, laminin, elastin, entactin, cartilage proteoglycans, etc [1]. Accumulating evidence suggests that MMP-7 is necessary for physiological processes associated with tissue remodeling, such as embryonic development, wound healing, trophoblast implantation, and organ morphogenesis [2,3]. In addition, the enzymatic activity of MMP-7 in degrading ECM potentially contributes to the invasion and metastasis of tumor cells by increasing proliferation, by hydrolyzing the ECM and promoting migration and angiogenesis, and by affecting cell apoptosis [4][5][6]. Along these lines, Sugita et al. [7] reported that the imunnostaining score of MMP-7 increases with the histological grade of chondrosarcomas. MMP-7 may thus serve as a useful indicator in the diagnosis of chondrosarcoma [8]. However, the mechanism of MMP-7 regulation and its biological functions in chondrosarcoma metastasis in vivo remain unclear.
Oncotarget 9141 www.impactjournals.com/oncotarget The major signaling pathway found to regulate MMP-7 expression in larynx carcinoma is epithelial growth factor receptor (EGFR) signaling pathway [9]. PI3-K/AKT signaling pathway mediated the upregulation of MMP-7 via FoxO1-activating manner in EGFstimulated Hep-2 cells [9]. Moreover, Shi et al. [10] reported that MMP-7 expression was upregulated by activating AP-1 and stat3 in catecholamine-stimulated human gastric cancer cells, which promote the invasion and metastasis of gastric cancer. Apart from AP1 and stat3, the forkhead box transcription factor (FOXC1) was identified to mediate MMP-7 upregulation in human breast cancer cells [11].
Interstitial fluid flow is a relevant mechanical signal in cartilage and bone (patho)physiology [12]. Interstitial fluid flow and associated fluid shear stress induce MMP-1 and MMP-12 expression in human chondrosarcoma cells [13,14]. Although MMP-7 is elevated in advanced stages of chondrosarcoma disease [7] as well as in shear-activated chondrosarcoma cells [13], the signaling mechanism by which MMP-7 activation may contribute to chondrosarcoma invasion and metastasis has yet to be investigated. We herein demonstrate that fluid shear stress stimulates the synthesis of cAMP and IL-1β, which in turn activate PI3-K/AKT, ERK1/2 and p38 mitogen-activated protein kinase (MAPK)-dependent pathways. PI3-K, ERK1/2 and p38 transactivate the phosphorylation of c-Jun and NF-κB, which in turn bind to the MMP-7 promoter and mediate MMP-7 mRNA and protein synthesis in human shearstimulated chondrosarcoma cells. We further demonstrate the contribution of MMP-7 activation to lung colonization in vivo which may help us gain insights into therapeutic strategies aiming to combat chondrosarcoma metastasis.

MMP-7 is upregulated in human chondrosarcoma tissues and shear-activated chondrosarcoma cells
Prior work revealed that MMP-7 is detected in human chondrosarcoma but not normal cartilage [7]. Because of the limited number of tissue specimens (a total of 28) examined in the previous study [7], we first wished to confirm these data. Consistent with prior findings [7], MMP-7 immunostaining was markedly elevated (~3-fold) in human chondrosarcoma tissues relative to normal bone controls (Fig.1A). Given that interstitial fluid flow and associated fluid shear stress are relevant mechanical signals in cartilage and bone (patho)physiology, we next evaluated the effects of fluid shear on MMP-7 expression in human chondrosarcoma cells, using SW1353, HS.819.T and CH2879 chondrosarcoma cell lines as model systems.
Our data reveal that cell exposure to a fluid shear stress level of 2 dyn/cm 2 for 48 h markedly induced the MMP-7 mRNA expression and activity in SW1353 and HS.819.T cells (Figs. 1B, 1C). In light of these observations, we examined whether shear stress has ability to promote lung colonization of human chondrosarcoma cells in vivo. To this end, shear-activated CH2879 chondrosarcoma cells along with their shear-conditioned medium were injected via the tail vein into NOD/SCID/IL2 receptor gamma knockout mice; mice were sacrificed 35 days following tail-vein injection. H.E. staining was used to determine the number of micrometastases, whereas qPCR was used to quantify the content of human DNA (and thus tumor colonization) in the lungs of mice. The results reveal that solid tumors were formed in the lungs of mice after 35 days (Fig. 1D). The extent of lung colonization was significantly higher in shear-activated versus static control chondrosarcoma cells. These data suggest that fluid shear stress may potentially contribute to chondrosarcoma invasion and metastasis via induction of MMP-7.
cAMP and IL-1β regulate the shear-dependent upregulation of MMP-7 via activation of PI3-K/ AKT, ERK1/2 and p38 pathways in human chondrosarcoma cells We next aimed to delineate the signaling cascade of MMP-7 induction in shear-activated chondrosarcoma cells. Prior work has shown that exogenously added IL-1β upregulates MMP-7 expression in human LNCaP prostate cells and articular chondrocytes [15,16], whereas cAMP can induce various MMPs in diverse cell types [17]. Interestingly, we recently reported that fluid shear increases the accumulation of both cAMP and IL-1β in human chondrosarcoma cells [14]. Thus, we examined the potential roles of cAMP and IL-1β in MMP-7 expression and activity in sheared SW1353 cells. Incubation of SW1353 chondrosarcoma cells with either an adenylate cyclase inhibitor, SQ22536 (10 μM) or an anti-IL-1β antibody (1 μg/ml) just prior to the onset of shear stress exposure abolished shear-induced MMP-7 mRNA synthesis and activity ( Fig. 2A).
To validate the involvement of these signaling pathways in MMP-7 regulation in sheared chondrosarcoma cells, experiments were performed by transfecting SW1353 cells with siRNAs targeting AKT, ERK1/2 or p38. These molecular interventions effectively knocked down the protein expression of AKT, ERK1/2 or p38 relative to cells transfected with a scramble siRNA control (Figs. 2C, S1C). Knockdown of AKT, ERK1/2 or p38 markedly repressed MMP-7 mRNA expression and enzymatic activity (Fig. 2C). anti-MMP-7 antibody. Strong immunostaining was detected in ~90% of cancerous tissues, whereas no immunostaining was present in normal bone tissues. SW1353 B. or HS.819.T cells C. were subjected to fluid shear stress (2 dyn/cm 2 ) or static conditions (0 dyn/cm 2 ) for 48 h. MMP-7 mRNA and protein levels were determined by qRT-PCR and zymography, respectively. GAPDH and MMP-11 total protein amount served for internal control in qRT-PCR and zymography assays, respectively. D. CH2879 cells were exposed to either static or shear (2 dyn/cm 2 ) conditions for 48 h. Cells suspended in their corresponding conditioned medium were injected into mice via tail vein (t = 0 weeks); the conditioned medium was injected via the tail vein every 3 days for 5 weeks. The right lung lobes from each animal were fixed, stained with hematoxylin and eosin, and examined for signs of lung micrometastases (indicated by arrowheads) (upper panel). Quantification of the number of micrometastases present in lungs of mice following tail vein injection in the absence or presence of staticor shear-conditioned medium as described above; n = 10 mice per group (lower left panel). Presence of human DNA quantified in lungs of mice injected with CH2879 chondrosarcoma cells via qPCR of hLINE-1 DNA. n = 10 mice per group. Data represent the mean ± S.E. of 3 independent experiments. *p < 0.05 with respect to normal bone tissues or static controls.

c-Jun and NF-κB regulate shear-induced MMP-7 mRNA synthesis in human chondrosarcoma cells
We next sought to decipher the critical transcription factor(s) responsible for MMP-7 synthesis in sheared chondrosarcoma cells. The promoter of mmp-7 gene contains several consensus sequences, including those for AP-1 and NF-κB [19,20]. Because of the elevated AKT, ERK1/2 and p38 phosphorylation levels in shear stress-, forskolin-and IL-1β-stimulated chondrosarcoma cells, we examined the potential contributions of AKT, ERK1/2 and p38 to regulating the activities of c-Jun and NF-κB. Application of fluid shear to human SW1353 cells induces phosphorylation of c-Jun at Ser 63 and p65 at both Ser 536 and Ser 276 (Figs. 3A, S2A). Cell treatment with SQ22536 (10 μM) or an anti-IL-1β antibody (1 μg/ml) repressed the shear-induced phosphorylation of c-Jun and NF-κB down to basal levels (Figs. 3A, S2A). Akin inhibitory effects on the phosphorylation of c-Jun were noted in shear stress-, forskolin-or IL-1β-activated SW1353 cells that were pre-treated with LY294002   To establish the involvement of c-Jun and NF-κB in the regulation of shear-induced MMP-7 mRNA synthesis, a series of MMP-7 promoter constructs were generated using the luciferase reporter plasmid, pGL3-basic vector (Fig. 4A, left panel). As a first step, SW1353 cells were transiently transfected with a construct encompassing the 5'-flanking region of the human MMP-7 gene from −1997 to +39 bp prior to their exposure to fluid shear for 48 h. Shear stress induced a pronounced (~8 fold) increase in the MMP-7 promoter activity in SW1353 cells (Fig. 4A). A similar 8-fold upregulation was detected upon transfection of cells with plasmids containing deletions from −1997 to −1597 bp (−1597/+39), thereby suggesting that DNA region between −1997 and −1597 bp upstream of the transcriptional start site is not crucial to the induction of shear-induced MMP-7 promoter activity (Fig. 4A). However, subsequent deletion from −1597 to −1197 bp (−1597/+60) markedly diminished the luciferase activity (Fig. 4A). Bioinformatics analysis of the consensus sequence in (−1597/−1197) region revealed the presence of a NF-κB site, which may be responsible for shear-induced MMP-7 synthesis. Indeed, introduction of a point mutation into one of the aforementioned NF-κB binding site (−1507/−1498) significantly diminished shear-activated luciferase activity relative to the reported wild type (Fig. 4B). Using similar experimental methods, we identified another AP-1 binding site (−68/−58), which is important for shear-induced MMP-7 transcription (Figs. 4A, 4B).
The potential involvement of NF-κB and c-Jun in the induction of MMP-7 in shear-activated chondrocytes was next disclosed by gel and supershift assay. Incubation of nuclear extracts from sheared versus untreated SW1353 chondrosaroma cells with the biotinylated c-Jun or NF-κB probe leads to the formation of the c-Jun or NF-κB specific DNA-protein complex (Fig. 4C). Furthermore, incubating nuclear extracts from shearactivated SW1353 cells with an anti-c-Jun or anti-p65 antibody prior to the addition of c-Jun or NF-κB probe results in a marked supershift of the complex (Fig. 4D).

Shear-induced MMP-7 promotes chondrosarcoma cell motility and invasion
Prior work has suggested that MMP-7 correlates with the degree of malignancy in human chondrosarcoma [7]. We and others have reported the involvement of MMP-7 in the migration and invasion of different tumor cell types [7,8,21]. We herein sought to delineate the mechanism by which fluid shear stress potentiates chondrosarcoma cells motility and invasion. In view of our observations showing that endogenous cAMP and In the ChIP assays, the anti-RNA polymerase II antibody was used as a positive control. DNA purified from immunoprecipitated (IP) and preimmune (Input) specimens was subjected to qPCR amplification using primers for the mmp-7 promoter. All experiments are representative of three independent experiments, all revealing similar results (E-J) The data represent the means ± S.E. of three independent experiments. *p < 0.05 with respect to the static or vehicle treatment control. #p < 0.05 compared with fluid shear stress, forskolin or IL-1β treatment alone.
Oncotarget 9148 www.impactjournals.com/oncotarget IL-1β regulate shear-induced MMP-7 activation, we examined the potential effects of exogenously added forskolin (10 μM) and IL-1β (100 ng/ml) on SW1353 cell motility and invasion. Both forskolin and IL-1β augmented SW1353 cell motility and invasion as shown in Transwell assays (Figs. 5A-5D). Interestingly, incubation of SW1353 cells with the PI3-K inhibitor LY294002 (10 μM), the ERK1/2 inhibitor U0126 (10 μM), the p38 inhibitor SB203580 (10 μM), the JNK inhibitor SP600125 (10 μM) or the NF-kB inhibitor QNZ (2 μM) abrogated the ability of forskolin or IL-1β (100 ng/ml) to augment SW1353 cells migration and invasion (Figs. 5A-5D). To further establish the relationship between shear stress and tumor migration and invasion, shear conditioned medium was collected and used as chemoatractant. The results reveal that sheared medium markedly increased the migration and invasion of human SW1353 cells (Figs. 5E, 5F). In light of these findings, we also assessed the potential contribution of MMP-7 to the migration and invasion of human chondrosarcoma cells. In select experiments, SW1353 cells were exposed to either static or shear (2 dyn/cm 2 ) for 48 h. Cells suspended in their corresponding conditioned medium were then subjected to transwell experimens E, F. In separate experiments, SW1353 cells were incubated with rhMMP-7 (1 μg/ml) or vehicle control before being seeded to the transwell device G, H. In distinct experiments, SW1353 cells were transfected with either MMP-7 cDNA or the empty vector before being seeded to transwell device I, J. *p < 0.05 with respect to static-, vehicle-treated or vector-transfected control cells. #p < 0.05 as compared with forskolin or IL-1β treated cells. www.impactjournals.com/oncotarget As a first step, we treated human SW1353 cells with rhMMP-7 (1 μg/ml). The results indicate that treatment of SW1353 cells with rhMMP-7 increased their motility and invasion in vitro (Figs. 5G, 5H). Of note, MMP-7 overexpression by MMP-7 cDNA plasmid transfection also enhanced the migration and invasion of human SW1353 cells. To extend these in vitro observations to the in vivo setting, mice were injected via tail vein with CH2879 chondrosarcoma cells treated with rhMMP-7 or transfected with either MMP-7 cDNA or the empty vector. rhMMP-7 treatment or ectopic expression of MMP-7 markedly increased lung colonization in vivo, as evidenced by the higher number of micrometastases and higher human DNA content in the lung of mice (Figs. 6A,  6B). To further support and confirm these in vivo data, live animal imaging experiments were carried out and

DISCUSSION
MMP-7 is one of the few MMPs that is secreted by tumor cells, but not stromal cells [22]. MMP-7 expression [7,23,24] correlates with the degree of malignancy in chondrosarcoma [7]. Interestingly, MMP-7 is localized at the invasive protrusions of tumor cells [7]. In line with these observations, we found that MMP-7 is overexpressed in human chondrosarcoma relative to appropriate normal controls. MMP-7 expression is also upregulated in shearactivated chondrosarcoma cells. In view of these findings and given that fluid shear is a relevant biomechanical signal in bone and cartilage (patho)physiology, we herein delineated the signaling pathways of MMP-7 induction in shear stress-activated human chondrosarcoma cells. Moreover, we report the critical roles of shear stress in promoting chondrosarcoma lung colonization in mice via the induction of the enzymatic activity of MMP-7 (Fig. 7).
Interstitial fluid flow in bone and cartilage is driven not only by mechanical loading but also by vascular pressure differences. Vascular pressure-driven interstitial fluid flow exists at all times in bone and cartilage because of transcortical pressure gradients [25], and is expected to be elevated in tumors in cancer patients [26]. Although interstitial flow moves around the cell-matrix interface in all directions rather than only on the apical side, Oncotarget 9151 www.impactjournals.com/oncotarget both interstitial flow and shear fluid flow can drive cell responses via mechanical (i.e., shear stress on the cell surface) and non-mechanical effects (i.e., transport effects). It is noteworthy that shear fluid flow studies have been widely performed to investigate the mechanobiology of bone and cartilage-derived cells [25,27].
MMPs have been implicated in the malignant transformation of human chondrocytes, and their expression is associated with chondrosarcoma recurrence [28,29]. MMPs also promote the invasive potential of diverse tumor cell types [30][31][32]. Recent work reveals a link among fluid shear stress, MMP induction and cell invasion. For instance, Qazi et al. [33] showed that fluid shear stress enhances the invasive potential of glioma cells via regulating the activity of MMP-1 and MMP-2. We recently reported that shear-induced MMP-12 promotes the invasion and lung colonization of human chondrosarcoma cells [13]. High fluid shear stress has also the ability to modulate the mesenchymal transformation and invasion of aortic valve endothelial cells [34]. In line with these observations, Milkiewicz et al. [35] further established the link between shear stress and angiogenesis via stimulating the expression of VEGF in Sprague-Dawley rats. Besides tumor cells, fluid shear stress has ability to modulate the endothelial cell invasion into three-dimensional collagen matrices [36]. In agreement with these prior reports, our data reveal that shear stress exposure increases the lung colonization of human chondrosarcoma cells via MMP-7 activation (Fig. 1D). MMP-7 is tightly regulated under physiological conditions and is elevated during the course of chondrosarcoma mesenchymal transformation [7]. Powell et al. [37] reported that MMP-7 overexpression increases the potential invasion of human prostate cancer cells (DU-145) into SCID mice. Witty et al. [38] also detected differences in the invasive potentials of MMP-7-transfected human colon cancer cells versus control cells. These in vitro findings were further corroborated by clinical observations showing that MMP-7 was overexpressed in six out of eight colon carcinomas [39]. Although there is no further evidence suggesting the direct relationship between MMP-7 and metastasis, a clear involvement of MMP-7 has been shown in tumor growth, invasion and spread via degrading its substrates including casein, elastin, laminin, proteoglycans, osteopontin, fibronectin and type IV collagen [40][41][42]. Nevertheless, MMP-7 might not regulate tumor cell metastasis via direct degradation of ECM. In some cases, human pro-MMP1, 2, and 9 can be activated by MMP-7, which further facilitate the metastasis of tumor cells [43][44][45]. Our data reveal Oncotarget 9152 www.impactjournals.com/oncotarget that rhMMP-7 promotes the lung colonization of human chondrosarcoma cells (Fig. 7A). This finding was further substantiated by data showing that MMP-7 cDNA plasmid transfection enhanced the invasion and metastasis of human chondrosarcoma cells (Fig. 7B). Along these lines, MMP-7 activation may function as powerful machinery for ECM degradation, which facilitates local invasion and metastasis.
The underlying mechanisms of MMP-7 activation were not fully deciphered in previous investigations. We herein demonstrate the pivotal role of endogenous cAMP in fluid shear stress-induced MMP-7 synthesis, which was substantiated by the use of adenylate cyclase inhibitor SQ22536 that essentially abrogated MMP-7 induction in shear-activated chondrosarcoma cells (Fig. 2A). In line with our results, Hanemaaijer et al. [17] reported that forskolin affected the production of MMPs in a celltype-specific way. However, they did not extend their study to MMP-7. Apart from cAMP, IL-1β has a critical role in inducing the expression of MMP-7 in shearactivated human chondrosarcoma cells (Fig. 2E). In line with our data, Klein et al. [15] demonstrated that IL-1β secreted from monocytic cells increases the expression of MMP-7 in the prostatic cell line LNCaP. Moreover, IL-1β is capable of inducing MMP-7 expression in human articular chondrocytes [16]. Therefore, both cAMP and IL-1β are key signaling molecules in mediating shear-induced MMP-7 expression in human chondrosarcoma cells.
We and others [14] have identified the two major MAPKs (ERK1/2, and p38) and PI3-K in transmitting cAMP- [46][47][48] and IL-1β-dependent signaling [47,49], which result in MMP synthesis [50,51]. In light of these findings [46][47][48][49]52], we evaluated the potential contributions of these signaling pathways to shear-induced MMP-7 synthesis. Fluid shear stress activates PI3-K/AKT, ERK1/2, and p38 pathways in human chondrosarcoma cells via a cAMP-and IL-1β-dependent mechanism, as evidenced by the inhibition of AKT, ERK1/2 and p38 phosphorylation by a cAMP inhibitor and an anti-IL-1β neutralizing antibody. In addition, activation of PI3-K/ AKT, ERK1/2, and p38 pathways leads to synthesis of MMP-7 in human SW1353 chondrosarcoma cells. In agreement with our data, several studies suggest that the ERK1/2 signaling pathway plays a key role in regulating MMP-7 expression. Kawabata et al. [53] demonstrated that transfection of ERK1/2 siRNA led to a significant reduction of pro-MMP-7 protein production in human HT-29 colorectal adenocarcinoma cells. In concert with this finding [53], ERK1/2 activation induces MMP-7 expression in human brain gliomas [54], colon [55] and pancreatic cancer cells [21,56]. ERK1/2 activation could provide a source for upregulation of MMP-7 expression in human chondrosarcoma cells, but ERK1/2 activation may not be sufficient to achieve full activity of MMP-7. Apart from ERK1/2, PI3-K [57] and p38 [55,58] have been involved in MMP-7 regulation in rat aortic vascular smooth muscle cells, human colon cancer and LOVO cells. Our data using pharmacological inhibitors and siRNA sequences specific for PI3-K, ERK1/2 and p38 reveal the pivotal role of these signaling pathways in mediating the effects of fluid shear stress on MMP-7 induction.
In light of our previous work showing that exposure of human chondrocytes to fluid shear stress transactivates the NF-κB p65 subunit [59,60] and given that the expression of MMP-7 may be dependent on NF-κB [61], we evaluated the potential role of the NF-κB pathway in MMP-7 induction in human chondrosarcoma cells. We determined that fluid shear induces NF-κB phosphorylation at both Ser-276 and Ser-536. Moreover, inhibition of NF-κB markedly suppresses shear-induced MMP-7 expression, thereby establishing that the key role of canonical NF-κB pathway in this process. This finding is in concert with an earlier report suggesting a key role for NF-κB in the regulation of MMP-7 expression induced by helicobacter pylori infection in human gastric adenocarcinoma cells [62]. However, the NF-κB site may not be sufficient to achieve full transcription activity of MMP-7 synthesis. AP-1 (activator protein-1, formed by heterodimers of the two protooncogene families c-Jun and c-fos) has a binding site at the promoter region of MMP-7, which may cooperate with the NF-κB element to mediate fluid shear stress-dependent stimulation of MMP-7 transcriptional activity (Fig. 7). We herein report for the first time that both transcriptional factors are significantly stimulated by fluid shear stress, and in turn bind to their respective cis-elements, thereby inducing MMP-7 expression. This finding is further corroborated by our observations showing that PI3-K, ERK1/2 and p38 are able to regulate the phosphorylation of both NF-κB and c-Jun. In line with our data, c-Jun was also phosphorylated by PI3-K [63], ERK1/2 [64] and p38 [65] in human bronchial epithelial cells, mouse embryonic fibroblasts and human T lymphocytes. In addition, AP-1 has shown its pivotal roles in inducing MMP-7 expression in human gastric cancer cells [10]. Through the use of pharmacological inhibitors, promoter constructs, and ChIP assays, we demonstrated here the functional role of the NF-κB p65 subunit and c-Jun in shear-induced MMP-7 expression in human chondrosarocma cells.
In summary, we have elucidated the signaling pathway by which fluid shear stress upregulates the expression of MMP-7 in human chondrosarcoma cells.

Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR assays were performed on the iCycler iQ detection system (Bio-Rad) using total RNA, the iScript one-step RT-PCR kit with SYBR green (Bio-Rad) and primers. The GenBank accession numbers and forward (F-) and reverse (R-) primers are as follows: MMP-7 (NM_002423), F-ATGTGGAGTGCCA GATGTTG, R-GCCAATCATGATGTCAGCAG; The GenBank accession numbers and forward (F-) and reverse (R-) primers for GAPDH are provided in our previous publications [59,69]. GAPDH was used as internal control. Reaction mixtures were incubated at 50°C for 15 min followed by 95°C for 5min, and then 35 PCR cycles were performed with the following temperature profile: 95°C 15s, 58°C 1 min, 68°C 1 min, 77°C 20s. Data were collected at the (77°C 20 s) step to remove possible fluorescent contribution from dimer-primers [59,69]. Gene expression values were normalized to GAPDH.

Western blot analysis
SW1353 or HS.819.T cells from static and sheared specimens were lysed in a radioimmune precipitation assay buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a cocktail of proteinase inhibitors (Pierce Chemical Company). The protein content of the cell lysates was determined using bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Company). Total cell lysates (4 μg) were subjected to SDS-PAGE, transferred to a membrane, and probed with a panel of specific antibodies. Each membrane was probed using one antibody only. β-actin was used as loading control. All Western hybridizations were performed at least in triplicate using a different cell preparation each time.

Measurement of cAMP and IL-1β concentration in medium
cAMP and IL-1β levels in both static and sheared medium were determined using the corresponding kits following the manufacturer's instructions [14]. The concentration of total protein in the medium was used as loading control, and the results were expressed as pmol cAMP or pg IL-1β per μg of total protein.

Transfection
SW1353 chondrosarcoma cells were transfected with 100 nM of a siRNA oligonucleotide sequence specific for AKT, ERK1/2 or p38. In control experiments, cells were transfected with 100 nM of scramble siRNA. In promoter assays, SW1353 chondrosarcoma cells were transfected with 1.6 μg/slide of the MMP-7 promoter reporter construct together with the pRL-SV40 vector. Transfected cells were allowed to recover for at least 12 h in growth medium, and then incubated overnight in medium containing 1% Nutridoma-SP before their exposure to shear stress. In live animal imaging experiments, CH2879 cells were transfected with www.impactjournals.com/oncotarget MMP-7 cDNA-mcherry plasmids and further established stable transfected cell lines for tail vein injection.

Promoter assay
Firefly and Renilla luciferase activities were measured by use of the Dual-Luciferase Report Assay kit (Promega). Firefly luciferase activities were normalized to the Renilla luciferase controls. Data are expressed as ratios of shear to static normalized firefly luciferase activity unless otherwise stated.

EMSA and supershift assay
A 5′-biotinylated oligonucleotide probes (5′-GGAAATGCCTT-3′; 5′-ATGAGTCACCT-3′) were synthesized containing the NF-κB and c-Jun cis-element present on the MMP-7 promoter. EMSAs were performed with a commercially available nonradioisotopic EMSA kit (LightShift Chemiluminescence EMSA kit; Pierce). Briefly, nuclear extracts (1-2 μg) were incubated in 10 × binding buffer (supplemented with 50 ng of poly (dI-dC), 2.5% glycerol, 0.05% Nonidet P-40, 5 mM MgCl 2 , and 0.25 mg of bovine serum albumin), containing 20 fmol of biotinylated, double-stranded probes for NF-κB or c-Jun for 30 min on ice. For competition binding, a 200-fold excess of unlabeled (cold) probe was incubated with nuclear extracts before the inclusion of the biotinylated one. For supershift assays, the nuclear extracts were preincubated for 30 min on ice with an anti-p65 antibody. The biotinylated oligonucleotide probe specific for NF-κB or c-Jun was then added to the reaction mixture and incubated for another 30 min on ice. To exclude the possibility of nonspecific binding, a 5′-biotinylated random probe (5′-AGTCCGTTGAA-3′; 5′-GAGCATTATCC-3′) designed using a random sequence generator was used in shift and supershift assays. The protein-DNA complexes were resolved on a native 6% polyacrylamide retardation gel in 0.5 × Tris borate-EDTA running buffer at 10 mA for 1 h, transferred to a nylon membrane (Pierce), visualized using the LightShift Chemiluminescence kit (Pierce) and exposed to Kodak x-ray film (Pierce).
In select experiments, SW1353 cells were treated rhMMP-7 (1 μg/ml) or vehicle controls. The lower chamber contains medium supplemented with 10% FBS, which serves as chemoattractant. 48 h later, cells on the upper side were removed with a cotton swab. Then, the membrane was fixed with 3.7% formaldehyde and stained with 0.1% crystal violet in PBS (-). Under low magnification microscope (× 100), five horizons (up, down, left, right, center) were examined on each membrane. The number of cells went through the polycarbonate membrane was counted and averaged for each field of vision to indicate the migratory ability of tumor cells. In invasion experiments, the polycarbonate membrane was pre-coated with Matrigel (Becton Dickinson, Bedford, MA, USA) before seeding SW1353 cells in the upper inserts. Following the same protocol as described above, the number of cells, which went through the matrigel, is indicative of their invasive capacity.

Immunohistochemistry
Human bone tissue microarrays (US Biomax Inc., Rockville, MD, USA) included 69 chondrosarcoma tissue cores and 24 normal bone tissue cores were fixed on tissue microarray slides. Slides were first deparaffinized with xylene, rehydrated in a graded series of ethanol and submerged in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. MMP-7 level was determined using immunohistochemical staining kit, following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).

Tail vein injections
NOD/SCID/IL2 receptor gamma knockout mice (10 per experimental group; obtained from Jackson Lab, Strain 005557) were injected via the tail vein with 10 5 CH2879 chondrosarcoma cells in a volume of 100 μl of serum free medium followed by tail vein injection with either recombinant human MMP-7 (100 μl of 10 μg/ml) or vehicle control at the day of the cell injection (t = 0 weeks) and weekly thereafter for 5 weeks. In select experiments, cells were transfected with MMP-7 cDNA or MMP-7 cDNA-mcherry plasmids before injecting to the tail vein of mice. In other experiments, CH2879 cells, exposed to either static or shear (2 dyn/cm 2 ) conditions for 48 h, were suspended in their corresponding conditioned medium and injected into mice via tail vein (t = 0 weeks); the conditioned medium was injected via the tail vein every 3 days for 5 weeks. Mice were euthanized 5 weeks post cell injection.

Luciferase assays and live animal imaging
The CH2879 cells transfected with MMP-7 cDNAmcherry plasmids were injected to mice via tail vein. After 35 days post cell injection, mice were anesthetized and scanned using Bruker in vivo imaging systems (MS FX PRO, Carestream, U.S.A).

Histopathology of lung tissue
Lung samples for pathology were fixed in 10% buffered formalin. The left lung lobe was used for hLINE analysis and the remaining lobes were embedded in a single cassette and sectioned for histopathological analysis of metastatic foci [74,75]. Samples were sectioned by cryostats at 10 μm, and stained with hematoxylin and eosin using standard techniques. Tissue collection, histopathology analysis, and grading were performed by a pathologist (DLH). Metastatic foci were counted in 3 lung histology sections from each mouse and scored as follows: no lung colonization = 0 foci; mild = 1-10 foci; moderate = 11-20 foci; severe > 20 foci.

Statistics
Data represent the mean ± S.E. of at least 3 independent experiments. Statistical significance of differences between means was determined by Student's