uPA‐derived peptide, Å6 is involved in the suppression of lipopolysaccaride‐promoted inflammatory osteoclastogenesis and the resultant bone loss

Abstract Introduction Chronic inflammatory diseases such as rheumatoid arthritis and periodontitis frequently cause bone destruction. Inflammation‐induced bone loss results from the increase of bone‐resorbing osteoclasts. Recently, we demonstrated that urokinase type plasminogen activator (uPA) suppressed lipopolysaccaride (LPS)‐inflammatory osteoclastogenesis through the adenosine monophosphate‐activated protein kinase (AMPK) pathway, whereas its receptor (uPAR) promoted that through the Akt pathway. Methods We investigated the effects of uPA‐derived peptide (Å6) in the LPS‐induced inflammatory osteoclastogenesis and bone destruction. Results We found that Å6 attenuated inflammatory osteoclastogenesis and bone loss induced by LPS in mice. We also showed that Å6 attenuated the LPS‐promoted inflammatory osteoclastogenesis by inactivation of NF‐κB in RAW264.7 mouse monocyte/macrophage lineage cells. Furthermore, we showed that Å6 attenuated the Akt phosphorylation, and promoted the AMPK phosphorylation. Conclusion Å6 is involved in the suppression of LPS‐promoted inflammatory osteoclastgensis and bone destruction by regulating the AMPK and Akt pathways. These findings provide a basis for clinical strategies to improve the bone loss caused by inflammatory diseases.


Introduction
Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) have the proteolytic function which converts plasminogen (Plg) to plasmin [1], and then plasmin not only degrade fibrin and any ECM proteins but also activate matrix metalloproteinases and growth factors [2]. uPA and uPAR also promotes the intracellular signaling by the interaction with transmembrane proteins such as integrins, and mediate cellular adhesion, differentiation, proliferation, and migration [3]. These functions of uPA and uPAR are associated with the development of inflammatory diseases such as rheumatoid arthritis, periodontitis, cancer, and fibrosis [4][5][6][7][8][9][10]. Recently, we demonstrated that uPA suppressed inflammatory osteoclastogenesis through the adenosine monophosphate-activated protein kinase (AMPK) pathway [11]. Conversely, uPAR promoted inflammatory osteoclastogenesis through the Akt pathway, and the blocking of uPAR attenuated them [12]. Å 6 is an 8-mer capped peptide derived from uPA (amino acid 136-143, KPSSPPEE), and inhibits the interaction of uPA with uPAR [13]. It has been reported that Å 6 inhibits tumor growth, tumor metastasis and angiogenesis [13][14][15]. Å 6 also inhibits hypoxia-induced retinal neovascularization and choroidal neovascularization [16,17]. Several clinical studies have been shown that Å 6 was well tolerated, and no toxicity in Phase 1 and 2 clinical trials [18][19][20][21]. Although the molecular mechanism of Å 6 remains to be clarified, the inhibition of uPA and uPAR interaction by Å 6 may affect the amelioration of various diseases.
We herein reported the effects of uPA-derived peptide, Å 6 on the lipopolysaccaride (LPS)-induced inflammatory OC formation and the resultant bone loss.

Material and Methods
The animal experiments in this study were approved by the Animal Research Committee of Doshisha Women's Collage of Liberal Arts (Approval ID: Y15-025). All experiments were performed in accordance with relevant guidelines and regulations.

Animals
C57B6J mice littermates were housed in groups of two to five in filter-top cages with a fixed 12 h light, 12 h dark cycle.
Bone destruction by the administration of LPS in mice LPS (25 mg/kg) and Å 6 (50 mg/kg) were administered subcutaneously into the shaved back of the male mice. The administration was carried out weekly for up to 4 weeks.

Bone histology
Bone histomorphometry of femurs in male mice were performed as previously described [12]. Each femur was removed and fixed in 4% paraformaldehyde for 2 days, and then demineralized with 10% EDTA for 14 days before embedding in paraffin. Paraffin-embedded tissue was serially sectioned at 4-7 mm distances. Then, the sections were stained with TRAP by using TRAP kit (Sigma-Aldrich).
For the quantitative evaluation of the intensity of TRAPstaining in decalcified sections of femurs from the mice, the TRAP-stained images obtained from separate fields on the specimens were analyzed by using ImageJ 1.43u.

Measurement of bone mineral density
Bone mineral density (BMD) was measured as previously described [11,22]. The BMD of femurs from mice at the indicated time was evaluated by using peripheral quantitative computed tomography with a fixed X-ray fan beam of 50-mm spot size, at 1 mA and 50 kVp (LaTheta LCT-100S; Aloka, Tokyo, Japan).
Cell culture and OC differentiation RAW264.7 mouse monocyte/macrophage lineage cells were maintained in a-MEM supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin at 378C in a humidified atmosphere of 5% CO 2 /95% air. OC formation was induced as previously described [11,12]. RAW 264.7 cells were cultured for 3 days with LPS (1 mg/ml) and M-CSF (100 ng/ml) in the absence or presence of Å 6 (100 mM) in 48-well plates.
siRNAs study RAW 264.7 cells were transfected with uPA or uPAR siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. A nonspecific siRNA (Santa Cruz Biotechnology) was employed as the control.

Dual luciferase reporter assay
Dual luciferase reporter assay was performed as previously described [11]. pGL4.32 (luc2P/NF-kB/Hygro) vector contains five copies of NF-kB response element (NF-kB-RE) that Effects of Å6 on inflammatory bone loss Y. Kanno et al.

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derives transcription of the luciferase reporter gene luc2P (Promega, Madison, WI, USA). RAW264.7 cells were cotransfected with pGL4.32 (luc2P/NF-kB/Hygro) vector and the internal control vector pGL4.74 (hRluc/TK) using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. At 24 h posttransfection, the cells were stimulated with described reagents, and then assayed for luciferase activity using the Dual-Glo luciferase assay system (Promega) according to the manufacturer's protocol.

ELISA
RAW 264.7 cells were cultured for 24 h with LPS (1 mg/ml).
After the indicated incubation periods, the conditioned medium was collected, and the TNF-a in the medium was then measured using a TNF-a mouse antibody pair (Invitrogen). The absorbance of the ELISA samples was measured at 450 nm using Multiskan JX (Thermo Labsystems, Waltham, MA).

Statistical analysis
All data are expressed as mean AE SEM. The significance of the effects of each treatment (p < 0.05) was determined by analysis of variance [24] followed by the least significant difference test.

Å6 attenuated inflammatory osteoclastogenesis and bone destruction induced by LPS in mice
To clarify the effects of Å 6 in the inflammatory osteoclastogenesis and bone destruction, we examined the bone mineral density (BMD) in the mice by the administration of lippolysaccaride (LPS), which is a well-known pathogen of inflammatory bone loss [25]. Å 6 attenuated the decrease of BMD induced by LPS (Fig. 1A). Additionally, Å 6 attenuated the increase of TRAP-positive area induced by LPS ( Fig. 1B and C).

Å6 attenuated OC differentiation of macrophage RAW264.7 cells promoted by LPS
We also examined that the effects of Å 6 in LPS-induced the OC differentiation of RAW264.7 cells. RAW264.7 cells simultaneously treated with LPS and M-CSF ( Fig. 2A, center panel) looked more clearly positive against TRAP staining than the cells treated with M-CSF alone ( Fig. 2A, left panel), but did not appear to be typically and predominantly enlarged multi-nuclear cells containing more than three nuclei in each cell like as maturely differentiated OCs stimulated with LPS and M-CSF. In addition, the cell number of TRAP-positive and enlarged

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RAW264.7 cells under the treatment with LPS and M-CSF was moderately decreased by administration of Å 6 ( Fig. 2A, right panel). Additionally, the stimulation with LPS and M-CSF more clearly induced the expression of OC markers, NFATc1 and TRAP than stimulation with M-CSF alone (Fig. 2B). In addition, the LPS-promoted OC differentiation of RAW264.7 cells under the M-CSF treatment was clearly suppressed by administration of Å 6. These data suggest that Å 6 treatment attenuated the LPS-promoted OC differentiation of macrophage under the M-CSF treatment.
Å6 attenuated TNF-a secretion induced by LPS from macrophage RAW264.7 cells It has been reported that LPS-stimulated osteoclastogenesis is mediated by TNF-a [26,27]. To clarify the effects of Å 6 in the LPS-induced TNF-a production, we examined that the TNF-a production in RAW264.7 cells. Å 6 attenuated the LPS-induced TNF-a secretion from RAW264.7 cells (Fig. 3).

Å6 attenuated NF-kB activation induced by LPS in macrophage RAW264.7 cells
We examined the effects of Å 6 on the LPS-induced NF-kB transcriptional activity through the use of a functional promoter assay with NF-kB-responsive element. Å 6 attenuated the LPS-induced NF-kB activation (Fig. 4A). We also confirmed that Å 6 attenuated the LPS-induced IkBa degradation (Fig. 4B). These data strongly suggest that Å 6 inhibited the LPS-activated NF-kB signaling.

Å6 inhibited the Akt pathway, but activated the AMPK pathway
It has been reported that the Akt pathway induces OC differentiation [28,29]. Conversely, adenosine monophosphate-activated protein kinase (AMPK) acts as a negative regulator during OC differentiation [30]. Therefore, we examined whether or not Å 6 is associated with the Akt and AMPK pathways in RAW264.7 cells. We showed that Å 6 attenuated the Akt phosphorylation, but promoted the AMPK phosphorylation (Fig. 5A). Next, we examined that the effects of AMPK inhibitor, compound C [31] in the Å 6-attenuated the OC differentiation induced by LPS. Compound C inhibited the Å 6-attenuated OC differentiation of RAW264.7 cells induced by LPS (Fig. 5B). Compound C also inhibited the Å 6-attenuated the expression of OC markers, NFATc1 and TRAP induced by LPS (Fig. 5C). Furthermore, compound C inhibited the Å 6-attenuated the TNF-a production induced by LPS (Fig. 5D).

No effects of Å6 on the LPS-induced inflammatory OC differentiation in the uPA or uPAR knockdown conditions
In previous study, we demonstrated that uPA knockdown promoted the LPS-induced OC differentiation [11]. Conversely, uPAR knockdown attenuated them [12]. Here, we examined that the effects of Å 6 on the LPS-induced OC differentiation in the uPA or uPAR knockdown condition. First, we confirmed that uPA siRNA suppressed the uPA expression but control siRNA did not at protein level in the RAW264.7 cells (Fig. 6A). Å 6 inhibited the LPS-induced OC differentiation, TNF-a production, and IkBa degradation in the control condition, whereas Å 6 had no effects on them in the uPA knockdown condition (Fig. 6B-E). Next, we examined that the effects of Å 6 on the LPS-induced OC differentiation in the uPAR knockdown condition. We confirmed that uPAR siRNA suppressed the uPAR expression but control siRNA did not at protein level in the RAW264.7 cells (Fig. 6F). Å 6 inhibited the LPS-induced OC differentiation and TNF-a production in the control condition, whereas Å 6 had no effects on them in the uPAR knockdown condition (Fig. 6G-I).

Discussion
We herein showed the uPA-derived peptide, Å 6 attenuated LPS-induced inflammatory osteoclastogensis and bone loss in mice (Fig. 1). We also showed that Å 6 attenuated the LPSpromoted OC differenatiation (Fig. 2). Furthermore, Å 6 attenuated the production of TNF-a (Fig. 3), which is positively associated with the LPS-induced OC differentiation [26,27]. These data strongly suggest that Å 6 is involved in the suppression of LPS-induced inflammatory osteoclastogenesis and bone loss by attenuation of secretion of the inflammatory cytokine from macrophages that homed into the LPS-induced inflammatory tissue. We previously demonstrated that uPA-activated AMPK attenuated the LPS-induced NF-kB activation, and is involved in the suppression of LPS-induced inflammatory osteoclastogenesis and bone loss [11]. Conversely, uPARactivated Akt is involved in the promotion of LPS-induced inflammatory osteoclastogenesis and bone loss [12]. We herein showed that Å 6 activated the AMPK signaling  The expression of TRAP and NFATc1 in RAW264.7 cells with control or uPAR siRNA was examined by a Western blot analysis. The histogram on the right panel shows quantitative representations of NFATc1 or TRAP obtained from densitometry analysis after normalization to the levels of GAPDH expression (n ¼ 3). (I) RAW264.7 cells were cultured with either control or uPAR siRNA for 24 h in the absence or presence of LPS (1 mg/ml) or Å6 (100 mM) as indicated. The TNF-a content in the conditioned media of RAW264.7 cells transfected with control or uPAR siRNA was determined by using ELISA as described in Materials and Methods (n ¼ 3). Ã p < 0.01, ÃÃ p < 0.05, NS, not significant. (Fig. 5A), and attenuated the LPS-induced NF-kB activation (Fig. 4). We also showed that the inhibition of AMPK inhibited the Å 6-attenuated OC differentiation induced by LPS ( Fig. 5B and C). These data suggest that the Å 6attenuated inflammatory osteoclastogenesis is associated with the AMPK activation. On the other hand, Å 6 inhibited the Akt signaling (Fig. 5A). The activation of Akt is known to inhibit the AMPK pathway [32]. It has been reported that uPA promotes the Akt activation [33], and the downregulation of uPA inhibits the Akt signaling [34,35]. In addition, we previously demonstrated that uPAR deficiency or uPAR blocking attenuated the Akt pathway [8,12,36]. Here, we demonstrated that Å 6 inhibited the LPS-induced OC differentiation, and TNF-a production in the control condition, whereas Å 6 had no effects on them in the uPA or uPAR knockdown conditions (Fig. 6). These results suggested that Å 6 functioned as an attenuator of LPSinduced osteoclastogenesis through the disruption of interaction between uPA and uPAR.
Thus, LPS induced NF-kB activation, resulting in inflammatory osteoclastogenesis and bone destruction. On the other hand, uPAR and uPA-activated plasmin activated the Akt and AMPK pathways, respectively. In addition, the uPAR-induced Akt activation inhibited AMPK pathway. Å 6 attenuated the uPAR-activeted Akt pathway, possibly resulting in the upregulaton of AMPK activation. The resultant suppression of NF-kB activity inhibited inflammatory osteoclastogenesis and bone destruction (Fig. 7). Å 6 has multiple functions, such as inhibition of angiogenesis, cell growth, cell migration, cell invasion [13]. Angiogenesis, cell growth, cell migration, and cell invasion are known to link to inflammatory bone destruction [37,38]. These functions of Å 6 may also affect the suppression of inflammatory osteoclastogenesis and bone loss. Furthermore, it has been reported that Å 6 was no toxicity in Phase 1 and 2 clinical trials [18][19][20][21], Å 6 might be available for the therapy of inflammatory bone destruction.
In conclusion, uPA-derived peptide, Å 6 is involved in the suppression of LPS-promoted inflammatory osteoclastogenesis and the resultant bone loss. These findings provide a basis for therapeutic strategies for the inflammatory bone disease. Figure 7. The proposed mechanism of the Å6-attenuated inflammatory osteoclastogenesis and bone destruction induced by LPS. LPS induced NF-kB activation, resulting in inflammatory osteoclastogenesis and bone destruction. On the other hand, uPAR and uPA-activated plasmin activated the Akt and AMPK pathways, respectively. In addition, the uPAR-induced Akt activation inhibited AMPK pathway. Å6 attenuated the uPAR-activeted Akt pathway, possibly resulting in the upregulaton of AMPK activation. The resultant suppression of NF-kB activity inhibited inflammatory osteoclastogenesis and bone destruction.