Summary
Mesenchymal stem cell (MSC)-mediated coupling of osteogenesis and angiogenesis is a critical phenomenon in bone formation. Herein, we investigated the role and mechanism of SGMS1 in the osteogenic differentiation of MSCs and, in combination with osteogenesis and angiogenesis, to discover new therapeutic targets for skeletal dysplasia and bone defects. SGMS1 addition accelerated MSC osteogenic differentiation, whereas SGMS1 silencing suppressed this process. Moreover, SGMS1 overexpression inhibited ceramide (Cer) and promoted sphingomyelin (SM) levels. SM treatment neutralized the suppressive effect of shSGMS1 on osteogenesis. SGMS1 restrained PP2A activity by regulating Cer/SM metabolism and thus enhanced the levels of phosphorylated Akt, Runx2, and vascular endothelial growth factor (VEGF). Furthermore, SGMS1 transcription was regulated by Runx2. SGMS1 increased MSC-mediated angiogenesis by promoting VEGF expression. SGMS1 addition promoted rat bone regeneration in vivo. In conclusion, SGMS1 induces osteogenic differentiation of MSCs and osteogenic-angiogenic coupling through the regulation of the Cer/PP2A/Akt signaling pathway.
Subject areas: Orthopedics, Molecular biology, Cell biology, Stem cells research
Graphical abstract
Highlights
-
•
SGMS1 promotes osteogenic differentiation and osteogenesis-angiogenesis coupling
-
•
SGMS1 facilitates osteogenesis in a calvarial defect rat model
-
•
Cer/PP2A/Akt signaling participates in osteogenic differentiation
-
•
SGMS1 regulates Cer/PP2A/Akt signaling
Orthopedics; Molecular biology; Cell biology; Stem cells research
Introduction
Skeletal dysplasia (SD) refers to a group of diseases characterized by abnormal bone formation owing to intrinsic disorders in bone growth, development, and/or differentiation.1 The overall prevalence of SD is at least one case per 1,000 births.2 The clinical manifestations of SD vary, involving abnormalities in growth, bone density, or bone morphology, reflecting complex etiological mechanisms. Treatment options for SD are limited. In the past few decades, >400 genes that can cause SD have been discovered to better understand the cellular and biological pathways involved in skeletal development,3 particularly congenital defects involving key regulators of bone formation in mesenchymal stem cells (MSCs) that may lead to rare genetic disorders of the bone.4 MSCs have attracted much attention in recent years owing to their ability to induce osteogenic differentiation and secrete growth factors.5 Osteogenic differentiation of MSCs is a complex, multistage process essential for normal bone formation, and this process is affected by multiple endogenous and environmental elements as well as multiple signaling pathways.6 Elucidating the mechanism of regulating MSC osteogenic differentiation may aid in the development of novel therapies for the clinical treatment of SD or other diseases involving bone regeneration.
Lipid rafts are specific microdomains in the plasma membrane that contain high levels of sphingomyelin (SM) and cholesterol and are considered important signaling platforms.7 SM is one of the major sphingolipid types and accounts for approximately 85% of the total sphingolipid content and 10%–20% of the total phospholipid content in the cell membrane.8 SM is generally distributed in the bone tissue, skin epidermis, and myelin sheath in nerve tissue. Abnormalities in SM may cause bone mineralization defects, including severe bone abnormalities, severe skeletal and dental mineralization defects, and epiphyseal dysplasia of the spine.9 SM synthetase (SMS) plays an important role in SM synthesis by transferring the phosphatidyl head group of phosphatidylcholine to the primary hydroxyl group of ceramide (Cer). SMS exists as two isomers: SMS1 (also known as SGMS1) and SMS2 (also known as SGMS2). In the osteoblast lineage, both SGMS1 and SGMS2 genes were highly expressed.10 Yoshikawa et al. suggested that SGMS2 regulated osteoclast differentiation by inducing the receptor activator of nuclear factor-κB ligand expression in mouse primary osteoblasts.11 Wesley et al. reported that SGMS1 deficiency reduced bone formation owing to impaired osteoblast differentiation.10 These findings suggested that SGMS1 is essential for bone formation. In our previous study, we demonstrated that SGMS1 and Cer/SM homeostasis played a vital role in senescence, maintenance of stemness, and differentiating ability of dental pulp stem cells regulated by receptor tyrosine kinase-like orphan receptor 2, the pathogenic gene for a rare SD.12 However, whether SGMS1 regulates MSC osteogenic differentiation, its mechanism, and its effect on SD development remains to be elucidated.
Protein phosphatase 2A (serine/threonine phosphatase, PP2A) comprises a constant subunit (A subunit), a variable subunit (B subunit), and a catalytic subunit (C subunit).13 The A subunit includes two subtypes of protein phosphatase 2 scaffold subunits α (PPP2R1A, Aα) and β (PPP2R1B, Aβ), which act as scaffold proteins and control the assembly of the B and C subunits, thereby regulating the enzymatic function of PP2A.14 Inactivation of the α-isoform of the PP2A catalytic subunit (PP2A Cα) facilitated bone formation by increasing osterix in MC3T3-E1 cells.15 Moreover, PP2A is involved in the regulation of many important physiological processes, such as apoptosis, growth, and signal transduction. PP2A inhibition expedited aortic valvular interstitial cell osteogenic differentiation through p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) signaling.16 PP2A Cα impeded the level of osteogenic genes and osteoblast differentiation in C3H10T1/2 cells.17 Nevertheless, whether PP2A affects the osteogenic differentiation of MSCs and the specific underlying mechanism remain unclear.
The present study investigated the biological function of SGMS1 in the osteogenic differentiation of MSCs and MSC-mediated osteogenesis-angiogenesis coupling and attempted to unravel the underlying mechanisms involved.
Results
SGMS1 facilitated the osteogenic differentiation of MSCs
MSCs were cultured in the osteogenic medium (OM) for 14 days to induce osteogenic differentiation according to a previously described method.18 To ascertain the effect of SGMS1 on osteogenesis, we compared the SGMS1 expression levels between the normal medium (NM) and OM groups. Compared with the NM group, SGMS1 mRNA and protein expression levels were upregulated in the OM group (Figures 1A and 1B), suggesting that SGMS1 might play a vital role in osteogenesis. Alkaline phosphatase (ALP) activity is a classic marker of early osteogenesis. The addition of SGMS1 further enhanced OM-stimulated ALP activity (Figure 1C). As ALP is an important marker of osteogenic differentiation, the formation of calcium nodules was assessed via alizarin red S staining. SGMS1 amplification was evaluated to further promote the formation of mineralized nodules in OM, which yielded results consistent with those of ALP activity (Figure 1D). Moreover, the mRNA and protein levels of osteogenesis-related genes (osteocalcin [OCN], Col-1, and osteopontin [OPN]) were enhanced by the addition of SGMS1 to OM (Figures 1E and 1F). These results suggested the potential role of SGMS1 in regulating the osteogenic differentiation of MSCs.
Figure 1.
SGMS1 facilitates osteogenic differentiation of MSCs
(A and B) mRNA and protein expression levels of SGMS1 assessed via qPCR and western blot assays, respectively.
(C and D) ALP and ARS staining of MSCs (100 μm; bar).
(E) mRNA and protein (F) expression levels of OCN, Col-1, and OPN assessed via qPCR and western blot assays, respectively. Data are represented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ALP: alkaline phosphatase; ARS: Alizarin red S; MSC: mesenchymal stem cell; OCN: osteocalcin; OPN: osteopontin; qPCR: quantitative polymerase chain reaction; SGMS1: sphingomyelin synthase 1; NM: normal medium; OM: osteogenic medium.
Deficiency of SGMS1 altered the MSC phenotype
To investigate the role of SGMS1 further in the osteogenic differentiation of MSCs, MSCs were infected with shSGMS1 to silence SGMS1 expression. The ALP activity and calcium levels were increased in OM, and these changes were partially neutralized by shSGMS1 (Figures 2A and 2B). Moreover, transfection with shSGMS1 partly reversed the OM-induced increase in OCN, Col-1, and OPN in MSCs (Figures 2C and 2D). Furthermore, immunofluorescence (IF) confirmed that the OM-triggered upregulation of osteogenesis-related gene expression (Runx2) was suppressed by SGMS1 depletion (Figure 2E). To summarize, SGMS1 depletion attenuated the ability of MSCs to undergo osteogenic differentiation.
Figure 2.
SGMS1 deficiency alters the MSC phenotype
MSCs were cultured in OM for 14 days to induce osteogenic differentiation.
(A and B) Differentiation was confirmed with ARS and ALP staining (100 μm; bar).
(C and D) mRNA and protein expression levels of OCN, Col-1, and OPN determined via qPCR and western blot assays, respectively.
(E) Immunofluorescence staining showing Runx2 expression (100 μm; bar). Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ALP: alkaline phosphatase; ARS: Alizarin red S; MSC: mesenchymal stem cell; OCN: osteocalcin; OPN: osteopontin; qPCR: quantitative polymerase chain reaction; SGMS1: sphingomyelin synthase 1; NM: normal medium; OM: osteogenic medium.
SGMS1 is involved in the osteogenic differentiation of MSCs by regulating Cer/SM metabolism
Cer and SM are involved in various cellular functions, including signaling pathways for cell growth, differentiation, and apoptosis.19 Pulsatilla chinensis saponins disturb the balance of Cer/SM by decreasing SGMS1 levels,20 suggesting that SGMS1 can affect Cer/SM metabolism. The amplification of SGMS1 enhanced the inhibitory effect of OM on Cer levels (Figure 3A). SGMS1 knockdown partly reversed the inhibitory effect of OM on Cer levels. The effect of SGMS1 on SM was opposite to that on Cer levels (Figure 3B). Furthermore, SM treatment eliminated shSGMS1-mediated rescue of ALP activity and calcium levels in OM-cultured MSCs (Figures 3C and 3D). Moreover, the suppressive effect of shSGMS1 on OCN, Col-1, OPN, and Runx2 expression was counteracted with SM treatment in OM-cultured MSCs (Figures 3E–3G). Taken together, SGMS1 might affect the osteogenic differentiation potential of MSCs by regulating the metabolic balance of Cer/SM.
Figure 3.
Involvement of SGMS1 in the osteogenic differentiation of MSCs by regulating Cer/SM metabolism
MSCs were cultured in OM for 14 days to induce osteogenic differentiation.
(A and B) Cer and SM expression levels detected using LC-MS/MS.
(C and D) Osteogenic differentiation verified via ARS and ALP staining (100 μm; bar).
(E and F) mRNA and protein expression levels of OCN, Col-1, and OPN determined via qPCR and western blot assays, respectively.
(G) Immunofluorescence staining to assess Runx2 expression (100 μm; bar). Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ALP: alkaline phosphatase; ARS: Alizarin red S; Cer: ceramide; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MSC: mesenchymal stem cell; OCN: osteocalcin; OPN: osteopontin; qPCR: quantitative polymerase chain reaction; SGMS1: sphingomyelin synthase 1; SM: sphingomyelin; NM: normal medium; OM: osteogenic medium.
Effect of SGMS1 on PP2A activity by regulating Cer/SM metabolism and p-Akt, Runx2, and VEGF expression
C16 Cer could induce PP2A activation. Cer is converted into SM by SMS.21 The Cer/SM balance is crucial to the fate of many cell types because Cer and SM exhibit opposite effects in terms of cell activity.22 Therefore, we hypothesized that SGMS1 might affect PP2A activity by regulating Cer/SM metabolism. Unsurprisingly, Cer partially reversed the effect of SGMS1 amplification on the inhibition of PP2A activity, and SM counteracted the effect of SGMS1 knockdown on PP2A activity stimulation in OM-induced MSCs (Figure 4A). PP2A regulates various cellular processes via Akt dephosphorylation.23 Akt is a widely recognized osteogenic activator. Zhang et al. indicated that the Akt pathway is involved in the osteogenic differentiation of MSCs.24 Therefore, we inferred that PP2A might regulate the osteogenic differentiation of MSCs through in vitro signaling. To verify this hypothesis, we established PP2A-overexpressed and -knockdown cells to detect their effect on p-Akt (Serine 473). As shown in Figure 4B, PP2A silencing increased, and PP2A amplification counteracted the effect of OM on p-Akt induction in MSCs. Western blot results showed that the levels of p-Akt, Runx2, and vascular endothelial growth factor (VEGF) were enhanced by PP2A inhibition and diminished by PP2A overexpression in OM-cultured MSCs, which were partially reversed with MK2206 and SC79 treatments, respectively (Figure 4C). These data suggest that the involvement of SGMS1 in the osteogenic differentiation of MSCs might affect PP2A activity by regulating the metabolic balance of Cer/SM and consequently regulating p-Akt, Runx2, and VEGF levels.
Figure 4.
SGMS1 affects PP2A activity by regulating Cer/SM metabolism and p-Akt, Runx2, and VEGF expression
MSCs were cultured in OM for 14 days to induce osteogenic differentiation.
(A) PP2A activity assessed using a commercial kit.
(B) Western blot assay to measure PP2A, Akt, and p-Akt protein expression levels in MSCs.
(C) Western blot assay to detect the expression of p-Akt, Akt, Runx2, and VEGF in MSCs. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cer: ceramide; MSC: mesenchymal stem cell; SGMS1: sphingomyelin synthase 1; SM: sphingomyelin; VEGF: vascular endothelial growth factor; NM: normal medium; OM: osteogenic medium; PP2A: protein phosphatase 2A.
PP2A is implicated in the regulation of SGMS1 on the osteogenic differentiation of MSCs
Next, we probed whether SGMS1 affected the osteogenic differentiation potential of MSCs through the regulation of PP2A. As shown in Figures 5A and 5B, SGMS1 overexpression reversed PP2A amplification as well as reduced ALP activity and calcium levels in OM-stimulated MSCs. SGMS1 deficiency reduced the increased ALP activity and calcium levels caused by PP2A silencing in OM-stimulated MSCs. Moreover, SGMS1 addition partially neutralized the repressive effect on OCN, Col-1, OPN, and Runx2 expression caused by PP2A amplification in OM-stimulated MSCs. Moreover, PP2A knockdown further enhanced OCN, Col-1, OPN, and Runx2 expression in OM-induced MSCs, which was abolished by SGMS1 silencing (Figures 5C–5E). These findings suggest that the SGMS1/PP2A axis is implicated in the osteogenic differentiation potential of MSCs.
Figure 5.
PP2A was implicated in the regulation of SGMS1 in the osteogenic differentiation of MSCs
MSCs were cultured in OM for 14 days to induce osteogenic differentiation.
(A and B) Osteogenic differentiation of MSCs assessed via ALP and ARS staining (100 μm; bar).
(C and D) mRNA and protein expression levels of OCN, Col-1, and OPN determined by via qPCR and western blot assays, respectively.
(E) Immunofluorescence staining performed to assess Runx2 expression (100 μm; bar). Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ALP: alkaline phosphatase; ARS: Alizarin red S; MSC: mesenchymal stem cell; OCN: osteocalcin; OPN: osteopontin; qPCR: quantitative polymerase chain reaction; SGMS1: sphingomyelin synthase 1; NM: normal medium; OM: osteogenic medium; PP2A: protein phosphatase 2A.
Transcription factor Runx2 accelerated SGMS1 transcription in the osteogenic differentiation of MSCs
The addition of Runx2 upregulated SGMS1 expression, and the depletion of Runx2 downregulated SGMS1 expression in both the NM and OM (Figures 6A and 6B). Bioinformatics tools revealed that the transcription factor Runx2 could bind to the SGMS1 gene promoter (Figure 6C). Immunoprecipitation with specific Runx2 antibodies successfully pulled down the SGMS1 promoter but not the α-satellite, suggesting that Runx2 binds directly to the SGMS1 promoter (Figure 6D). Compared with that of the negative control, the vector containing the E2 binding site had no significant difference in luciferase activity, whereas the vector containing the E1 binding site had enhanced luciferase activity, indicating that the E1 binding site was a functional binding site (Figure 6E). These data indicated that Runx2 transcriptionally activated SGMS1 to promote its expression in MSCs.
Figure 6.
Transcription factor Runx2 accelerated SGMS1 transcription in the osteogenic differentiation of MSCs
MSCs were cultured in OM for 14 days to induce osteogenic differentiation.
(A and B) mRNA and protein expression levels of SGMS1 determined via qPCR and western blot assays, respectively.
(C) Binding motifs of Runx2 and the top two binding sites obtained using JASPAR.
(D and E) Relationship between Runx2 and SGMS1 verified using chromatin immunoprecipitation (ChIP) and luciferase experiments. Data are represented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001. MSC: mesenchymal stem cell; qPCR: quantitative polymerase chain reaction; SGMS1: sphingomyelin synthase 1; NM: normal medium; OM: osteogenic medium.
SGMS1 silencing impeded osteogenesis-angiogenesis coupling
VEGF is a critical growth factor that regulates blood vessel development and angiogenesis. Osteogenesis-angiogenesis coupling directly affects osteoblast function in bone formation. Therefore, we investigated whether SGMS1 affects angiogenesis and promotes bone formation through VEGF regulation. The culture supernatants from MSCs were used to culture human umbilical vein endothelial cells (HUVECs) for related experiments. As shown in Figures 7A and 7B, VEGF expression was upregulated in HUVECs cultured with OM-induced MSC supernatants. SGMS1 deletion inhibited the elevated expression of VEGF, and the addition of VEGF partially offset the inhibitory effect of Lv-shSGMS1. Moreover, the cell migration, invasion, and tubule formation abilities of HUVECs increased after they were cultured with OM-induced MSC-containing supernatants. SGMS1 knockdown reduced the MSC-induced angiogenesis potential of HUVECs, which was reversed with VEGF addition (Figures 7C–7E). These findings emphasized that SGMS1 could regulate osteogenesis-angiogenesis coupling by regulating VEGF.
Figure 7.
SGMS1 silencing impeded osteogenesis-angiogenesis coupling
MSCs were cultured in OM for 14 days to induce osteogenic differentiation, and the conditional medium was collected to induce HUVECs.
(A and B) VEGF mRNA and protein expression levels detected in HUVECs.
(C–E) Migration (100 μm; bar), invasion (200 μm; bar), and tubule formation (100 μm; bar) of HUVECs. Data are represented as mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001. HUVEC: human umbilical vein endothelial cell; MSC: mesenchymal stem cell; VEGF: vascular endothelial growth factor; SGMS1: sphingomyelin synthase 1; NM: normal medium; OM: osteogenic medium.
SGMS1 promoted in vivo bone regeneration
We investigated whether the effect of SGMS1 on MSCs can be replicated in vivo. Compared with the control group, the SGMS1 overexpression group showed that the bone regeneration area increased at 4 and 8 weeks postoperatively using microcomputed tomography images (Figure 8A). Moreover, SGMS1 addition facilitated the formation of bone island, new blood vessels, and fibrous tissue, as observed in the H&E and Masson’s trichrome staining images (Figure 8B). Furthermore, IF assay results indicated that SGMS1 addition promoted the expression of Runx2 and CD31 (Figure 8C). These data indicate that SGMS1 increased wound healing ability in the bone of rats with calvarial defects.
Figure 8.
SGMS1 promotes bone regeneration
(A) Microcomputed tomography images and analysis of the calvarial defect in rats.
(B) H&E and Masson’s trichrome staining to detect bone repair (500 or 100 μm; bar).
(C) Runx2 and CD31 expression levels detected using the IF assay. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01. H&E: hematoxylin-eosin; IF: immunofluorescence; SGMS1: sphingomyelin synthase 1; BMD: bone mineral density; BV: bone volume; TV: total volume of the bone.
Discussion
SD is a group of heterozygous genetic disorders characterized by bone malformations.25 Endochondral and endomembrane ossification are two processes of bone formation that begin with the coagulation of mesenchymal cells in the parts of the body that eventually form bone elements.26 The coagulate that forms the skull undergoes intramembrane ossification, wherein mesenchymal cells directly differentiate into osteoblasts, thereby transforming the clots into true bone tissue. MSCs, also known as multipotent stromal cells, have the potential to undergo osteogenic differentiation in specific microenvironments and regulate homeostasis of bone metabolism in vivo.27,28 Accumulating evidence suggests that osteogenesis-angiogenesis coupling could effectively promote bone regeneration.29 In the present study, in vitro experiments were performed with MSCs to verify the effects of SGMS1 on their osteogenic differentiation potential and related vascular coupling, as well as to identify any novel promising targets for the treatment of SD and bone defects. Interestingly, we found that SGMS1 promoted osteogenic differentiation and osteogenic-angiogenesis coupling. Mechanically, we determined that SGMS1 affected PP2A activity by regulating the metabolic balance of Cer/SM, thereby regulating p-Akt, Runx2, and VEGF expression in OM-induced MSCs. Moreover, SGMS1 induced by Runx2 contributed to the osteogenic differentiation of MSCs. Furthermore, the addition of SGMS1 expedited osteogenesis-angiogenesis coupling through VEGF regulation. Meanwhile, in vivo studies confirmed that SGMS1 promoted bone repair and angiogenesis in skull defect models. The mechanism of SGMS1 function on MSCs is illustrated in Figure 9.
Figure 9.
An illustration summarizing the findings of this study
Upregulation of SGMS1 expression disturbed the metabolic balance between Cer and SM; the expression of Cer was downregulated, whereas that of SM was upregulated. Downregulation of Cer reduced PP2A activity and increased p-Akt, thereby promoting VEGF-mediated angiogenesis and Runx2-mediated osteogenic differentiation and accelerated bone regeneration. In addition, Runx2 promoted SGMS1 transcription to form a positive feedback regulatory loop.
SGMS1 has been studied in hepatocellular carcinoma,30 steatohepatitis,31 and ovarian cancer,32 among others. Additionally, osteoblast-specific loss of SGMS1 leads to delayed ossification. SGMS1 plays a critical role in bone development by coregulating osteoblast development with BMP2 signaling.10 Similarly, Ramsay et al. indicated that SGMS knockout inhibited mouse osteogenesis, as reflected in the suppression of osteogenesis-associated proteins and reduction of mineralization.33 Consistent with previous studies, we observed that SGMS1 can promote osteogenic differentiation of MSCs. SGMS1 overexpression enhanced and SGMS1 depletion decreased ALP activity; formation of calcified nodules; and OCN, Col-1, OPN, and Runx2 levels in OM-cultured MSCs. Altogether, our findings suggest that SGMS1 can facilitate the osteogenic differentiation of MSCs.
Cer and SM are the main components of sphingolipid. Cer is a bioactive lipid involved in various cellular processes, including necrosis, apoptosis, and autophagy-dependent cell death. In contrast, SM regulates other cellular processes, such as cell migration and proliferation, by altering receptor-mediated signaling in lipid microdomains.9 Cer/SM balance has been a critical aspect for the determination of cell fate, proliferation, and survival. Moreover, as previously reported, increased Cer levels after tumor necrosis factor-α treatment lead to osteoblast apoptosis through the nuclear factor-κB pathway.34 C24:1 Cer levels in serum extracellular vesicles increased with age, which might cause senescence of bone-marrow-derived MSCs in human bones.35 Meanwhile, local SM catabolism was critical to the mineralization process in healthy bones.9 These studies confirm that Cer and SM are critical components for bone formation and resorption. Therefore, we hypothesized that SGMS1 might regulate the metabolic balance of Cer/SM and affect the osteogenic differentiation of MSCs. In this study, we found that Cer decreased and SM increased in OM-induced cells. SGMS1 overexpression decreased Cer expression levels and increased SM expression levels. SGMS1 knockdown had the opposite effect. Moreover, the inhibitory effect of SGMS1 knockdown on osteogenic differentiation was reversed following SM treatment.
Cer was previously described as a PP2A activator.36 The role of PP2A in suppressing osteogenic differentiation through p38 MAPK and ERK signaling has been confirmed in aortic valvular interstitial cells.16 PP2A in LepR+ MSCs was involved in embryonic and postnatal endochondral ossification via dephosphorylation of Runx2.37 Moreover, many studies have indicated that PP2A is a negative factor in osteoblast differentiation.38 Consistent with the previous findings, PP2A activity was reduced in OM, SGMS1 overexpression further inhibited PP2A activity, and Cer treatment partially reversed SGMS1 inhibition, thereby reducing PP2A activity. Moreover, SGMS1 knockdown increased PP2A activity that was decreased by OM, and SM treatment again decreased PP2A activity. Furthermore, SGMS1 overexpression significantly counteracted the inhibitory effect of PP2A on osteogenic differentiation. SGMS1 knockdown partially reversed the effect of shPP2A on promoting osteogenic differentiation. Numerous studies have demonstrated the activation of the Akt pathway in osteogenic differentiation.39 The PI3K-Akt signaling pathway regulates BMP2-induced osteogenic differentiation of MSCs.40 Kdm5c regulated osteogenesis and bone formation via the PI3K/Akt/HIF1α axis.41 PP2A, as a negative regulator of the Akt pathway, is involved in the progression of many diseases.42 In this study, p-Akt (Ser473) expression levels were increased in OM, and the deletion of PP2A further increased the expression levels of p-Akt, whereas PP2A overexpression weakened the effect of OM on increasing p-Akt levels. To further verify the effect of Akt signaling on PP2A-mediated osteogenic differentiation in OM-challenged MSCs, MK2206, a well-recognized Akt inhibitor, and SC79, an Akt agonist, were applied to inactivate and activate the Akt signaling. MK2206 reversed the effects of PP2A silencing on Runx2 and VEGF promotion. SC79 counteracted the inhibitory effect of PP2A on Runx2 and VEGF.
Transcription factors can accelerate or inhibit disease progression by altering the expression of other genes. Runx2 is a transcription factor that can activate or inhibit transcriptional activity. Recent studies have suggested that the mutual stabilization of ABL and TAZ regulates osteoblastogenesis through the transcription factor Runx2.43 In this study, we elucidated that the upregulated expression of SGMS1 was attributed to the activation of SGMS1 transcription in MSCs via Runx2; thus, Runx2 and SGMS1 act in a positive feedback loop. This finding confirmed the key role of Runx2 in the regulation of other genes and their biological activities, providing a reference for studying the role of genes with abnormal expression in osteogenic differentiation.
Osteogenesis-angiogenesis coupling is essential for maintaining bone mass and bone regeneration as blood vessels transport oxygen, substances, and nutrients essential for bone development.44 Neovascularization injury can inhibit trabecular bone regeneration and delay fracture healing. For example, bone density and strength decrease after vascular injury during surgery.45 VEGF is considered one of the most critical factors for bone regeneration.46 Moreover, the regulatory effect of VEGF on osteogenesis-angiogenesis coupling has been confirmed in several studies.47 Therefore, we hypothesized that SGMS1 might affect osteogenic-angiogenic coupling through VEGF regulation, which may further guide SD or bone defect treatment. Our study also showed that VEGF expression was enhanced in OM. SGMS1 depletion suppressed VEGF expression, which was abolished by VEGF addition. Moreover, VEGF counteracted the inhibitory effect of SGMS1 silencing on vascular endothelial cell migration, invasion, and tubule formation. Taken together, these results suggest that SGMS1 promotes osteogenic-angiogenic coupling by regulating VEGF. In vivo studies further demonstrated that SGMS1 promoted bone regeneration and angiogenesis.
In summary, this study highlights the contribution of SGMS1 to promoting the osteogenesis of MSCs and MSC-dependent angiogenesis. Runx2-induced SGMS1 accelerated the osteogenic differentiation of MSCs by regulating the Cer/PP2A/Akt signaling axis in vitro. Additionally, SGMS1 promotes MSC-regulated angiogenesis by regulating VEGF expression. In vivo experiments confirmed the effect of SGMS1 on bone regeneration. These findings suggest that SGMS1 might be a promising therapeutic target for treating SD and bone defects.
Limitations of the study
No clinical trials were conducted in this study, and we will try to improve this limitation in future studies. In addition, the upstream mechanism of the SGMS1 upregulation in SD requires further investigation.
STAR★Methods
Key resources table
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| SGSM1 Rabbit monoclonal antibody | Abcam | ab171943; RRID:AB_2479279 |
| OCN Rabbit polyclonal antibody | Abcam | ab93876; RRID:AB_10675660 |
| Col-1 Rabbit monoclonal antibody | Abcam | ab260043; RRID:AB_305411 |
| OPN Rabbit polyclonal antibody | Abcam | ab63856; RRID:AB_1663286 |
| PP2A Rabbit polyclonal antibody | Proteintech | 10321–1-AP; RRID:AB_2284352 |
| Akt Mouse monoclonal antibody | Proteintech | 60203-2-Ig; RRID:AB_2919902 |
| p-Akt Rabbit Recombinant antibody | Proteintech | 80455-1-RR; RRID:AB_2923906 |
| Runx2 Rabbit Polyclonal antibody | Proteintech | 20700-1-AP; RRID:AB_2722783 |
| VEGF Rabbit polyclonal antibody | Abcam | ab46154; RRID:AB_2212642 |
| GAPDH Mouse monoclonal antibody | Abcam | ab8245; RRID:AB_2107448 |
| Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP | Invitrogen | Cat#31460; RRID: AB_228341 |
| Goat Anti-Mouse IgG H&L (HRP) | Abcam | Cat#ab6789; RRID:AB_955439 |
| Bacterial and virus strains | ||
| shSGMS1 | GenePharma, Suzhou, China | 5′-TGACCCTCCACC TCGACATTA–3′ |
| shPP2A | GenePharma, Suzhou, China | 5′-CCGTTTGATGAG AGGCTGTTT-3′ |
| shRunx2 | GenePharma, Suzhou, China | 5′-CAGCACTCCATA TCTCTACTA-3′ |
| Chemicals, peptides, and recombinant proteins | ||
| FBS | GIBCO, Grand Island, NY, USA | A5669701 |
| Dexamethasone | Aladdin | D137736 |
| Dulbecco’s modified Eagle’s medium | Gibco | Cat#C11995500BT |
| Penicillin and streptomycin | Solarbio | Cat#P1400 |
| TRIzol | Invitrogen | Cat#15596026 |
| Critical commercial assays | ||
| cDNA Reverse Transcription Kit | Applied Biosystems | 4368814 |
| SYBR Green Master Mix kit | Takara | 639676 |
| Enhanced Chemiluminescence Kit | Millipore | CS1000 |
| PP2A Immunoprecipitation Phosphatase Assay Kit | Millipore | 17-313 |
| Experimental models: Cell lines | ||
| MSCs | Cyagen Biosciences | HUXMA-01001 |
| HUVECs | Procell Life Science&Technology Company | CL-0122 |
| Experimental models: Organisms/strains | ||
| Sprague Dawley rats | SiPeiFu Biotechnology Co., Ltd | N/A |
| Oligonucleotides | ||
| SGMS1 Forward: 5′-TAGCTCGAC CAATGGCTGCAA C-3′ | This paper | N/A |
| SGMS1 Reverse: 5′-TTCTCACGG TGGACAGGTGTC T-3′ | This paper | N/A |
| OCN Forward: 5′-CGCTACCTGTAT CAATGGCTGG-3′ | This paper | N/A |
| OCN Reverse: 5′-CTCCTGAAAGCC GATGTGGTC A-3′ | This paper | N/A |
| Col-1 Forward: 5′-GATTCCCTG GACCTA AAGGTG C-3′ | This paper | N/A |
| Col-1 Reverse:5′-AGCCTCTCCATC TTTGCCAGC A-3′ | This paper | N/A |
| OPN Forward: 5′-CGAGGTGATAGT GTGGTTTATGG-3′ | This paper | N/A |
| OPN Reverse: 5′-GCACCATTCAAC TCC TCG CTT TC-3′ | This paper | N/A |
| VEGF Forward: 5′-TTGCCTTGC TGCTCTACCTCC A-3′ | This paper | N/A |
| VEGF Reverse: 5′-GAT GGC AGT AGC TGC GCT GAT A-3′ | This paper | N/A |
| GAPDH Forward: 5′-GTC TCC TCT GAC TTC AAC AGC G-3′ | This paper | N/A |
| GAPDH Reverse: 5′-ACC ACC CTG TTG CTG TAG CCA A-3′ | This paper | N/A |
| Software and algorithms | ||
| GraphPad Prism v7 software | GraphPad | N/A |
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Cheng-hong Yin (yinchh@ccmu.edu.cn).
Material availability
This study did not generate new unique reagents.
Data and code availability
Western blot original data have been deposited at Mendeley and publicly available as of the date of publication. The DOI and accession numbers are listed in the key resources table. This paper does not report any original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Experimental model and study participant details
Cells
Human bone marrow MSCs were obtained from Cyagen Biosciences (Guangzhou, China). Human umbilical vein endothelial cells (HUVECs) were obtained from Procell Life Science &Technology Company (Wuhan, China). All the cell lines included in this study have been authenticated by STR profiling and tested for mycoplasma contamination. MSCs were cultured in an α-modified essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA) and 1% penicillin–streptomycin and incubated in a humid environment containing 5% CO2 at 37°C. HUVECs were obtained from Procell Life Science &Technology Company (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% FBS and 1% penicillin–streptomycin.
Animals
All animal experiments were conducted with the approval of the PLA General Hospital Ethics Committee (approval no.2018-X14-40). Thirty-six male Sprague Dawley rats were used in this study.
Method details
Cell lines and cell culture
Human bone marrow MSCs (HUXMA-01001; Cyagen Biosciences, Guangzhou, China) were cultured in an α-modified essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA) and 1% penicillin–streptomycin and incubated in a humid environment containing 5% CO2 at 37°C. Osteogenic differentiation was induced using a medium containing β-glycerol phosphate (10 mM), trisodium salt of 2-phospho-ascorbate (50 mM), dexamethasone (10 nM), and L-ascorbic acid (20 μM). The culture medium was replenished every 3 days. Human umbilical vein endothelial cells (HUVECs) were obtained from Procell Life Science &Technology Company (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% FBS and 1% penicillin–streptomycin.
Animal experiments
All animal experiments and protocols of this study were approved by the PLA General Hospital Ethics Committee (approval no.2018-X14-40). Thirty-six male Sprague Dawley rats (6 weeks old; 250-300g) were purchased from SiPeiFu Biotechnology Co., Ltd (Beijing, China). The rats were anesthetized via intraperitoneal injections of Zoletil (30 mg/kg body weight) and Rompun® (10 mg/kg body weight). After the skull was shaved, the shaven area was cleaned with an alcohol swab, following which the hair was trimmed, and the skin tissue was recovered. Skull defects of critical dimensions (4 mm inner diameter/5 mm outer diameter) were established using trephine burs. The experimental animals were randomly divided into three groups (n = 6): Control (20 μL of fibrin gel was implanted into the defects), Lv-NC (5 × 105 MSCs transfected with Lv-NC were mixed with fibrin gel and implanted into the defects), Lv-SGMS1 (5 × 105 MSCs transfected with Lv-SGMS1 were mixed with fibrin gel and implanted into the defects). After 4- or 8-week treatment, the rats were euthanized, and their skulls were removed and fixed in containers.
Cell transfection and treatment
Short hairpin RNAs (shRNAs) targeting SGMS1 (shSGMS1, 5′-TGA CCC TCC ACC TCG ACA TTA–3′), PP2A (shPP2A, 5′-CCG TTT GAT GAG AGG CTG TTT-3′), Runx2 (shRunx2, 5′-CAG CAC TCC ATA TCT CTA CTA-3′), and shNC were purchased and inserted into lentivirus vectors (GenePharma, Suzhou, China). SGMS1, PP2A, Runx2, and vascular endothelial growth factor (VEGF) overexpression lentivirus (Lv-SGMS1, Lv-PP2A, Lv-Runx2, and Lv-VEGF) and negative control (Lv-NC) were purchased from OBiO Technology (Shanghai, China). Cell transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). For drug treatment, 125 μM SM (#85615; Sigma-Aldrich), 10 μM SC79 (HY-18749; MCE, Monmouth Junction, NJ, USA), and 5 μM MK2206 (HY-108232; MCE) were used to treat the cells according to the experimental protocols. To determine the angiogenesis-related functions of MSCs, HUVECs were cultured under different conditions: (1) fresh medium (NM), (2) conditioned osteogenic medium from MSCs (OM), (3) conditioned medium from MSCs with Lv-shNC (OM + Lv-shNC), conditioned medium from MSCs with Lv-shSGMS1 (OM + Lv-shSGMS1), (4) conditioned medium from MSCs with Lv-shSGMS1 + Lv-NC (OM + Lv-shSGMS1 + Lv-NC), and (5) conditioned medium from MSCs with Lv-shSGMS1 + Lv-VEGF (OM + Lv-shSGMS1 + Lv-VEGF).
Quantitative polymerase chain reaction assay
Total RNA was extracted from cells using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA). Reverse transcription was carried out with 0.5 μg of RNA sample using a cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). A quantitative polymerase chain reaction (qPCR) assay was performed using the SYBR Green Master Mix kit (Takara, Tokyo, Japan) on an Applied Biosystems 7500 Fast Dx Real-Time PCR System (Life Technologies). Relative mRNA expression was calculated according to the 2−ΔΔCt method and normalized to that of GAPDH. The following primers were used for the assay: SGMS1 (forward: 5′-TAG CTC GAC CAA TGG CTG CAA C-3′; reverse: 5′-TTC TCA CGG TGG ACA GGT GTC T-3′); osteocalcin (OCN) (forward: 5′-CGC TAC CTG TAT CAA TGG CTG G-3′; reverse: 5′-CTC CTG AAA GCC GAT GTG GTC A-3′; reverse: 5′-CTC CTG AAA GCC GAT GTG GTC A-3′); collagen I (Col-1) (forward: 5′-GAT TCC CTG GAC CTA AAG GTG C-3′; reverse: 5′-AGC CTC TCC ATC TTT GCC AGC A-3′); osteopontin (OPN) (forward: 5′-CGA GGT GAT AGT GTG GTT TAT GG-3′; reverse: 5′-GCA CCA TTC AAC TCC TCG CTT TC-3′); and VEGF (forward: 5′-TTG CCT TGC TGC TCT ACC TCC A-3′, reverse: 5′-GAT GGC AGT AGC TGC GCT GAT A-3′) and GAPDH (forward: 5′-GTC TCC TCT GAC TTC AAC AGC G-3′, reverse: 5′-ACC ACC CTG TTG CTG TAG CCA A-3′).
Western blotting assay
Total protein was isolated from MSCs using radio immunoprecipitation buffer. The concentration of the extracted protein was determined using the bicinchoninic acid method. For separation of the extracted proteins, polyacrylamide gel electrophoresis was performed using 20-μg protein sample loaded onto 10% sodium dodecyl sulfate polyacrylamide gels, followed by electroblotting onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated overnight with primary antibodies at 4°C, followed by incubation with horseradish peroxide–conjugated secondary antibodies. Finally, the membranes were examined using the Enhanced Chemiluminescence Kit (Millipore) and analyzed using ImageJ software (National Institutes of Health, USA). Primary antibodies specific to SGMS1 (ab171943, 1:2000 dilution; Abcam, Cambridge, MA, USA), OCN (ab93876, 1:1000 dilution; Abcam), Col-1 (ab260043, 1:1000 dilution; Abcam), OPN (ab63856, 1:1000 dilution; Abcam), PP2A (10321–1-AP, 1:500 dilution; Proteintech, Wuhan, China), Akt (60203-2-Ig, 1:5000 dilution; Proteintech), phosphor(p)-Akt (Serine 473; 80455-1-RR, 1:5000 dilution; Proteintech), Runx2 (20700-1-AP, 1:500 dilution; Proteintech), VEGF (ab46154, 1:500 dilution; Abcam), and GAPDH (ab8245, 1:5000 dilution; Abcam) were used in the study.
Alizarin red S and alkaline phosphatase staining assays
Fourteen days after osteogenic induction, Alizarin red S (ARS) and alkaline phosphatase (ALP) staining assays were performed according to the manufacturer’s instructions. For the ARS staining assay, cells were fixed with 4% paraformaldehyde for 20 min, followed by incubation with the ARS solution (Cyagen Biosciences) for 30 min. Images were captured using a Nikon microscope (Nikon, Tokyo, Japan). For the ALP staining assay, cells were washed thrice (5 min each) with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the MSCs were incubated with ALP solution (CWBIO, Beijing, China) for 20 min. The stained cells were viewed under an optical microscope, and the images were captured.
Immunofluorescence staining
Immunofluorescence (IF) assay was performed to assess the expression level of Runx2 or CD31. Briefly, cells or tissues were fixed with 4% paraformaldehyde and permeabilized at room temperature. Subsequently, the samples were blocked for 30 min and incubated with anti-Runx2 (20700-1-AP, 1:200 dilution; Proteintech) and anti-CD31 (CL647-65058, 1:100 dilution; Proteintech) at 4°C for 16 h. Thereafter, the cells or tissues were incubated with fluorescent dye-conjugated secondary antibody. Next, the cells were washed with PBS and stained with DAPI (Beyotime, Shanghai, China). Images of the stained cells were captured using a confocal microscope (Nikon, Tokyo, Japan).
PP2A phosphatase activity assay
PP2A activity was assessed using the PP2A Immunoprecipitation Phosphatase Assay Kit (Millipore). The cells were lysed with the lysis buffer and subjected to ultrasound for 10 s, followed by centrifugation for 5 min at 2000× g. Next, the total protein extract was incubated with the anti-PP2A-C subunit antibody (4 μg) at 4°C for 18 h. Subsequently, 40 μL of Protein A agarose slurry was added and subjected to shock for 2 h. The beads were washed thrice with 700 μL of tris-buffered saline and once with 500 μL of Ser/Thr assay buffer, followed by incubation with 60 μL of diluted phosphopeptide and 20 μL of Ser/Thr assay buffer for 10 min at 30°C. After centrifugation, the absorbance of the samples was measured at 650 nm.
Liquid chromatography-tandem mass spectrometry
Cells were sonicated with 0.1 mL of methanol/butanol for 30 s. Then, 0.05 mL of 0.5 M phosphate buffer, 0.2 mL of water, and 0.7 mL of butanol were added. The samples were shaken well and subjected to ultrasound for 3 min and centrifuged for 5 min at 9000× g. After the upper phase was collected, the remaining solution was resuspended in 0.35 mL of hexane–ethyl acetate solution. The upper phase was again collected and mixed with the previously collected upper phase, followed by the addition of 0.7 mL of methanol. Subsequently, 10% volume of that solution was used to measure SM and Cer levels using a liquid chromatography-tandem mass spectrometry system (Ultimate 3000 LC system; Thermo Fisher Scientific, Rockford, IL, USA).48
Transwell assay
Cell invasion ability was measured using Transwell insert chambers (8-μm pore size; Corning) coated with Matrigel. Briefly, HUVECs were resuspended in serum-free medium and seeded in the upper chamber. A complete medium supplemented with 10% FBS was added to the lower chamber. After 48 h, the cells were stained with 0.1% crystal violet. The stained cells were observed under a light microscope, and images were captured.
Wound healing assay
For cell migration assay, HUVECs were seeded into 6-well plates, after which the cell monolayer was scratched using a pipette tip to create a wound. Cell movement and migration were monitored under a microscope. Photographs were collected immediately and 24 h after scratching.
Tubule formation assay
HUVECs were seeded at a density of 1.5 × 104 per well into Matrigel-coated 96-well plates and incubated in the above medium (as mentioned in the Transwell assay section). After 4 h of cell culture, tubule formation in the cells was observed under an inverted microscope.
Chromatin immunoprecipitation (ChIP)
Chromatin was crosslinked, isolated, and digested with micrococcal nuclease to obtain DNA fragments. The chromatin extracts were immunoprecipitated using anti-Runx2 (Proteintech). IgG was used as mock ChIP controls. The Runx2-bound DNA of the SGMS1 gene promoter was evaluated using real-time quantitative PCR assay with gene promoter-specific primers.49
Dual-luciferase reporter assays
Different sequences of the SGMS1 promoter containing the Runx2-binding sites (E1 and E2) were synthesized and inserted into the pmirGLO luciferase vector and divided into the following groups: pmirGLO-E1; pmirGLO-E2; pmirGLO-E1 + E2; and pmirGLO-none-E1 or pmirGLO-none-E2 (control group). MSCs were cotransfected with pmirGLO-E1, pmirGLO-E2, pmirGLO-E1 + E2, pmirGLO-none-E1, or pmirGLO-none-E2, and negative control (NC) empty vector or Runx2. The relative luciferase activity was measured using a dual luciferase reporting system 48 h post-transfection.
Microcomputed tomography analysis
The calvaria of the rats were fixed in 4% paraformaldehyde for 3 days. The microscopic structure of the rat skull was subsequently analyzed using a microcomputed tomography (CT) system (Scanco Medical; Brüttisellen, Switzerland) and related software according to previous methods.50 Bone mineral density (mg/cm2) and the ratio of new bone volume to existing tissue volume were analyzed and calculated.
Histological analysis
The skull tissue was fixed and decalcified in 10% EDTA for histological analysis. The specimen was then cut in half, following which it was dehydrated, paraffin-embedded, and sectioned into 4-μm-thick slices. The slices were stained with hematoxylin & eosin (H&E) and Masson’s trichrome.
Quantification and statistical analysis
The data are presented as the mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism v7 software. Data comparisons were performed using Student’s t test or one-way analysis of variance. We marked with ∗p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Acknowledgments
We acknowledge the MDKN Ltd. for professional editing of this manuscript. This work was supported by China Postdoctoral Science Foundation (no. 2022T150445), the Beijing Hospitals Authority Youth Program (no. QML20211401), Beijing Nova Program (no. 20220484231), and China Higher Education Innovation Fund (nos. 2021JH039 and 2022BC079).
Author contributions
C.-H.Y., D.-L.Z., K.Y., and Y.-Y.L. conceived and designed the whole study; K.Y., Y.-Y.L., S.W., J.W., Y.-L.X., and Z.-L.L. performed the experiments; Y.-S.Y., Y.-P.W., Y.-Q.S., and J.Z. analyzed the data; K.Y., Y.-Y.L., W.-Q.C., and D.-L.Z. contributed to discussion; K.Y. and Y.-Y.L. wrote the original version of this paper. All authors read and approved the final manuscript.
Declaration of interests
The authors declare that they have no conflict of interests.
Published: March 5, 2024
Footnotes
Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.109358.
Contributor Information
Dong-liang Zhang, Email: zhangdongliang@mail.ccmu.edu.cn.
Cheng-hong Yin, Email: yinchh@ccmu.edu.cn.
Supplemental information
References
- 1.Marzin P., Cormier-Daire V. New perspectives on the treatment of skeletal dysplasia. Ther adv endocrinol. 2020;11 doi: 10.1177/2042018820904016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Zhang L., Yang Y., Zhang L., Ma J., Sun R., Tian Y., Yuan X., Liu B., Yu T., Jiang Z. Identification of long non-coding RNA in formaldehyde-induced cardiac dysplasia in rats. Food Chem. Toxicol. 2023;174 doi: 10.1016/j.fct.2023.113653. [DOI] [PubMed] [Google Scholar]
- 3.Mortier G.R., Cohn D.H., Cormier-Daire V., Hall C., Krakow D., Mundlos S., Nishimura G., Robertson S., Sangiorgi L., Savarirayan R., et al. Nosology and classification of genetic skeletal disorders: 2019 revision. Am. J. Med. Genet. 2019;179:2393–2419. doi: 10.1002/ajmg.a.61366. [DOI] [PubMed] [Google Scholar]
- 4.Valenti M.T., Dalle Carbonare L., Mottes M. Osteogenic differentiation in healthy and pathological conditions. Int. J. Mol. Sci. 2016;18:41. doi: 10.3390/ijms18010041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Kong Y., Zhao Y., Li D., Shen H., Yan M. Dual delivery of encapsulated BM-MSCs and BMP-2 improves osteogenic differentiation and new bone formation. J. Biomed. Mater. Res. 2019;107:2282–2295. doi: 10.1002/jbm.a.36737. [DOI] [PubMed] [Google Scholar]
- 6.Qu Z., Guo S., Fang G., Cui Z., Liu Y. AKT pathway affects bone regeneration in nonunion treated with umbilical cord-derived mesenchymal stem cells. Cell Biochem. Biophys. 2015;71:1543–1551. doi: 10.1007/s12013-014-0378-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Ando J., Kinoshita M., Cui J., Yamakoshi H., Dodo K., Fujita K., Murata M., Sodeoka M. Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy. Proc. Natl. Acad. Sci. USA. 2015;112:4558–4563. doi: 10.1073/pnas.1418088112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Ding T., Li Z., Hailemariam T., Mukherjee S., Maxfield F.R., Wu M.P., Jiang X.C. SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis. J. Lipid Res. 2008;49:376–385. doi: 10.1194/jlr.M700401-JLR200. [DOI] [PubMed] [Google Scholar]
- 9.Qi T., Li L., Weidong T. The role of sphingolipid metabolism in bone remodeling. Front. Cell Dev. Biol. 2021;9 doi: 10.3389/fcell.2021.752540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Matsumoto G., Hashizume C., Watanabe K., Taniguchi M., Okazaki T. Deficiency of sphingomyelin synthase 1 but not sphingomyelin synthase 2 reduces bone formation due to impaired osteoblast differentiation. Mol. Med. 2019;25:56. doi: 10.1186/s10020-019-0123-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Yoshikawa Y., Yoshizawa T., Domae E., Hirai Y., Kamada A., Okazaki T., Ikeo T. Knockdown of sphingomyelin synthase 2 inhibits osteoclastogenesis by decreasing RANKL expression in mouse primary osteoblasts. Biomed. Res. 2019;40:189–196. doi: 10.2220/biomedres.40.189. [DOI] [PubMed] [Google Scholar]
- 12.Dong X.Y., Huang Y.X., Yang Z., Chu X.Y., Wu J., Wang S., He X., Gao C.Y., Chen X., Yang K., Zhang D.L. Downregulation of ROR2 promotes dental pulp stem cell senescence by inhibiting STK4-FOXO1/SMS1 axis in sphingomyelin biosynthesis. Aging Cell. 2021;20 doi: 10.1111/acel.13430. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Peng S., He T., Liu Y., Zheng L., Zhong Y., Niu Z., Zhang M., Yang S. Lnc-PPP2R1B Mediates the Alternative Splicing of PPP2R1B by Interacting and Stabilizing HNRNPLL and Promotes Osteogenesis of MSCs. Stem Cell Rev. Rep. 2023;19:1981–1993. doi: 10.1007/s12015-023-10559-5. [DOI] [PubMed] [Google Scholar]
- 14.Licheva M., Raman B., Kraft C., Reggiori F. Phosphoregulation of the autophagy machinery by kinases and phosphatases. Autophagy. 2022;18:104–123. doi: 10.1080/15548627.2021.1909407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Okamura H., Yoshida K., Ochiai K., Haneji T. Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix. Bone. 2011;49:368–375. doi: 10.1016/j.bone.2011.06.004. [DOI] [PubMed] [Google Scholar]
- 16.Xie F., Li F., Li R., Liu Z., Shi J., Zhang C., Dong N. Inhibition of PP2A enhances the osteogenic differentiation of human aortic valvular interstitial cells via ERK and p38 MAPK pathways. Life Sci. 2020;257 doi: 10.1016/j.lfs.2020.118086. [DOI] [PubMed] [Google Scholar]
- 17.Kim K.-M., Kim D.-Y., Lee D.-S., Kim J.-W., Koh J.-T., Kim E.-J., Jang W.-G. Peroxiredoxin II negatively regulates BMP2-induced osteoblast differentiation and bone formation via PP2A Cα-mediated Smad1/5/9 dephosphorylation. Exp. Mol. Med. 2019;51:1–11. doi: 10.1038/s12276-019-0263-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Pan F.-f., Shao J., Shi C.-J., Li Z.-p., Fu W.-m., Zhang J.-f. Apigenin promotes osteogenic differentiation of mesenchymal stem cells and accelerates bone fracture healing via activating Wnt/β-catenin signaling. Am. J. Physiol. Endocrinol. Metab. 2021;320:E760–E771. doi: 10.1152/ajpendo.00543.2019. [DOI] [PubMed] [Google Scholar]
- 19.Stoffel W., Hammels I., Jenke B., Schmidt-Soltau I., Niehoff A. Neutral sphingomyelinase 2 (SMPD3) deficiency in mice causes chondrodysplasia with unimpaired skeletal mineralization. Am. J. Pathol. 2019;189:1831–1845. doi: 10.1016/j.ajpath.2019.05.008. [DOI] [PubMed] [Google Scholar]
- 20.Su D., Liao Z., Feng B., Wang T., Shan B., Zeng Q., Song J., Song Y. Pulsatilla chinensis saponins cause liver injury through interfering ceramide/sphingomyelin balance that promotes lipid metabolism dysregulation and apoptosis. Phytomedicine. 2020;76 doi: 10.1016/j.phymed.2020.153265. [DOI] [PubMed] [Google Scholar]
- 21.Takanashi Y., Funai K., Eto F., Mizuno K., Kawase A., Tao H., Kitamoto T., Takahashi Y., Sugimura H., Setou M., et al. Decreased sphingomyelin (t34: 1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study. BMC Cancer. 2021;21:1232–1314. doi: 10.1186/s12885-021-08948-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Taniguchi M., Okazaki T. Role of ceramide/sphingomyelin (SM) balance regulated through “SM cycle” in cancer. Cell. Signal. 2021;87 doi: 10.1016/j.cellsig.2021.110119. [DOI] [PubMed] [Google Scholar]
- 23.Zhao L., Liu Z., Deng X., Wang J., Sun L., Fan L., Zhang Y. Polyphyllin VII induces mitochondrial apoptosis by regulating the PP2A/AKT/DRP1 signaling axis in human ovarian cancer. Oncol. Rep. 2021;45:513–522. doi: 10.3892/or.2020.7879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Zhang Y., Cao X., Li P., Fan Y., Zhang L., Ma X., Sun R., Liu Y., Li W. LncRNA NKILA integrates RXFP1/AKT and NF-κB signalling to regulate osteogenesis of mesenchymal stem cells. J. Cell Mol. Med. 2020;24:521–529. doi: 10.1111/jcmm.14759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Reinsch B., Grand K., Lachman R.S., Kim H.K.W., Sanchez-Lara P.A. Expanding the phenotypic spectrum of RPL13-related skeletal dysplasia. Am. J. Med. Genet. 2021;185:2776–2781. doi: 10.1002/ajmg.a.61965. [DOI] [PubMed] [Google Scholar]
- 26.Geister K.A., Camper S.A. Advances in skeletal dysplasia genetics. Annu. Rev. Genom. Hum. Genet. 2015;16:199–227. doi: 10.1146/annurev-genom-090314-045904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Chu D.T., Phuong T.N.T., Tien N.L.B., Tran D.K., Thanh V.V., Quang T.L., Truong D.T., Pham V.H., Ngoc V.T.N., Chu-Dinh T., Kushekhar K. An Update on the Progress of Isolation, Culture, Storage, and Clinical Application of Human Bone Marrow Mesenchymal Stem/Stromal Cells. Int. J. Mol. Sci. 2020;21 doi: 10.3390/ijms21030708. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Xu G.P., Zhang X.F., Sun L., Chen E.M. Current and future uses of skeletal stem cells for bone regeneration. World J. Stem Cell. 2020;12:339–350. doi: 10.4252/wjsc.v12.i5.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Tan Z., Zhou B., Zheng J., Huang Y., Zeng H., Xue L., Wang D. Lithium and copper induce the osteogenesis-angiogenesis coupling of bone marrow mesenchymal stem cells via crosstalk between canonical Wnt and HIF-1α signaling pathways. Stem Cell. Int. 2021;2021:1–15. doi: 10.1155/2021/6662164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Lu H., Zhou L., Zuo H., Le W., Hu J., Zhang T., Li M., Yuan Y. Overriding sorafenib resistance via blocking lipid metabolism and Ras by sphingomyelin synthase 1 inhibition in hepatocellular carcinoma. Cancer Chemother. Pharmacol. 2021;87:217–228. doi: 10.1007/s00280-020-04199-6. [DOI] [PubMed] [Google Scholar]
- 31.Koh E.H., Yoon J.E., Ko M.S., Leem J., Yun J.-Y., Hong C.H., Cho Y.K., Lee S.E., Jang J.E., Baek J.Y., et al. Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis. Gut. 2021;70:1954–1964. doi: 10.1136/gutjnl-2020-322509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Jing F., Jing C., Dai X., Zhou G., Di S., Bi X., Dai T., Qin T., Hong L. Sphingomyelin synthase 2 but not sphingomyelin synthase 1 is upregulated in ovarian cancer and involved in migration, growth and survival via different mechanisms. Am. J. Transl. Res. 2021;13:4412–4421. [PMC free article] [PubMed] [Google Scholar]
- 33.Ramsay A.L., Alonso-Garcia V., Chaboya C., Radut B., Le B., Florez J., Schumacher C., Fierro F.A. Modeling Snyder-Robinson Syndrome in multipotent stromal cells reveals impaired mitochondrial function as a potential cause for deficient osteogenesis. Sci. Rep. 2019;9:15395–15410. doi: 10.1038/s41598-019-51868-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Chae H.J., Chae S.W., Kang J.S., Bang B.G., Cho S.B., Park R.K., So H.S., Kim Y.K., Kim H.M., Kim H.R. Dexamethasone suppresses tumor necrosis factor-α-induced apoptosis in osteoblasts: possible role for ceramide. Endocrinology. 2000;141:2904–2913. doi: 10.1210/endo.141.8.7604. [DOI] [PubMed] [Google Scholar]
- 35.Khayrullin A., Krishnan P., Martinez-Nater L., Mendhe B., Fulzele S., Liu Y., Mattison J.A., Hamrick M.W. Very long-chain C24: 1 ceramide is increased in serum extracellular vesicles with aging and can induce senescence in bone-derived mesenchymal stem cells. Cells. 2019;8:37. doi: 10.3390/cells8010037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Dobrowsky R.T., Kamibayashi C., Mumby M.C., Hannun Y.A. Ceramide activates heterotrimeric protein phosphatase 2A. J. Biol. Chem. 1993;268:15523–15530. [PubMed] [Google Scholar]
- 37.Yen Y.-T., Chien M., Wu P.-Y., Hung S.-C. PP2A in LepR+ mesenchymal stem cells contributes to embryonic and postnatal endochondral ossification through Runx2 dephosphorylation. Commun. Biol. 2021;4:658. doi: 10.1038/s42003-021-02175-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Son H.E., Jang W.G. Cip2A modulates osteogenic differentiation via the ERK-Runx2 pathway in MG63 cells. Biofactors. 2021;47:658–664. doi: 10.1002/biof.1760. [DOI] [PubMed] [Google Scholar]
- 39.Zhao S.-J., Kong F.-Q., Jie J., Li Q., Liu H., Xu A.-D., Yang Y.-Q., Jiang B., Wang D.-D., Zhou Z.-Q., et al. Macrophage MSR1 promotes BMSC osteogenic differentiation and M2-like polarization by activating PI3K/AKT/GSK3β/β-catenin pathway. Theranostics. 2020;10:17–35. doi: 10.7150/thno.36930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Gao S., Chen B., Zhu Z., Du C., Zou J., Yang Y., Huang W., Liao J. PI3K-Akt signaling regulates BMP2-induced osteogenic differentiation of mesenchymal stem cells (MSCs): A transcriptomic landscape analysis. Stem Cell Res. 2023;66 doi: 10.1016/j.scr.2022.103010. [DOI] [PubMed] [Google Scholar]
- 41.Li Y., Ding L., Zhang Y., Zhou B., Man G.C.W., Wang M., Li J., Liu Y., Lin W., Wang H. Histone demethylase Kdm5c regulates osteogenesis and bone formation via PI3K/Akt/HIF1α and Wnt/β-catenin signaling pathways. Genes Dis. 2024;11:64–66. doi: 10.1016/j.gendis.2023.02.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Liu T., Gatto N.M., Chen Z., Qiu H., Lee G., Duerksen-Hughes P., Fraser G., Wang C. Vegetarian diets, circulating miRNA expression and healthspan in subjects living in the Blue Zone. Precis. Clin. Med. 2020;3:245–259. doi: 10.1093/pcmedi/pbaa037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Suo J., Feng X., Li J., Wang J., Wang Z., Zhang L., Zou W. VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription. Sci. Adv. 2020;6 doi: 10.1126/sciadv.aba4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Granero-Moltó F., Weis J.A., Miga M.I., Landis B., Myers T.J., O'Rear L., Longobardi L., Jansen E.D., Mortlock D.P., Spagnoli A. Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cell. 2009;27:1887–1898. doi: 10.1002/stem.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Huang B., Wang W., Li Q., Wang Z., Yan B., Zhang Z., Wang L., Huang M., Jia C., Lu J., et al. Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone. Nat. Commun. 2016;7 doi: 10.1038/ncomms13885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Tao Y., Li R., Shen C., Li J., Zhang Q., Ma Z., Wang F., Wang Z. SENP1 is a crucial promotor for hepatocellular carcinoma through deSUMOylation of UBE2T. Aging (Albany NY) 2020;12:1563–1576. doi: 10.18632/aging.102700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Ji H., Cui X., Yang Y., Zhou X. CircRNA hsa_circ_0006215 promotes osteogenic differentiation of BMSCs and enhances osteogenesis–angiogenesis coupling by competitively binding to miR-942-5p and regulating RUNX2 and VEGF. Aging (Albany NY) 2021;13:10275–10288. doi: 10.18632/aging.202791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Taniguchi M., Nagaya S., Yuyama K., Kotani A., Igarashi Y., Okazaki T. Ceramide Metabolism Regulated by Sphingomyelin Synthase 2 Is Associated with Acquisition of Chemoresistance via Exosomes in Human Leukemia Cells. Int. J. Mol. Sci. 2022;23 doi: 10.3390/ijms231810648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Miao Y., Liu G., Liu L. Histone methyltransferase SUV39H2 regulates LSD1-dependent CDH1 expression and promotes epithelial mesenchymal transition of osteosarcoma. Cancer Cell Int. 2021;21:2. doi: 10.1186/s12935-020-01636-7. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 50.Chen L., Zhang R.-Y., Xie J., Yang J.-Y., Fang K.-H., Hong C.-X., Yang R.-B., Bsoul N., Yang L. STAT3 activation by catalpol promotes osteogenesis-angiogenesis coupling, thus accelerating osteoporotic bone repair. Stem Cell Res. Ther. 2021;12:108–116. doi: 10.1186/s13287-021-02178-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Western blot original data have been deposited at Mendeley and publicly available as of the date of publication. The DOI and accession numbers are listed in the key resources table. This paper does not report any original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.