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Introduction
Multiple myeloma (MM) is characterized by the excessive production of monoclonal proteins [ 1 ]. Although the current progress in MM treatment has significantly improved survival of the patients, multiple myeloma bone disease (MMBD) still affects the quality of life of 80% of newly diagnosed MM patients [ 2 ]. The healthy bone is maintained by a balance between osteoblasts (OBs) which contribute to bone formation, and osteoclasts (OCs) which contribute to bone resorption. MM cells affect the bone marrow microenvironment, resulting in heightened bone resorption and diminished bone formation [ 3 ]. In patients with MMBD, OCs exhibit increased numbers and enhanced activity, while OBs are inhibited. This imbalance leads to overactive bone resorption and the development of prominent bone damage [ 4 ]. In general, the cytokines secreted by MM cells and non-tumor cells in MM microenvironment have inhibitory effects on OBs and promoted effects on OCs. For OC activation, Nuclear factor-κB ligand (RANKL), macrophage inflammatory protein-1 A (MIP-1α), interleukin 3 (IL-3) and interleukin 6 (IL-6), matrix metalloproteinase-9 (MMP-9) and so on, are the key mediators of bone resorption in MM [ 5 , 6 ]. In addition, MM and the tumor microenvironment derived excessive Dickkopf-Related Protein 1 (DKK1), interleukin-7 (IL-7) and transforming growth factor b (TGFβ) contributes to OB inhibition [ 7 ].
RND3 belongs to a subgroup of the Rho family and is a Rho-GTPase. Unlike RND1 and RND2, RND3 can bind GTP but lacks GTPase activity [ 8 ]. RND3 plays a potential role in a variety of diseases. RND3 could inhibit cell cycle pRb checkpoints [ 9 ]. In colorectal cancer, RND3 silencing caused cell cycle arrest [ 10 ]. The dysregulation of RND3 was associated with the development and metastasis of numerous cancers [ 11 ]. In prostate cancer, the overexpression of RND3 could induce the tumor cell apoptosis [ 12 ]. However, the potential role of RND3 in MM, especially in MMBD, was not investigated. Here, we elucidated that MM endogenous RND3 was a negative regulator of MMBD. RND3 downstream cell signaling affected both OC and OB in MM bone marrow microenvironment.
Methods and materials
Patient samples and omics data
All experiments including human specimens were approved by the West China Hospital of Sichuan University. Informed consent was obtained from all participants. The Gene Expression dataset GSE755 , GSE13591 , GSE26760 of MM patients was downloaded from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/ .) [ 13 ]. Differential expressed gene analysis was performed using the “limma” package in R (4.4.0).
Cell culture
Human MM cell lines RPMI-8226, AMO.1, ARD, KMS11, LP1, and murine MM cell line 5TGM1 were cultured in RPMI-1640 medium (Corning, USA) supplemented with 10% fetal bovine serum (Excellbio, China). Human OCs were cultured as previously described [ 14 ]. In brief, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were seeded into serum-free culture medium and cultured for 2 h. The medium was then removed, and osteoclast induction medium composed of 10% fetal bovine serum (FBS), 25 ng/mL macrophage colony-stimulating factor (M-CSF, R&D, USA), and 50 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL, PeproTech, USA) was added. The bone marrow cavity of mice was repeatedly rinsed with PBS solution, and the washing fluid was collected to prepare a cell suspension. Subsequently, the cell suspension was seeded into a 6-well plate at a concentration of 1 × 10⁶, and mouse mesenchymal stem cells were cultured with a special medium (OriCell, China). Once the cell confluence reached 80%, the original medium was discarded and replaced with osteoblast induction medium (Pricella, China). The OB induction medium contained 10% FBS, 0.1 µM dexamethasone, 0.2 mM vitamin C, and 10 mM β-glycerophosphate.
Resorption assay and electron microscopy
PBMCs were seeded on autoclaved bovine bone slices and cultured for 21 days according to the aforementioned OC culture method. Subsequently, cells on the surface of the bovine bone slices were washed away. After drying and gold plating, the surfaces of the bovine bone slices were imaged using a scanning electron microscope (Zeiss, EVO 10).
Lentivirus infection
The pLKO.1 control vector (MilliporeSigma, catalogue number SHC002) and pLKO.1 plasmids containing human-RND3 shRNAs (TRCN0000047713 and TRCN0000047715) and mouse-RND3 shRNAs (RCN0000077332) were used to transduce 293 T cells for the production of control virus and shRNA virus.
Western blot
The cells were lysed, and an equal amount of protein sample was loaded into each lane of the sodium dodecyl sulphate-polyacrylamide gels. The proteins were then transferred onto the nitrocellulose membrane and incubated overnight with the primary antibody. The antibodies utilized included RND3 (ProMab, USA, #30838), actin (Proteintech, China, #2D4H5), GAPDH (Proteintech, China, #1E6D9), ROCK1 (Proteintech, China, #21850-1-AP), ROCK2 (Proteintech, China, #21645-1-AP), Phospho-Smad2/3 (Affinity, China, #AF3367), DKK1 (Proteintech, China, # 21112-1-AP), STAT3 (Proteintech, China, # 10253-2-AP), Phospho-Stat3 (CST, USA, #9134), TBP (Proteintech, China, # 66166-1-Ig), and MMP-9 (Hua Bio, China, #ET1704-69).
Immunofluorescence staining and con-focal microscopy
Immunofluorescence staining of bone marrow biopsies was performed using the TSA Fluorescence Dual Staining Kit (Abclonal, RK05902), which involved antigen retrieval, blocking with 3% H₂O₂, antibody incubation, and subsequent nuclear staining with DAPI. For the immunofluorescence assay of cells, MM cells were first seeded into 6-well plates pre-coated with slides and cultured overnight to allow cell attachment. The cells attached to the slide were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 1% BSA for 1 h. Then, the cells were incubated with primary antibody (diluted 1:200) overnight at 4 °C, followed by incubation with fluorescently labeled secondary antibody (diluted 1:400). Images were acquired using a confocal microscopy (Olympus, FV3000).
Chromatin immunoprecipitation
The Magna ChIP kit (MilliporeSigma, #17–10085) was used for ChIP testing following the manufacturer’s instructions.
Quantitative reverse transcription PCR
Cell RNA was extracted using the FastPure cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China, RC112-01). Reverse transcription was carried out following the manufacturer’s instructions using the HiScript II Q RT SuperMix (Vazyme, China, R223-01). qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme, China). Primers are shown in Supplemental Table 1.
Luciferase reporter assay
According to previous studies, the MMP-9 promoter fragment was cloned into the PGL3-Basic vector [ 15 ]. The MMP-9 promoter vector plasmid and the ExFect Transfection Reagent (Vazyme, China) were simultaneously transfected into ARD cells using the ExFect Transfection Reagent. The Dual Luciferase Reporter Gene Assay Kit (Yeasen, 11402ES60) was used according to the manufacturer’s instructions. Finally, the luciferase activity was detected using a multimode microplate reader (Synergy).
Enzyme linked immunosorbent assay (ELISA)
The Human MMP-9 ELISA Kit (Neobioscience, China, EHC115.48) was used to detect MMP-9 in the cell culture supernatant according to the manufacturer’s guidelines. The Human κ - light chain (Abcam, USA, #ab157709) to detect human M protein in mouse serum. The detection of DKK1 was carried out using the Human Dkk-1 ELISA Kit (Multi sciences, China, # EK1111).
Staining assay of osteoclasts and osteoblasts
The OCs were examined with the tartrate-resistant acid phosphatase (TRAP) stain kit (Nanjing Jiancheng Bioengineering Institute, China) following the manufactuer’s guidelines. Mature osteoclasts exhibited the morphology of purplish-red multinucleated giant cells after TRAP staining. ALP staining of OBs was performed using TRAP/ALP staining Kit (wako, Japan, PMC-AK04F-COS) kits. OBs showed a blue-black color after ALP staining. Meanwhile, a 0.2% alizarin red S solution (Solarbio, China) was used to detect calcium nodules formed by mature OBs according to the manufacturer’s instructions. After staining, the nodules appeared orange-red.
Immunohistochemistry (IHC)
Immunohistochemistry was performed as previously described [ 16 ]. The RND3 antibody (ProMab, USA, #30838) was diluted at a ratio of 1:200. The IHC Profiler plugin in Image J was used for automated scoring and quantification of immunohistochemical staining images.
ROCK activity assay
The ROCK Activity Assay was carried out with manufacturer’s instructions (Cell Biolabs, USA, STA-416).
Immunocoprecipitation
Cell lysates were incubated overnight with protein A/G agarose beads (MedChemExpress, USA) and antibodies at 4 °C. The next day, they were washed with PBS, re-suspend in the loading buffer, boil for 10 min, and subjected to western blot.
Protein-protein interaction prediction
The protein sequences of RND3 and ROCK1 were retrieved from the UniProt website [ 17 ], and then AlphaFold3 was used for prediction [ 18 ].
Animal study
Animal research was approved by the Animal Ethics Committee of West China Hospital of Sichuan University. The human MM xenograft mouse model was established in 8-week-old C-NKG mice (Cyagen, China) by intravenous injection of human MM cells (1 × 10 6 cells per mouse). The mouse scapula was scanned and imaged using Micro-CT (Quantum GX, PerkinElmer).
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 8.0.2, and the two-tailed Student’s t-test was used to compare differences between the two groups. A significance level of p ≤ 0.05 was considered statistically significant.
Results
Identification of RND3 as a myeloma bone disease associated gene
To explore intra-MM gene expressions that related to MMBD, we used primary MM gene expression profiles with clinical information. The patients were divided into 2 groups, one with MMBD at diagnosis while the other without. As shown in '. 1 A, in 3 independent datasets, RND3 was differentially regulated in the two groups. The patients with MMBD at diagnosis had low RND3 expression. Further analysis showed that primary MM cells and human MM cell lines expressed RND3 (Fig. 1 B and C). Using the patients’ bone marrow biopsies, we found that primary MM cells expressed RND3 (Fig. 1 D). Next, we collected primary MM cells and MM patients’ biopsies from West China Hospital, Sichuan University. The patients were categorized into 2 groups, with or without MMBD at the MM diagnosis. IHC was performed to detect RND3 expression in MM bone marrow (Fig. 1 E, Supplementary Fig. 5). The RND3 expression was quantified and clearly suggested the patients without MMBD had higher RND3 expression than the patients with MMBD (Fig. 1 F). Primary MM cells RND3 qPCR assay also suggested that the patients with MMBD had high RND3 expression (Fig. 1 G). Overall, our findings identified a correlation between RND3 expression in MM cells and the severity of MMBD.
Fig. 1 RND3 expression and multiple myeloma bone disease. A ) The results of differential gene analysis of the GSE755 , GSE13591 , GSE26760 datasets showed that the expression of RND3 was negatively correlated with bone diseases. B ) Expression of RND3 was detected in 8 primary MM samples. C ) RND3 was expressed in five human multiple myeloma (MM) cell lines, namely RPMI-8226, ARD, KMS11, AMO.1, and LP1. D ) The results of immunofluorescence showed that RND3 was expressed in the bone marrow sections of MM patients. Green represented RND3, red represented CD138, and blue represented nuclear staining. E ) The expression of RND3 in bone marrow sections of MM patients was detected by IHC. F ) IHC quantitative analysis showed that the expression of RND3 in the bone marrow (BM) of patients with bone disease was lower than that in non-bone disease patients. G ) RND3 expression in the bone marrow fluid of patients was examined via qPCR. The findings indicated that patients with bone disease exhibited lower RND3 levels. RN-KD = RND3 knockdown; CT-KD = control knockdown;
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Myeloma RND3 regulated both osteoclast and osteoblast
To investigate the function of RND3 in MMBD regulation, we established MM cell lines with consistent RND3 knocking down (Fig. 2 A). Control knocked-down MM cells (CT-KD) or RND3 knocked-down MM cells (RN-KD) were cocultured with OC precursors as described in method section. RN-KD MM stimulated more mature OC than CT-KD (Fig. 2 B, Supplementary Fig. 1 A). Resorption assay also suggested that increased functional OCs was formed after RN-KD MM coculture (Fig. 2 C, Supplementary Fig. 1B). Of note, intra-MM RND3 expression also affect OB formation under coculture. As shown in Fig. 2 D, alizarin red staining results demonstrated that OBs co-cultured with RN-KD MM cells formed more calcium nodules. ALP assay results similarly showed that OB activity in the RN-KD group was significantly inhibited compared with the CT-KD group (Fig. 2 E). Next, we intravenously inoculated CT-KD or RN-KD MM cells in immune deficient mice. Circulating M protein indicated that RN-KD MM group had slow tumor growth in vivo, compared to CT-KD (Supplementary Fig. 2 A). However, RN-KD MM inoculated mice had more severe MMBD than mice with CT-KD (Fig. 2 F, Supplementary Fig. 2B). After the animal study, we isolated bone biopsies from the mice and conducted the TRAP assay. RN-KD inoculated mice had increased number of OC in bone (Fig. 2 G). To summary, our data suggested that MM RND3 promoted OC and inhibited OB in vitro and in vivo.
Fig. 2 Myeloma RND3 regulate the activations of osteoclast and osteoblast in vitro and in vivo . ( A ) Western blot results demonstrated that RND3-knockdown cell lines were successfully established in the human-derived MM cell lines AMO.1 and ARD, as well as the murine -derived MM cell line 5TGM1. ( B ) After peripheral blood mononuclear cells were induced by RANKL (50 ng/mL) and M-CSF (50 ng/mL) to form pre-OCs, they were co-cultured with CT-KD and RN-KD ARD MM cells respectively. The results of TRAP staining showed that co-culture with RN-KD MM cells could induce a larger number of mature OC cells. ( C ) The results of the bone resorption assay showed that, compared with the co-culture with CT-KD, the OCs in the group co-cultured with RN-KD ARD MM cells could generate more bone resorption pits on the bovine bone slices. ( D ) Mice pre-OBs were co-cultured with murine CT-KD and RN-KD 5TGM1 cells. The results of alizarin red staining indicated that, in comparison with the CT-KD culture group, co-culturing with RN-KD MM cells yielded fewer OBs ( E ) According to the ALP staining results, compared to co-culturing with CT-KD MM cells, co-culturing with RN - KD MM cells led to a smaller number of generated OBs. ( F ) The results of Micro-CT demonstrated that, compared with the mice in the CT-KD group, the area of scapular bone destruction in the mice inoculated with RN-KD MM cells was larger. ( G ) The results of TRAP staining of mice bone sections showed that there were more active OCs in the mice with RN-KD MM. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)
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RND3 mediated regulation of ROCK-related pathways in myeloma
Next, we investigated the molecular basis of RND3 in MMBD regulation. RND3 was recognized as an endogenous antagonist of RhoA signaling transduction, and ROCK, as a direct target of RhoA [ 19 ]. Transcriptome analysis showed that ROCK1/2 were up-regulated in RN-KD cells (Fig. 3 A). ROCK1/2 belonged to serine/threonine kinases and played important roles in processes such as cell migration and proliferation [ 20 ]. RN-KD cells had higher ROCK1/2 enzyme activity than CT-KD (Fig. 3 B). Immune fluorescent staining followed by confocal microscopy suggested co-localization of RND3 and ROCK1 (Fig. 3 C, Supplementary Fig. 3 A). Alphafold3 predicted the protein interaction between RND3 and ROCK1(Fig. 3 D). And the interaction between ROCK1 and RND3 was further confirmed by immunoprecipitation(Fig. 3 E, Supplementary Fig. 3B). The interaction between ROCK1 and RND3 might promote ROCK1 degradation. In RN-KD cells, ROCK1 had longer half-life than that in CT-KD (Fig. 3 F). We also observed down-regulation of ROCK1 ubiquitination in RN-KD cells (Fig. 3 G). Past studies have shown that the transcription factor SRF is a downstream target of RhoA [ 21 ]. Therefore, we found that RhoA nuclear translocation was significantly increased in RN-KD cells (Fig. 3 H, Supplementary Fig. 3 C). ChIP-qPCR experiments confirmed that the transcription factor RhoA can bind to the promoters of ROCK1/2 to regulate transcription (Fig. 3 I). To summary, our findings suggested an interaction between ROCK1/2 and RND3. RND3 could directly or indirectly affect the expression of ROCK1/2 in MM cells; specifically, the expression levels of ROCK1/2 were upregulated after RND3 knockdown.
Fig. 3 RND3 regulate ROCK1/2 in myeloma cells. A ) After performing transcriptome sequencing on CT-KD ARD MM and RN-KD ARD MM cells and conducting differential gene analysis, the results showed that the expression levels of ROCK1 and ROCK2 were up-regulated in the RN-KD group. B ) After counting the same number of CT-KD and RN-KD ARD MM cells, they were lysed and the ROCK enzyme activity was measured. The results showed that the ROCK enzyme activity of RN-KD MM cells was higher. C ) Immunofluorescence results indicated co-localization between ROCK1 (green) and RND3 (red) within AMO.1 cells. The right panel presented the result of fluorescent light intensity quantification, which showed the colocalization of ROCK1 and RND3 signals; D ) Alphafold3 predicted the interaction between RND3 and ROCK1 proteins, E ) The ARD MM experiment was used to perform co-immunoprecipitation experiments, which confirmed the protein-protein interaction between RND3 and ROCK1 F ) Cycloheximide (25 µg/mL) was used to treat MM cells for 0 h, 2 h, and 5 h, respectively. Western blot analysis of ROCK1 showed that ROCK1 degradation was slower in RN-KD ARD MM cells compared to CT-KD ARD MM cells. G ) The ubiquitination level of ROCK1 in RN-KD ARD MM cells was significantly lower than that in CT-KD ARD MM cells. H ) Western blot experiments demonstrated that compared with CT-KD cells, nuclear translocation of SRF was significantly increased in RN-KD MM cells. Meanwhile, the expression of RhoA, ROCK2, and ROCK1 was upregulated in RN-KD MM cells within AMO.1 and ARD models. I ) ChIP-qPCR experiments confirmed that SRF can bind to the promoters of ROCK1/2 using ARD MM experiment. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)
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RND3/ROCK1 in myeloma regulate MMP-9 expression
To demonstrate how intra-MM cell signaling affect OC and OB activity in MM bone marrow, we hypothesized that the OC- and OB-regulation was mediated by MM-derived cytokines. Thus, we performed a qPCR-array to examine a panel of cytokines’ expression in CT-KD versus RN-KD cells (Fig. 4 A). Those cytokines were shown to regulate OC and OB activity [ 14 ]. Among those differentially regulated cytokines, MMP-9 was extremely up-regulated in RN-KD MM cells. Next, we confirmed MMP-9 overexpression in RN-KD cells at mRNA level (Fig. 4 B, Supplementary Fig. 3D) and protein level (Fig. 4 C, Supplementary Fig. 3E). Using reporter construct, we showed MMP-9 promoter activity was up-regulated in RN-KD cells (Fig. 4 D). Past studies have shown that overexpression of ROCK1 and ROCK2 up regulates the downstream effector pSMAD2/3 through the TGF-β pathway [ 22 ]. We showed that SMAD2/3 bound to MMP-9 promoter area. In contrast to CT-KD cells, a greater amount of SMAD2/3 exhibited binding to the MMP9 promoter in RN-KD cells. ( Fig. 4 E, Supplementary Fig. 3 F ) . Western blot results showed that in AMO.1 and ARD cells, knockdown of RND3 had no effect on the expression level of SMAD2/3, but increased the phosphorylation of SMAD2/3 and the content of downstream MMP9 ( Fig. 4 F ) . We detected the content of p-SMAD2/3 in the nucleus and cytoplasm respectively, and found that the level of p-SMAD2/3 was upregulated in RN-KD cells (both in nucleus and cytoplasm) ( Fig. 4 G ). At last but not at least, SMAD2/3 inhibitor NCB-0846 repressed SMAD2/3 phosphorylation and down-regulated MMP-9 expression (Fig. 4 H). In summary, we have elucidated a signaling pathway that ultimately affects MMP-9 via RND3. The MMP9 produced by this pathway is likely secreted by multiple myeloma cells and thereby promotes osteoclast activity.
Fig. 4 RND3-ROCK1/2 axis regulate MMP9 expression in myeloma cells. A ) The qPCR array showed the up-regulation of MMP9 and DKK1 in RN-KD MM cells compared with CT-KD ARD MM cells. B ) According to the qPCR results, it was evident that the MMP-9 transcription level in RN-KD ARD MM was elevated. C ) The culture supernatants of CT-KD and RN-KD were detected by ELISA. The content of MMP-9 in the culture supernatant of RN-KD ARD MM was higher. D ) The dual-luciferase reporter gene assays showed that the transcriptional level of MMP-9 in RN-KD ARD MM was higher than that in CT-KD. E ) The Ch-IP assays demonstrated that Smad2/3 could bind to the MMP-9 promoter, and compared with CT-KD, RN-KD ARD MM cells exhibited a greater amount of such binding. F ) Western blot results showed that compared with CT-KD MM cells, the expression of RhoA, ROCK1, ROCK2, MMP9, and pSMAD2/3 (Thr 8) was upregulated in RN-KD MM cells (AMO.1 and ARD). G ) The Western blot results showed that, compared with CT-KD, both the nucleus and cytoplasm of RN-KD MM cells contained more pSMADsmad2/3 (Thr 8). H ) After ARD MM cells were treated with the pSMAD2/3 inhibitor NCB-0846 (1 µM) for 48 h, the expression level of MMP9 was significantly downregulated. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)
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RND3/ROCK2 regulate DKK1 expression in myeloma
As shown in Fig. 4 A qPCR array, DKK1 was also up-regulated in RN-KD cells. Previous study suggested that DKK1, widely regarded as a factor inhibiting OBs in MM, was regulated by transcription factor STAT3 [ 23 ]. First, we confirmed DKK1 overexpression in RN-KD cells (Fig. 5 A, Supplementary Fig. 3G). Phosphorylation of STAT3 at S727 regulate DKK1 transcription activity. We also detected pSTAT3 up-regulation in RN-KD cells (Fig. 5 B). Immune precipitation assay suggested an interaction between ROCK2 and STAT3 (Fig. 5 C). In RN-KD cells, phosphorylated STAT3 were up-regulated in both cytosal and nucleus (Fig. 5 D). We also performed the chromosome immmune precipitation to show that STAT3 bound to DKK1 promoter (Fig. 5 E). STAT3 inhibitor KD025 decreased DKK1 expression (Fig. 5 F, 5 G). Meanwhile, the pSTAT3 inhibitor HY-13,818 significantly reduced the expression level of DKK1 (Fig. 5 H). In summary, we conclude that RND3 can ultimately exert an effect on multiple myeloma bone disease (MMBD) via DKK1.
Fig. 5 RND3-ROCK2 axis regulate DKK1 expression in myeloma cells. A ) The culture supernatants of CT-KD ARD cells and RN-KD ARD cells were detected by ELISA. The content of DKK1 in the culture supernatant of RN-KD MM was higher. B ) The Western blot results showed that, compared with CT-KD, the expression levels of DKK1 and p-STAT3 (Ser 727) in RN-KD were upregulated using ARD and AMO.1 MM experiment. C ) The Co-IP results showed that there was a protein-protein interaction between STAT3 and ROCK2 using ARD MM experiment. D ) The Western blot results showed that the expression of p-STAT3 (Ser 727) was upregulated in both the cytoplasm and nucleus of RN-KD ARD MM cells. E ) The results of the Ch-IP experiment demonstrated that STAT3 could bind to the DKK1 promoter using AMO.1 MM experiment. F ) After treatment with the ROCK2 inhibitor KD025 (10 μm, 24 h), the expression of DKK1 and other signaling molecules were examined by western blotting in AMO.1 MM cells. G ) After KD025treatment, DKK1 in the culture supernatant was examined by ELISA in AMO.1 MM cells. H ) Treatment with the pSTAT3 inhibitor HY-13818 (20 μm, 24 h) significantly downregulated DKK1 expression in AMO.1 MM cells. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)
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ROCK Inhibition alleviated myeloma bone disease
Based on above data, ROCK1/2 kinase played a central role in MMBD regulation. Next, we examined ROCK inhibition in MMBD treatment. Y-27,632, a pan-inhibitor of ROCK1/2 was used to treat MM cells, and inhibited MMP-9 and DKK1 expressions (Fig. 6 A, Supplementary Fig. 3H, 6B). Addition of Y-27,632 inhibited RN-KD MM conferred OC activation (Fig. 6 C, Supplementary Fig. 4), and OB repression (Fig. 6 D). Furthermore, we treated MM mice with Y-27,632 (5 mg/kg). The results showed that Y-27,632 significantly alleviated bone disease in vivo (Fig. 6 E, Supplementary Fig. 6), and HE staining indicated no obvious lesions in the hearts and livers of Y-27,632-treated mice (Fig. 6 F). The related mechanism was as follows: first, RND3 directly affected the transcription of ROCK1 and ROCK2 through the RhoA/SRF/ROCK pathway; second, the protein-protein interaction between RND3 and ROCK1 influenced the degradation of ROCK1. Those molecule mechanisms ultimately altered the phosphorylation levels of downstream SMAD2/3 and STAT3, leading to changes in the expression of OC-promoting factor MMP-9 and OB-inhibiting factor DKK1. Consequently, these changes affected the balance of OBs and OCs in the MM tumor microenvironment and the severity of MMBD.
Fig. 6 Pan-ROCK inhibitor Y-27632 alleviate myeloma bone disease . A ) The Western blot results showed that after treatment with the ROCK inhibitor Y-27632 (5μM) 48 h, the downstream molecules including MMP-9, p-smad2/3 (Thr 8), p-STAT3 (Ser 727) and DKK1 were inhibited in ARD. B ) After treatment with Y-27632, the content of MMP-9 and DKK1 in the culture supernatant decreased significantly in ARD. C ) In the co-culture system of pre-OCs and ARD MM, the number of mature OCs significantly decreased after the addition of Y-27632. However, in the co-culture system of pre-OBs and ARD MM, the number of mature OBs increased after the addition of Y-27632. D ) Alizarin red staining results showed that Y-27632 significantly increased the number of osteoblasts (OBs) in vitro after treatment. E )The bone disease in mice was alleviated after treatment with Y-27632 (5 mg/kg). F ). Y-27632 had no obvious toxic and side effects on the heart, liver and kidney of mice. G ) Graphic model of RND3 function in MM bone diseases. (*p<0.05 (Student’s t test). RN-KD=RND3 knockdown; CT-KD=control knockdown)
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Discussion
It has long been acknowledged that the balance between OC- mediated bone resorption and OB-mediated bone formation is crucial for maintaining healthy bone structure, and disruption of such balance leads to MMBD [ 24 ]. The growth of tumor cells in bone marrow initiates the skeletal-related events in MM patients. Therefore, we sought to understand intra-MM molecules and cell signaling that led to MMBD heterogeneity in different patients. In this study, we identified that RND3 expression in MM cells played an upstream role in MMBD regulation. Previous research efforts have unearthed several genes associated with MMBD, such as CST6 [ 25 ], MAST4 [ 14 ], hnRNPA2B1 [ 26 ] and CD166 [ 27 ]. These findings highlighted the complexity of the molecular mechanisms underlying MMBD. Each of these genes exerts its influence by modulating the expression of downstream cytokines, which play pivotal roles in skewing the OC-OB balance within the MM microenvironment.
In bone diseases, bone loss occurs because OCs excessively secrete soluble factors and proteases, which in turn degrade the collagen network in bones [ 28 ]. Matrix metalloproteinases (MMPs) are a family of proteases responsible for cleaving the extracellular matrix (ECM) [ 29 ]. In bones, the ECM protects the bone from damage or degradation. Many pathologies associated with an imbalance in bone remodeling are mainly caused by the overexpression of MMPs and abnormalities in the ECM, ultimately leading to osteolysis [ 30 ]. And MMP-9 was the most abundant among the MMPs, which was widely involved in the recruitment of osteoclasts and the cleavage of the ECM [ 31 ]. Sfiridaki et al. found that the content of MMP-9 in the serum of patients with osteolytic lesions was significantly higher than that in the serum of MM patients with normal bone structure [ 32 ]. Our study considered that in the regulatory effect of RND3-mediated MMBD, MMP-9 was a crucial downstream target. Although the detailed molecular mechanisms by which RND3 impacted MMP-9 expression were not yet fully understood, our data clearly indicated the involvement of the ROCK-related pathways. RND3 appeared to suppress the expression and enzymatic activities of ROCK1 and ROCK2. This suppression likely led to the disruption of the phosphorylation of key downstream molecules such as Smad2/3. Given that activated Smad2/3 could promote the transcription of MMP-9, the inhibition of ROCK by RND3 might ultimately have resulted in decreased MMP-9 production. However, the molecular basis of how RND3 regulated ROCK activity remained to be determined.
In addition to MMP-9, DKK1 also played a critical role in the pathogenesis of MMBD. DKK1, secreted by MM cells, was known to be a potent inhibitor of osteoblast function [ 33 ]. By competitively binding to the receptors in the Wnt pathway, DKK1 inhibited the signaling pathway, which in turn suppressed OB activity and new bone formation [ 34 ]. Studies found that there was a positive correlation between the concentration of DKK1 in the serum of MM patients and the amount of osteolytic bone lesions [ 35 ]. Our research demonstrated that RND3 influenced DKK1 expression specifically through the ROCK2-STAT3 axis. While RND3 could impact the expression and activity of both ROCK1 and ROCK2, it was ROCK2, not ROCK1, that played a pivotal role in regulating DKK1. Inhibition of RND3 led to an upregulation of both ROCK1 and ROCK2. However, we revealed that only the increased ROCK2 activity was responsible for promoting the phosphorylation of STAT3. Activated STAT3 then bound to the promoter of DKK1, and enhanced its transcription. As a result, more DKK1 was produced and released into the bone microenvironment. This finding regarding the specific role of ROCK2 in the RND3-mediated regulation of DKK1 expression is crucial for understanding the intricate molecular mechanisms underlying MMBD. In addition, we found that MM cells proliferated more slowly after knockdown of RND3 in vivo and in vitro (Supplementary Fig. 7).
Currently, the treatment methods for MMBD all focus on drugs targeting OCs, such as bisphosphonates (BPs) and denosumab. BPs such as zoledronic acid can affect the function of OCs and promote the apoptosis of OCs, thereby inhibiting bone resorption [ 36 ]. Zoledronate acid demonstrated excellent therapeutic effects in clinical trials, effectively reducing the incidence of skeletal-related events (SREs) [ 37 ]. However, patients who receive BPs for a long time are at risk of developing osteonecrosis of the jaw. Denosumab is a newly developed humanized antibody that inhibits the formation of OCs by directly targeting RANKL and also affects bone resorption [ 38 ]. A phase 3 study demonstrated that denosumab was superior to zoledronic acid in terms of the time to the first SREs [ 39 ]. However, it also carried the risk of jaw osteonecrosis [ 40 ]. Our research on the in vitro and in vivo efficacy of ROCK inhibitors, such as Y-27,632, against MMBD offers a potential approach for current treatments. By inhibiting ROCK1 and ROCK2, these inhibitors had the potential to alleviate the dysregulation of cytokine expression and contribute to the restoration of the balance between OC and OB. Nevertheless, determining the long-term safety and efficacy of these inhibitors in clinical settings, optimizing dosing, and exploring combinations with other anti-MM therapies are crucial areas for future study.
In conclusion, our study on RND3 in MMBD provided novel insights into the molecular mechanisms. By identifying RND3 as a key regulator and demonstrating the potential of targeting the ROCK pathway, we had laid a foundation for future research aimed at developing more effective treatments for MMBD. Future studies should focus on elucidating the detailed molecular mechanisms, validating RND3 as a biomarker, and optimizing therapeutic strategies targeting the RND3-ROCK axis.
Supplementary Information
Supplementary Material 1
Supplementary Material 2
Publisher’s note
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Wenjiao Tang and Qianwen Gao contributed equally to this manuscript.
Acknowledgements
The authors would like to thank the Department of Hematology, West China Hospital, Sichuan University for their technical support.
Author contributions
Y.Z. initiate this study and prepare the manuscript; T.N. and W.T. provided critical suggestions; Q.G. and W.T. performed the majority of the experiments and prepare the manuscript; H.L., H.Y., J.H., Y.Z., J. W., L.L., Z.M., Y.F., S.H., X.L., X.Z., X.W., and L.Z. performed experiments and data analyses.
Funding
This work was supported by grants to Y.Z. from the National Natural Science Foundation of China (No. 82470196), and the Sichuan University Faculty Start Fund. This work was supported by grant to W.T. from the National Natural Science Foundation of China (No. 82500274). This work was also supported by grants to N.T. from Incubation Program for Clinical Trials (No. 19HXFH030), Achievement Transformation Project (No. CGZH21001), 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (No. ZYJC21007), Translational Research Grant of NCRCH (No. 2021WWB03), Chengdu Science and Technology Program (No. 2022-YF05-01444-SN), Key Research and Development Program of Sichuan Province (No. 2023YFS0031), and National Key Research and Development Program of China (No. 2022YFC2502600, 2022YFC2502603).
Data availability
Data will be made available on request.
Declarations
Ethics approval and consent to participate
The use of the samples was approved by the Ethical Committee of West China Hospital, Sichuan University.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
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