Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1
2021; Cell Press; Volume: 23; Linguagem: Inglês
10.1016/j.omtn.2021.01.031
ISSN2162-2531
AutoresMengmeng Liang, Xiaofan Yin, Shuai Zhang, Hongbo Ai, Fei Luo, Jianzhong Xu, Ce Dou, Shiwu Dong, Qinyu Ma,
Tópico(s)Bone Metabolism and Diseases
ResumoActivated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs. Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs. IntroductionBone is a dynamic organ that undergoes constant turnover throughout the human lifespan. Bone remodeling involves the removal of old bone tissue by osteoclast bone resorption, followed by the formation of bone matrix via osteogenic differentiation that subsequently becomes mineralized.1Hadjidakis D.J. Androulakis I.I. Bone remodeling.Ann. N Y Acad. Sci. 2006; 1092: 385-396Crossref PubMed Scopus (872) Google Scholar Bone remodeling is accomplished by basic multicellular units (BMUs), the discrete temporary anatomic structures assembled by osteoblasts and osteoclasts.2Jilka R.L. Biology of the basic multicellular unit and the pathophysiology of osteoporosis.Med. Pediatr. Oncol. 2003; 41: 182-185Crossref PubMed Scopus (119) Google Scholar The bone-resorbing osteoclasts play essential roles in physiological bone remodeling. Abnormal osteoclast functions lead to pathological bone disorders such as rheumatoid arthritis, postmenopausal osteoporosis, and cancer-induced osteolysis.3Steffen U. Schett G. Bozec A. How autoantibodies regulate osteoclast induced bone loss in rheumatoid arthritis.Front. Immunol. 2019; 10: 1483Crossref PubMed Scopus (40) Google Scholar, 4Wang N. Agrawal A. Jørgensen N.R. Gartland A. P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis.Sci. Rep. 2018; 8: 3507Crossref PubMed Scopus (31) Google Scholar, 5Liang M. Ma Q. Ding N. Luo F. Bai Y. Kang F. Gong X. Dong R. Dai J. 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Scully S. et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4583) Google Scholar The intercellular communication between osteoclasts and osteoblasts is termed "coupling."9Howard G.A. Bottemiller B.L. Turner R.T. Rader J.I. Baylink D.J. Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism.Proc. Natl. Acad. Sci. USA. 1981; 78: 3204-3208Crossref PubMed Scopus (212) Google Scholar The coupling signals transmitted from osteoclasts to osteoblasts promote the transition from bone resorption to bone formation.10Sims N.A. Martin T.J. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit.Bonekey Rep. 2014; 3: 481Crossref PubMed Google Scholar,11Florencio-Silva R. Sasso G.R. Sasso-Cerri E. Simões M.J. Cerri P.S. Biology of bone tissue: structure, function, and factors that influence bone cells.BioMed Res. Int. 2015; 2015: 421746Crossref PubMed Scopus (826) Google Scholar Earlier studies reported that osteoblasts can regulate osteoclast differentiation through tight interplay between RANKL and osteoprotegerin, which are secreted from osteoblasts.12Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M.T. Martin T.J. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.Endocr. Rev. 1999; 20: 345-357Crossref PubMed Scopus (1951) Google Scholar Conversely, osteoclast-derived RANK also regulates osteogenesis and couples bone resorption and bone formation.13Chen X. Zhi X. Wang J. Su J. RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation.Bone Res. 2018; 6: 34Crossref PubMed Scopus (84) Google Scholar,14Ma Q. Liang M. Wu Y. Ding N. Duan L. Yu T. Bai Y. Kang F. Dong S. Xu J. Dou C. Mature osteoclast-derived apoptotic bodies promote osteogenic differentiation via RANKL-mediated reverse signaling.J. Biol. Chem. 2019; 294: 11240-11247Abstract Full Text Full Text PDF PubMed Scopus (29) Google ScholarMicroRNAs (miRNAs) are small non-coding RNAs that can suppress transcription activity of target mRNAs through binding to their 3′ UTR.15Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29004) Google Scholar In bone remodeling, miRNAs regulate the differentiation of osteoblasts and osteoclasts, as well as the orchestration of bone homeostasis.16Lian J.B. Stein G.S. van Wijnen A.J. Stein J.L. Hassan M.Q. Gaur T. Zhang Y. MicroRNA control of bone formation and homeostasis.Nat. Rev. Endocrinol. 2012; 8: 212-227Crossref PubMed Scopus (455) Google Scholar Recent studies demonstrated that miRNAs exist in the extracellular space, which are protected from RNase degradation mainly due to their encapsulation in extracellular vesicles (EVs).17Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. et al.Circulating microRNAs as stable blood-based markers for cancer detection.Proc. Natl. Acad. Sci. USA. 2008; 105: 10513-10518Crossref PubMed Scopus (6292) Google Scholar,18Lawrie C.H. Gal S. Dunlop H.M. Pushkaran B. Liggins A.P. Pulford K. Banham A.H. Pezzella F. Boultwood J. Wainscoat J.S. et al.Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.Br. J. Haematol. 2008; 141: 672-675Crossref PubMed Scopus (1454) Google Scholar EVs are divided into small EVs (sEVs, <200 nm) and large EVs (lEVs), with specific lipid bilayer membrane structure, and they contain proteins, lipids, and a variety of RNAs such as messenger RNA (mRNAs) and miRNAs.19Kalluri R. LeBleu V.S. The biology, function, and biomedical applications of exosomes.Science. 2020; 367: eaau6977Crossref PubMed Scopus (2132) Google Scholar Cargos of sEVs can be transferred from parental cells to recipient cells by direct fusion with the cell membrane or endocytosis to serve as essential mediators of intercellular communication.20Horibe S. Tanahashi T. Kawauchi S. Murakami Y. Rikitake Y. Mechanism of recipient cell-dependent differences in exosome uptake.BMC Cancer. 2018; 18: 47Crossref PubMed Scopus (124) Google Scholar sEV-mediated transfer of miRNAs is widely considered to be involved in a variety of physiological and pathological conditions such as tumor development, immune response, and angiogenesis.21Whiteside T.L. Exosomes and tumor-mediated immune suppression.J. Clin. Invest. 2016; 126: 1216-1223Crossref PubMed Scopus (330) Google Scholar,22Zeng Z. Li Y. Pan Y. Lan X. Song F. Sun J. Zhou K. Liu X. Ren X. Wang F. et al.Cancer-derived exosomal miR-25-3p promotes pre-metastatic niche formation by inducing vascular permeability and angiogenesis.Nat. Commun. 2018; 9: 5395Crossref PubMed Scopus (402) Google Scholar In bone, sEVs are also considered to be essential instruments for signal transduction between bone cells. Osteoclast-derived sEVs induce the differentiation of pre-osteoblasts via RANKL reverse signaling, whereas sEVs from osteoblasts are also shown to play regulatory roles in osteogenesis.23Ikebuchi Y. Aoki S. Honma M. Hayashi M. Sugamori Y. Khan M. Kariya Y. Kato G. Tabata Y. Penninger J.M. et al.Coupling of bone resorption and formation by RANKL reverse signalling.Nature. 2018; 561: 195-200Crossref PubMed Scopus (247) Google Scholar,24Cui Y. Luan J. Li H. Zhou X. Han J. Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.FEBS Lett. 2016; 590: 185-192Crossref PubMed Scopus (182) Google ScholarIn this study, we reveal an interaction pattern between osteoclasts and mesenchymal stem cells (MSCs). Osteoclast-derived miR-324 can be transferred to MSCs and thereby promotes osteogenic differentiation through targeting ARHGAP1, a negative regulator of osteogenic differentiation and mineralization. This interplay between osteoclasts and MSCs was further confirmed in vivo.ResultsCharacterization of osteoclast-derived sEVsTo investigate the sEVs derived from mature osteoclasts, bone marrow macrophages (BMMs) were isolated from 11-week-old male C57BL/6 mouse hindlimbs. BMMs were stimulated with M-CSF and RANKL for 96 h to generate mature osteoclasts. Tartrate-resistant acid phosphatase (TRAP) stain was performed, and the number of TRAP+ multinucleated cells was quantified to validate the maturation of osteoclasts (Figures 1A and 1B ). The gene array analysis further revealed that the expression of osteoclast-specific genes was significantly upregulated with the increasing induction time (Figure 1C). Osteoclast-derived sEVs (OC-sEVs) were isolated through a series of microfiltration and ultracentrifugation steps. Transmission electron microscopy (TEM) revealed the presence of round-shaped vesicles surrounded by lipid bilayer membranes, the typical characteristic of sEVs (Figure 1D). Nanoparticle tracking analysis showed that most vesicles ranged from 70 to 140 nm in diameter with a peak at 90 nm (Figure 1E). sEVs were enriched with the pan-EV markers such as CD81 and TSG101 and the absence of nuclear proteins such as Lamin A/C and histone 3 (Figure 1F).Osteoclast-derived sEVs promote osteogenic differentiation and mineralizationTo explore the effect of OC-sEVs on osteogenic differentiation, we first investigated the engulfment of OC-sEVs by recipient cells. Primary MSCs labeled with CellTracker CM-Dil dye were cultured with PKH67-labeled OC-sEVs. Confocal microscopy was used to analyze the co-incubation of sEVs and recipient MSCs, and the results showed that the sEVs can be engulfed by the recipient MSCs over time (Figure 2A). After MSCs were cultured with sEVs for 7 days, qPCR was performed to detect the expression of osteogenic markers. The results revealed a significant upregulation of osteogenic regulators Runt-related transcription factor 2 (Runx2) and Osterix (OSX, also known as Sp7), as well as osteogenic markers type I collagen (Col1a1) and Alpl in OC-sEV-cultured MSCs (Figure 2B). On the protein level, western blot analysis also showed that OC-sEV culturing significantly upregulated the expression of ALP, COL1A1, and RUNX2 in MSCs (Figure 2C). GW4869 is a potent neutral sphingomyelinases inhibitor, which prevents the formation of intraluminal vesicles to further block sEV production and release in numerous cell types.25Trajkovic K. Hsu C. Chiantia S. Rajendran L. Wenzel D. Wieland F. Schwille P. Brügger B. Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes.Science. 2008; 319: 1244-1247Crossref PubMed Scopus (2218) Google Scholar Osteoclastic conditioned medium (OC-CM) with or without GW4869 pretreatment of cells was used to culture MSCs, subsequently followed by detection of their osteogenic potency. An ELISA assay was performed to detect the interleukin (IL)-6, transforming growth factor (TGF)-β1, and BMP2 concentrations of supernatants from osteoclasts with or without GW4869 pretreatment. We discovered that the concentration of IL-6, TGF-β1, and BMP2 did not significantly change after GW4869 treatment (Figure S1). Intriguingly, OC-CM significantly induced osteogenic differentiation characterized by upregulation of osteogenic markers. However, pretreatment with GW4869 abolished the facilitating effect of OC-CM on osteogenic differentiation, suggesting that OC-sEVs in OC-CM play a dominant role in this process (Figures 2B and 2C). To examine the effect of OC-sEVs on osteogenic mineralization, alizarin red staining was performed after culturing MSCs for 21 days with OC-sEVs (Figure 2D). The results showed that both OC-sEVs and OC-CM treatment significantly promoted the mineralization of MSCs, while GW4869 pretreatment abolished the effect of OC-CM on inducing mineralization (Figures 2D and 2E). Taken together, these results suggested that OC-sEVs promote the expression of osteogenic markers and facilitate mineralization.Figure 2Osteoclast-derived sEVs promote osteogenic differentiation and mineralizationShow full caption(A) Representative confocal microscopy images show MSCs labeled with CellTracker CM-Dil dye that were cultured with PKH67-labeled sEVs for 24 h. Scale bar represents 10 μm. (B) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with OC-sEVs or osteoclast conditioned medium (OC-CM), with or without GW4869 pretreatment; n = 3. (C) Western blot analysis of COL1A1, RUNX2, ALP, and β-actin in indicated groups. (D) Representative images of alizarin red staining of MSCs cultured with OC-sEVs or OC-CM. Scale bar represents 100 μm. (E) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. The data in the figures represent the averages ± SD. ∗∗p < 0.01, for differences between the treatment and control groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)sEV-miR-324 derived from osteoclasts can be delivered into MSCs and induce osteogenic differentiationSince miRNAs released from sEVs are demonstrated to play an important role in intercellular communications, we speculated that miRNAs contained in OC-sEVs may play a role in affecting osteogenic differentiation. To explore this idea further, we first examined the expression profile of miRNAs in sEVs secreted during osteoclastogenesis. Total RNAs, including miRNAs, were extracted from isolated sEVs and subjected to miRNA microarray analysis. The comprehensive microarray analysis revealed that miR-1187, miR-128-3p, miR-324, and miR-130b-3p were highly expressed in OC-sEVs (Figure 3A). To examine the effect of miRNAs on osteogenic differentiation, each miRNA mimic was subsequently transfected into MSCs, and cells were cultured using osteogenic medium. An alkaline phosphatase (ALP) assay was performed after 14-day osteogenic induction, and results revealed that overexpression of miR-324 significantly induced the osteogenic differentiation characterized by increasing ALP activity, whereas overexpression of miR-130b-3p adversely reduced ALP activity (Figure 3B). To further confirm the effect of miR-324 of sEVs on osteogenic differentiation, miR-324 was knocked down or overexpressed in BMMs and RAW264.7 cells by transfection. Transfection efficiency was confirmed by qPCR (Figure S2A). Knockdown or overexpression of miR-324 in parental cells resulted in a significant reduction or upregulation of miR-324 in respective sEVs (Figure S2B). Additionally, qPCR analysis showed that miR-324 in lipid-bilayer sEVs was protected from RNase degradation (Figure S2C). Next, sEVs from miR-324 knockdown (anti-miR-324-sEVs) or overexpression (miR-324-sEVs) osteoclasts were added into the osteogenic medium of MSCs. Notably, miR-324, but not primary miR-324 (pri-miR-324), was markedly upregulated in MSCs cultured with miR-324-sEVs (Figures 3C and 3C). The effect of miR-324 in sEVs on the recipient MSC cell viability was also detected by a Cell Counting Kit-8 (CCK-8) assay. Results showed that miR-324-sEVs significantly increased cell viability, whereas treatment with anti-miR-324-sEVs decreased cell viability (Figure 3E). For evaluation of osteogenic markers, qPCR analysis showed that miR-324-sEVs significantly upregulated the expression of osteogenic markers, while treatment with anti-miR-324-sEVs had adverse effects (Figure 3F). On the protein level, western blot analysis confirmed the upregulation of osteogenic markers by miR-324-sEV treatment in MSCs (Figure 3G). Similarly, alizarin red staining revealed that miR-324-sEVs significantly increased osteogenic mineralization compared with mimic-negative control (NC)-sEVs, whereas anti-miR-324-sEVs decreased osteogenic mineralization compared with anti-NC-sEV treatment (Figures 3H and 3I). These data suggested that osteoclast-derived sEV-miR-324 promotes osteogenic differentiation of MSCs.Figure 3sEV-miR-324 derived from osteoclasts can be delivered into MSCs and induce osteogenic differentiationShow full caption(A) The expression profile of miRNAs in sEVs secreted during osteoclast differentiation. sEVs from BMMs were used as a normalization control. Red color represents higher expression, and blue color represents lower expression relative to the control. (B) ALP activity assay shows that overexpression of miR-324 promoted osteogenic differentiation; n = 3. (C and D) Relative expression levels of (C) miR-324 and (D) pri-miR-324 in MSCs cultured with sEVs from miR-324 overexpression (miR-324-sEVs) or knockdown (anti-miR-324-sEVs) osteoclasts; n = 3. (E) Cell viability evaluation of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs using a CCK-8 test at 1, 3, 5, and 7 days; n = 5. (F) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs; n = 3. (G) Western blot analysis of COL1A1, RUNX2, ALP, and β-actin in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. (H) Representative images of alizarin red staining of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. Scale bar represents 100 μm. (I) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. Data in the figures represent the averages ± SD. ∗p < 0.05, ∗∗p < 0.01, for differences between the treatment and control groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-324 directly binds to ARHGAP1 and induces osteogenic differentiationTo explore the target genes of miR-324 to regulate osteogenic differentiation, four mRNA target-predicting algorithms (miRanda, miRDB, miRWalk, and TargetScan) were utilized to identify the potential downstream targets of miR-324. Among the potential targets, ARHGAP1 was overlapped among all databases. It has been reported that ARHGAP1 served as a non-canonical negative regulator for osteogenic differentiation of human MSCs and was regulated by prostate cancer-derived miR-940.26Hashimoto K. Ochi H. Sunamura S. Kosaka N. Mabuchi Y. Fukuda T. Yao K. Kanda H. Ae K. Okawa A. et al.Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A.Proc. Natl. Acad. Sci. USA. 2018; 115: 2204-2209Crossref PubMed Scopus (148) Google Scholar In this study, the expression of ARHGAP1 was also knocked down or overexpressed in MSCs (Figure 4A). After 14-day osteogenic induction, the knockdown of ARHGAP1 increased ALP activity of MSCs whereas overexpression of ARHGAP1 had an adverse effect (Figure 4B). Additionally, qPCR analysis showed that knockdown of ARHGAP1 increased, whereas overexpression of ARHGAP1 decreased, expression of osteogenic markers in MSCs (Figure 4C). These results suggested that ARHGAP1 is a negative regulator for osteogenic differentiation of MSCs. In addition, miR-324 was also knocked down or overexpressed in MSCs by transfection with mimics and inhibitor, respectively (Figure 4D). qPCR analysis showed that overexpression of miR-324 significantly upregulated, whereas knockdown of miR-324 (anti-miR-324) downregulated, the expression osteogenic markers (Figure S3A). To establish whether ARHGAP1 was a target of miR-324, luciferase reporter plasmid containing the wild-type 3′ UTR of ARHGAP1 was generated and cotransfected with miR-324 mimics, while Renilla luciferase plasmid was used for normalization (Figure S3B). Notably, the luciferase activities of the 3′ UTR of ARHGAP1 were suppressed by miR-324 (Figure S3C). Moreover, overexpression of miR-324 in MSCs suppressed the expression of ARHGAP1, whereas knockdown of miR-324 resulted in upregulation of ARHGAP1 (Figure 4E). These results indicate that miR-324 regulates osteogenic differentiation through silencing ARHGAP1 in MSCs.Figure 4miR-324 directly binds to ARHGAP1 and induces osteogenic differentiationShow full caption(A) Relative mRNA expression levels of ARHGAP1 in MSC knockdown or overexpressed ARHGAP1; n = 3. CT represents empty control group without any treatment, and si-CT represents the random non-specific siRNA used for negative control. (B) Relative ALP activity of MSC knockdown or overexpressed ARHGAP1 after osteogenic induction for 14 days; n = 3. (C) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSC knockdown or overexpressed ARHGAP1; n = 3. (D) Relative expression levels of miR-324 in MSC knockdown or overexpressed miR-324; n = 3. (E) Relative expression levels of ARHGAP1 in MSC knockdown or overexpressed miR-324; n = 3. Data in the figures represent the averages ± SD. ∗∗p < 0.01, for differences between the treatment and control groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Osteoclast-secreted miR-324 silences ARHGAP1 during osteogenic differentiationWe then examined the luciferase activities of the ARHGAP1 3′ UTR in MSCs cultured with miR-324-sEVs. Notably, the luciferase activities of the ARHGAP1 3′ UTR were markedly decreased by miR-324-sEVs compared with mimic-NC-sEVs, whereas application of miR-324 inhibitor abrogated these effects (Figure 5A). These results suggested that ARHGAP1 expression in MSCs can be suppressed by miR-324 of sEVs released from osteoclasts. Additionally, miR-324-sEVs dramatically decreased ARHGAP1 expression and increased the expression levels of osteogenic markers (Figures 5B and 5C). It has been suggested that ARHGAP1 negatively regulates the downstream RhoA/ROCK signaling to inhibit cytoskeletal tension and reorganization during osteoblast differentiation, which determines the osteogenic commitment of stem cell lineage.27McBeath R. Pirone D.M. Nelson C.M. Bhadriraju K. Chen C.S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.Dev. Cell. 2004; 6: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3338) Google Scholar Notably, western blot analysis revealed that miR-324-sEVs markedly decreased the expression of ARHGAP1, while they increased the expression levels of downstream RhoA, ROCK, and the MYPT1 phosphorylation by ROCK kinase (Figure 5D). It has been reported that annexin V is an inhibitor of EV internalization.28Li J. Liu K. Liu Y. Xu Y. Zhang F. Yang H. Liu J. Pan T. Chen J. Wu M. et al.Exosomes mediate the cell-to-cell transmission of IFN-α-induced antiviral activity.Nat. Immunol. 2013; 14: 793-803Crossref PubMed Scopus (376) Google Scholar Treatment with annexin V or miR-324 inhibitor in miR-324-sEVs abolished their pro-osteogenic effects and suppression of ARHGAP1 (Figures 5B and 5C). Adenovirus overexpressed ARHGAP1 was designed and used to transfect MSCs, while these ARHGAP1-overexpresssing MSCs did not show a strong mineralization after culturing with miR-324-sEVs, suggesting that the positive effect of miR-324-sEVs on osteogenesis was abrogated by the restoration of ARHGAP1 (Figures 5B and 5C). Additionally, application of annexin V, miR-324-inhibitor, or restoration of the ARHGAP1 expression suppressed the positive regulation of miR-324-sEVs in the RhoA/ROCK pathway (Figure 5D). A CCK-8 assay was performed, and results showed that either treatment with annexin V or miR-324 inhibitor, or restoration of the expression of ARHGAP1, significantly reduced the recipient MSC viability (Figure 5E). Alizarin red staining and quantification analysis showed that transfection of miR-324-sEVs with miR-324 inhibitor or pretreatment of miR-324-sEVs with annexin V alleviated their function in facilitating osteogenic mineralization. Additionally, ARHGAP1 restoration in recipient MSCs suppressed these effects (Figures 5F and 5G). Taken together, the findings demonstrate that miR-324 of sEVs released from osteoclasts is sufficient to promote osteogenic differentiation by targeting ARHGAP1.Figure 5Osteoclast-secreted miR-324 silences ARHGAP1 during osteogenic differentiationShow full caption(A) Relative luciferase activity of reporter containing the 3′ UTR of ARHGAP1 in MSCs upon culturing with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, and miR-324-sEVs + miR-324 inhibitor; n = 5. (B) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (C) Relative expression levels of ARHGAP1 in indicated groups; n = 3. (D) Western blot analysis of ARHGAP1, RhoA, ROCK, p-MYPT1, and β-actin expression in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (E) Cell viability evaluation of MSCs in indicated groups at 0, 1, 3, 5 and 7 days; n = 3. (F) Representative images of alizarin red staining of MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1. Scale bar represents 100 μm. (G) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. The data in the figures represent the averages ± SD. ∗p < 0.05, ∗∗p < 0.01, for differences between the treatment and control groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)miR-324 of sEVs released from osteoclasts facilitates bone defect healing in vivoTo explore the pro-osteogenic effects of miR-324 of sEVs in vivo, we fabricated a novel sEV-modified scaffold by coating decalcified bone matrix (DBM) with isolated sEVs, and the pro-osteogenic potential of sEV-modified scaffolds was evaluated using a mouse calvarial defect model generated by cranial drilling (Figure 6A). This mouse model is useful to evaluate the impacts of cell- or EV-modified scaffolds on osteogenesis.29Wu Y. Cao L. Xia L. Wu Q. Wang J. Wang X. Xu L. Zhou Y. Xu Y. Jiang X. Evaluation of osteogenesis and angiogenesis of icariin in local controlled release and systemic delivery for calvarial defect in ovariectomized rats.Sci. Rep. 2017; 7:
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