Expression of RAS components was upregulated in the joints of SKGc mice and the PBMCs and BdCs of r-axSpA patients
To determine the expression of RAS components in the arthritic joints of an animal model of SpA, we injected SKG mice with curdlan to generate SKGc mice (Fig. 1a). The expression of RAS components in the joints of the SKGc mice was assessed using RT‒qPCR and Western blotting. The expression levels of AGT, ACE, AT1R, AT2R, and NEP were significantly higher in the joints of the SKGc mice than in those of the wild-type (WT) or SKG mice (p = 0.025, 0.034, 0.014, 0.020, and 0.011, respectively; Fig. 1b). In addition, Western blotting analysis results revealed that the protein levels of AGT, ACE, and AT1R in the ankle joints of the SKGc mice were higher than those in the WT or SKG mice (Supplementary Fig. 1).
We also compared the expression of RAS molecules between patients with r-axSpA and control participants. First, we identified the RAS molecules in the PBMCs of the patients with r-axSpA and the control participants. The expression levels of AGT, ACE, AT1R, AT2R, and NEP were significantly higher (p = 0.003, 0.0037, 0.005, 0.025, and 0.0306, respectively) in the PBMCs of the patients with r-axSpA than in those of the control participants (Fig. 1c). Next, we compared the expression of RAS molecules in BdCs between the patients with r-axSpA and the control participants. The expression levels of all RAS molecules, except for AT2R, were significantly higher (p = 0.027, 0.003, 0.05, and 0.032) in the r-axSpA patients than in the control participants (Fig. 1d).
ARBs, but not ACEis, inhibited bone erosion and systemic bone loss in SKGc mice
RAS affects the development of inflammation35. Hence, we investigated whether RAS modulators, such as ARBs and ACEis, influence the development of arthritis in SKGc mice. First, we monitored the severity of paw swelling in each limb and compared the clinical arthritis scores among the SKGc groups treated with vehicle, ARB, or ACEi (SKGc-saline, SKGc-ARB, or SKGc-ACEi, respectively) (Fig. 2a). No significant differences were observed between the groups (Fig. 2b). Next, we measured MPO activity using IVIS; however, we did not find any differences between the groups (Fig. 2c).
In addition, joint tissues were stained with hematoxylin and eosin to determine the degree of inflammatory cell infiltration in the ankle joints of the SKGc mice. Compared with the WT mice, the SKGc-saline mice showed increased inflammatory cell infiltration in the joints. There was no difference in the degree of inflammatory cell infiltration among the SKGc-saline, SKGc-ARB, and SKGc-ACEi mice, which is consistent with the clinical arthritis score and MPO activity (Supplementary Fig. 2). Thus, RAS modulators did not significantly affect the severity of arthritis in the SKGc mice.
Subsequently, we performed CT of arthritic joints to assess the effect of RAS modulation on the development of erosion and abnormal bone formation. The CT erosion score was significantly lower in the SKGc-ARB mice (p = 0.033) and higher in the SKGc-ACEi mice than in the SKGc-saline mice (p = 0.898) (Fig. 2d, e, left). CT bone formation scores decreased in the SKGc-ARB mice (p = 0.515) and increased in the SKGc-ACEi mice (p = 0.516) compared to those in the SKGc-saline mice (Fig. 2d, e, right).
Furthermore, bone CT was used to investigate the effect of RAS modulation on bone remodeling. Bone mineral density (BMD) was significantly higher in the SKGc-ARB group (p = 0.011) and lower in the SKGc-ACEi group (p = 0.462) than in the SKGc-saline group (Fig. 2f, g). Differences were not observed in BMD among the WT-saline, WT-ARB, and WT-ACEi groups treated with each RAS modulator (to determine whether these drugs affected BMD in a noninflammatory environment) (Supplementary Fig. 3a, b).
Next, we measured the expression levels of bone cell-related molecules in the joints of SKGc mice. The expression levels of OC differentiation markers, including TRAP, NFATc, and cathepsin K, were significantly lower in the SKGc-ARB mice than in the SKGc-saline mice (p = 0.006, 0.006, and 0.006, respectively); however, differences were not observed between the SKGc-ACEi and SKGc-saline mice (p = 0.522, 0.670, and 0.394, respectively) (Fig. 2h). Similarly, the expression levels of OB differentiation markers, including BMP2, RUNX2, and RANKL, were significantly lower in the SKGc-ARB mice (p = 0.006, 0.006, and 0.006, respectively) but higher in the SKGc-ACEi mice than in the SKGc-saline mice (p = 0.286, 0.088, and 0.394, respectively) (Fig. 2i).
ARBs and ACEis showed opposite effects on osteoclast differentiation of mouse primary bone marrow macrophages
To verify the direct role of RAS in bone cells, we applied RAS modulators in bone cell culture systems. First, we administered ARBs or ACEis to the BMMs of mice. TRAP staining revealed that OC differentiation was significantly inhibited by ARBs (p = 0.006) but significantly promoted by ACEis (p = 0.004) compared to that in the controls (Fig. 3a). ACEis increased the numbers of TRAP-positive multinucleated OCs and OCs with more than 30 nuclei per cell (p = 0.002; Supplementary Fig. 4). Differences in cell viability were not observed among the groups in the CCK-8 assay (Fig. 3b). RT‒qPCR analysis revealed that ARB significantly lowered (p = 0.006, 0.028, 0.009, 0.009, and 0.009, respectively) whereas ACEi increased (p = 0.006, 0.028, 0.009, 0.021, and 0.009, respectively) the expression levels of OC differentiation markers, including TRAP, NFATc, cathepsin K, DC-STAMP, and OC-STAMP, compared with those in the controls (Fig. 3c).
To investigate the signals acting upstream of the abovementioned factors, we measured the expression level of TRAF6 in RAW 264.7 cells treated with saline, ARBs, or ACEis. ARBs inhibited the expression of TRAF6, but ACEis did not (Fig. 3d). To further investigate the mechanism underlying the inhibition, we treated cells with MG132, a proteasome inhibitor, which restored TRAF6 expression (Fig. 3e), suggesting that the effect of RAS modulators on OCs was mediated via TRAF6 ubiquitination, resulting in degradation by the proteasome.
Ang 1-7 facilitated osteoclast differentiation from mouse primary bone marrow macrophages
The conflicting effects of ARBs and ACEis led us to hypothesize that contrary to previous reports22,36,37, Ang 1-7, rather than Ang II, might play a dominant role in OC differentiation. To verify this, we first confirmed that Ang 1-7 levels increased significantly (p = 0.021) after OC progenitors were treated with ACEis (Fig. 4a).
To examine the effect of Ang 1-7 on OC differentiation, we treated the cells with saline, Ang 1-7, or Ang II. TRAP staining revealed that Ang 1-7 enhanced OC differentiation (p = 0.004; Fig. 4b) and the expression of target molecules, including TRAP, NFATc, cathepsin K, DC-STAMP, OC-STAMP, OSCAR, and Blimp1 (p = 0.020, 0.018, 0.017, 0.019, 0.017, 0.019, and 0.019, respectively), to an extent similar to that observed with Ang II (Fig. 4c). Both Ang II and Ang 1-7 increased the expression of TRAF6, contrary to that observed with ARBs (Fig. 4d).
We hypothesized that the increase in OC differentiation after treatment with Ang II was caused by Ang 1-7 derived from Ang II and not by Ang II itself. Therefore, we simultaneously administered Ang II and an ACE2i to OC progenitor cells to inhibit conversion. This treatment diminished the promoting effect of Ang II on OC differentiation to a level similar to that of the control group (Fig. 4e). Furthermore, Ang 1-7 levels decreased when an ACE2i was used in combination with Ang II compared to Ang II alone (Supplementary Fig. 5). In addition, an NEP inhibitor (NEPi), administered with Ang I, completely blocked OC differentiation (p = 0.002; Fig. 4f), implying that NEP might be a key enzyme involved in Ang 1-7 production, at least in the bone milieu. OC differentiation was inhibited by NEPi treatment (p = 0.004) without changes in cell viability (Supplementary Fig. 6a, b), and the mRNA expression of target molecules, including TRAP, NFATc, cathepsin K, DC-STAMP, and OC-STAMP, decreased after NEPi treatment (p = 0.008, 0.008, 0.008, 0.008, and 0.009, respectively; Supplementary Fig. 6c). These results, combined with the finding that ACEis promoted OC differentiation, suggested that Ang 1-7, rather than Ang II, plays a major role in OC differentiation.
Furthermore, a Mas receptor (MasR) inhibitor (MasRi) was used to assess OC differentiation, as MasR is the major receptor of Ang 1-7. MasRi treatment significantly increased OC differentiation (p = 0.009; Fig. 4g). In addition, to determine whether Ang 1-7 acted on the AT1 receptor, we simultaneously treated OC progenitor cells with Ang 1-7 and ARB, which suppressed the Ang 1-7-induced promotion of OC differentiation (Fig. 4b, c). To further verify this finding, we simultaneously administered ACEis and ARBs to the cells. We observed that OC differentiation was inhibited without changes in cell viability (Supplementary Fig. 7a, b), and expression of the OC differentiation marker decreased (Supplementary Fig. 7c). Therefore, the Ang 1-7/AT1 axis is the major pathway involved in OC differentiation.
ARBs and ACEis exerted opposite effects on osteoblast differentiation from mouse primary osteoblast progenitor cells
To examine the effect of ARBs and ACEis on OB differentiation, we treated mouse OB progenitor cells with RAS modulators in an in vitro culture system. Intracellular ALP activity assays and alizarin red staining (ARS) were performed to assess OB differentiation and mineralization, respectively. Intracellular ALP activity in OBs was suppressed by ARB treatment (p = 0.021) but promoted by ACEi treatment (p = 0.11), suggesting that ARBs and ACEis exerted opposite effects on OB differentiation despite the lack of statistical significance (Fig. 5a). ARS analysis revealed that ARBs significantly inhibited (p = 0.021), whereas ACEis promoted (p = 0.043) mineralization (Fig. 5b) without changes in cell viability (Fig. 5c). Consistent with these findings, we observed that when compared with vehicle, ARBs significantly decreased the expression of bone formation markers, including BMP2, RUNX2, RANKL, and osteocalcin, in OBs (p = 0.021, 0.020, 0.021, and 0.021, respectively), whereas ACEis significantly increased their expression levels (p = 0.043, 0.248, 0.149, and 0.021, respectively) (Fig. 5d). Experiments using the human SaOS2 cell line yielded similar results (Supplementary Fig. 8a, b).
In addition, Ang 1-7 increased OB differentiation and mineralization to a degree similar to that of Ang II (Fig. 5e, f). When cells were costimulated by ARBs and ACEis, OB differentiation and mineralization were inhibited without changes in cell viability (Supplementary Fig. 9a–c).
ARBs inhibited osteoblast differentiation from BdCs of r-axSpA patients
We investigated whether RAS modulators affect the differentiation of OBs obtained from patients with r-axSpA. ARBs and ACEis were administered to the BdCs of biologic-naive r-axSpA patients and control participants. ALP, ARS, and Von Kossa staining revealed that the ARB significantly inhibited OB differentiation and mineralization in the biologic-naive r-axSpA patients (p = 0.0058, 0.042, 0.003, and 0.0144, respectively), whereas the ACEi did not (Fig. 6a, b, right panels). Significant changes were not observed in the OBs of the control participants treated with the ARB or ACEi (Fig. 6a, b, left panels). In addition, HA staining and bright field imaging showed that the ARB inhibited the mineralization of OBs in the r-axSpA patients but not in the control participants (Fig. 6c).