Osmr was regulated during osteogenic and adipogenic differentiation
The expression of Osmr was detected in various tissues of 8-week-old C57BL/6J mice, and the results showed that Osmr was affluent in heart, perirenal white fat, skeletal muscle, brown fat, bone, and lung (Additional file 1: Fig. S1A).
Then, we demonstrated by using qRT-PCR that Osmr level increased at day 3 through 13 and peaked at day 9 during osteogenic differentiation of BMSCs (Additional file 1: Fig. S1B). By contrast, it increased during the early stage of adipogenic differentiation of BMSCs, peaked at day 2, and decreased to a level below the baseline (day 0) at day 5 after adipogenic treatment (Additional file 1: Fig. S1C). In addition, Osmr level was significantly increased in the radial metaphysis of ovariectomized mice as compared to that in sham-operated mice (Additional file 1: Fig. S1D). Moreover, it was higher in the radius of 18-month-old mice than in that of 3-month-old mice (Additional file 1: Fig. S1E).
Silencing of OSMR in progenitor cells enhanced osteogenesis and inhibited adipogenesis
The role of OSMR in the differentiation of primary BMSCs was investigated. The efficacy of silencing lentivirus (Osmr-KD LV) in knocking down Osmr was verified in primary BMSCs at either mRNA level or protein level (Fig. 1A, B). The silencing of Osmr did not change the expression of leukemia inhibitory factor receptor (LIFR) (Fig. 1C). There was no significant change in cell viability (Fig. 1D) and proliferation (Additional file 1: Fig. S2A, B) of BMSCs following Osmr silencing, evaluated by using CCK-8 assay and 5-ethynyl-2′-deoxyuridine (EdU) staining, respectively. After osteogenic treatment, silencing of Osmr potentiated osteogenesis of BMSCs, as revealed by enhanced ALP staining at day 14 and alizarin red staining at day 21 as compared to the control (Fig. 1E, F). Accordingly, knockdown of Osmr increased the mRNA and protein levels of the key factors for osteogenesis (mRNAs: increased by 1.5–4.3-fold; proteins: increased by 2.1–2.6-fold), including Runx2, osterix, ALP, and osteopontin (OPN) in the cells 72 h after osteogenic treatment (Fig. 1G, H).
By contrast, in the presence of adipogenic medium, the formation of adipocytes was significantly alleviated in Osmr-silenced BMSCs, as evidenced by the decrease in the intensity of oil-red O staining (Fig. 1I, J). Consistently, the mRNA and protein levels of the key factors for adipogenesis including PPARγ, C/EBPα, FABP4, and adipsin were significantly decreased (mRNAs: decreased by 47–71%; proteins: decreased by 34–51%) in Osmr-silenced BMSCs 48 h and 72 h, respectively, after adipogenic treatment (Fig. 1K, L).
Additionally, we also investigated the role of OSMR in the differentiation of ST2 stromal cells. The two independent siRNAs targeting different regions of Osmr were proved to work well in ST2 cells by using qRT-PCR and Western blotting (Additional file 1: Fig. S3A, B). There was no significant change in the viability (Additional file 1: Fig. S3C) and proliferation (Additional file 1: Fig. S4A, B) of ST2 cells following Osmr silencing, evaluated by using CCK-8 assay and EdU staining, respectively. After osteogenic induction, Osmr siRNAs potentiated osteogenesis of ST2, as revealed by enhanced ALP staining (Additional file 1: Fig. S3D) and alizarin red staining (Additional file 1: Fig. S3E), and the increased mRNA/protein levels of osteogenic factors (Additional file 1: Fig. S3F, G). By contrast, silencing of Osmr in ST2 cells suppressed adipogenic differentiation and the expression of adipogenic factors (Additional file 1: Fig. S5A-D).
Enforced expression of OSMR in progenitor cells suppressed osteogenesis and promoted adipogenesis
Overexpression of OSMR in ST2 cells after transfection of Osmr expression construct was verified with qRT-PCR and Western blotting (Fig. 2A, B). CCK-8 assay and EdU staining showed that an increase in Osmr level in ST2 cells had no effect on cell viability (Fig. 2C) and proliferation (Additional file 1: Fig. S6A, B). In the presence of osteogenic induction, Osmr overexpression blunted osteogenesis, as revealed by reduced ALP staining and alizarin red staining of differentiated osteoblasts at day 14 and day 21, respectively, as compared to vector transfection (Fig. 2D, E). Accordingly, Osmr overexpression decreased the mRNA/protein levels of osteogenic factors 72 h after osteogenic treatment (Fig. 2F, G).
By contrast, Osmr overexpression stimulated the formation of adipocytes from ST2 cells, as evidenced by the dramatic increase in the intensity of oil-red O staining as compared to vector transfection (Fig. 2H, I). Consistently, the mRNA and protein levels of the adipogenic factors were higher in Osmr-overexpressing cells than in control cells 48 h and 72 h, respectively, after adipogenic treatment (Fig. 2J, K).
OSMR inactivated ERK signaling pathway and suppressed autophagy
We further explored the mechanism for the regulation of directional differentiation of stromal progenitor cells by OSMR. The levels of SHC1 and ERK1/2 after overexpression or silencing of Osmr were investigated. The results showed that the phosphorylated proteins of SHC1 and ERK1/2 were significantly decreased in Osmr-overexpressing ST2 cells, while they increased in Osmr-silenced cells (Fig. 3A, B).
As ERK signaling is a known activator of autophagy [34, 35], we further explored whether OSMR regulates autophagy. The mRNA and/or protein levels of the key molecules of autophagy, such as Unc-51 like kinase 1 (ULK1), Beclin1, ATG5, and ATG7, and the ratio of LC3II/LC3I were significantly decreased, while the level of P62 was remarkably increased in ST2 cells following Osmr overexpression (Fig. 3C, D). Conversely, after the knockdown of Osmr, the mRNA and/or protein levels of ULK1, Beclin1, ATG5, ATG7, and LC3II/LC3I were increased, while the level of P62 was reduced (Fig. 3E, F). Additionally, the level of LC3 puncta formation, which reflects the transient autophagosomal content based on the balance between the generation and degradation of autophagosomes [36], was analyzed in ST2 cells infected with GFP-LC3 adenovirus. We found that in the cells transfected with Osmr siRNA, the LC3 puncta formation was enhanced (Fig. 3G).
ERK signaling-mediated autophagy in stromal progenitor cells
U0126, the inhibitor of ERK signaling, was used to further analyze the effect of ERK signaling on autophagy and osteogenic differentiation. As expected, the phosphorylated protein level of ERK1/2 was downregulated in ST2 cells treated with U0126 (Fig. 4A). Moreover, the autophagy signaling was downregulated following the inactivation of ERK, as evidenced by the decreased levels of Beclin1, ATG5, ATG7, and LC3II/LC3I, and the increased level of P62 (Fig. 4B). Furthermore, in the presence of osteogenic medium, U0126 treatment blunted ALP staining and alizarin red staining of differentiated osteoblasts (Fig. 4C, D). Accordingly, the mRNA and protein levels of the key osteogenic factors were significantly declined (Fig. 4E, F). In addition, chloroquine (CQ), the autophagic pharmacological inhibitor, was also used to analyze the effect of autophagy on osteogenic differentiation. ALP staining and alizarin red staining of differentiated osteoblasts were attenuated after autophagy inactivation with chloroquine treatment (Fig. 4G, H). The mRNA and protein levels of the key osteogenic factors were significantly reduced in the cells treated with chloroquine (Fig. 4I, J).
OSMR-regulated osteogenic differentiation via ERK/autophagy signaling
To further investigate whether ERK/autophagy signaling mediates OSMR regulation of osteogenic differentiation, OSMR loss-of-function experiments were performed under the background of U0126 or chloroquine treatment in ST2 cells. We demonstrated that the stimulatory effect of Osmr siRNA on ERK/autophagy signaling was remarkably compromised in the presence of U0126 (Fig. 5A). Meanwhile, the stimulatory effect of Osmr siRNA on osteogenic differentiation was attenuated in the presence of U0126 or chloroquine, as evidenced by the blunted ALP staining (Fig. 5B) and the decreased levels of osteogenic factors in the cells treated with Osmr siRNA and U0126 or chloroquine vs. the cells treated with Osmr siRNA and vehicle (Fig. 5C, D).
OSMR silencing relieved the bone loss phenotype in OVX mice
We further explored if OSMR plays a role in the differentiation of progenitor cells and even in bone homeostasis in vivo. OVX and Sham mice were transplanted with BMSCs infected with either Osmr-KD LV or control LV, respectively. Histological analysis of the tibiae in the mice 4 weeks after surgery revealed that adipocytes in the marrow were more (Fig. 6A–C), and ALP-positive osteoblasts on the trabeculae were fewer (Fig. 6D, E) in “OVX/Ctrl LV” mice than in “Sham/Ctrl LV” mice. Transplantation of Osmr-silenced BMSCs to sham mice did not significantly alter marrow adipocyte number and adipocyte area, as well as ALP-positive osteoblasts (Fig. 6A–C). By contrast, the number and area of adipocytes in “OVX/Osmr-KD LV” mice were reduced (Fig. 6A–C) and the number of ALP-positive osteoblasts was increased (Fig. 6D, E) in “OVX/Osmr-KD LV group as compared to the “OVX/Ctrl LV” group. Moreover, 12 weeks after surgery, μCT analysis revealed that when compared to “Sham/Ctrl LV” mice, “OVX/Ctrl LV” mice showed a bone loss phenotype, as revealed by the decrease in bone volume percentage (BV/TV, 69% decrease), trabecular number (Tb.N, 40% decrease), trabecular thickness (Tb.Th, 27% decrease), and the increase in trabecular space (Tb.Sp, 64% increase). However, these parameters in “OVX/Ctrl LV” mice were ameliorated after transplantation of Osmr-silenced BMSCs in “OVX/Osmr-KD LV” mice (Fig. 6F–J). Briefly, BV/TV, Tb.N, and Tb.Th were increased by 83%, 24%, and 26%, respectively, and Tb.Sp was decreased by 15% in “OVX/Osmr-KD LV” group vs. “OVX/Ctrl LV” group.
OSMR in stromal progenitor cells regulated osteoclast differentiation
To explore whether OSMR in osteoblastic lineage regulates osteoclast formation, we investigated the correlation of RANKL and OPG to OSMR expression. The data showed that the mRNA and protein levels of RANKL were decreased and those of OPG were increased in the BMSCs underexpressing Osmr (Additional file 1: Fig. S7A-C). When cocultured with bone marrow osteoclast precursors, the BMSCs underexpressing Osmr largely inhibited osteoclast formation and downregulated the mRNA and/or protein expression levels of osteoclastogenic factors including NFATC1, CTSK, and TRAP (Additional file 1: Fig. S7D-G).
Furthermore, 2 weeks after surgery, the tibial marrow cells from “OVX/Ctrl LV” mice formed more osteoclasts and expressed higher levels of osteoclastogenic genes, while those from “Sham/Osmr-KD LV” group formed fewer osteoclasts and expressed lower levels of osteoclastogenic genes than those from “Sham/Ctrl LV” group when induced for differentiation (Fig. 7A–D). However, the induction of osteoclastogenesis in “OVX/Ctrl LV” mice was attenuated in the tibial marrow cells from “OVX/Osmr-KD LV” mice, as evidenced by the reduction in osteoclast number and mRNA and/or protein expression levels of osteoclastogenic genes (Fig. 7A–D).
Moreover, TRAP staining of the tibiae in the mice 4 weeks after surgery revealed that osteoclast number and surface on the trabeculae were more in “OVX/Ctrl LV” mice than in “Sham/Ctrl LV” mice. Transplantation of Osmr-silenced BMSCs to sham mice did not significantly alter osteoclast number and osteoclast surface (Fig. 7E–G). By contrast, the number and surface of osteoclasts in “OVX/Osmr-KD LV” mice were reduced as compared to the “OVX/Ctrl LV” group (Fig. 7E–G).