Intercellular adhesion molecule-1 inhibits osteogenic differentiation of mesenchymal stem cells and impairs bio-scaffold-mediated bone regeneration in vivo.
ABSTRACT: Mesenchymal stem cell (MSC) loaded bio-scaffold transplantation is a promising therapeutic approach for bone regeneration and repair. However, growing evidence shows that pro-inflammatory mediators from injured tissues suppress osteogenic differentiation and impair bone formation. To improve MSC-based bone regeneration, it is important to understand the mechanism of inflammation mediated osteogenic suppression. In the present study, we found that synovial fluid from rheumatoid arthritis patients and pro-inflammatory cytokines including interleukin-1?, interleukin-1?, and tumor necrosis factor ?, stimulated intercellular adhesion molecule-1(ICAM-1) expression and impaired osteogenic differentiation of MSCs. Interestingly, overexpression of ICAM-1 in MSCs using a genetic approach also inhibited osteogenesis. In contrast, ICAM-1 knockdown significantly reversed the osteogenic suppression. In addition, after transplanting a traceable MSC-poly(lactic-co-glycolic acid) construct in rat calvarial defects, we found that ICAM-1 suppressed MSC osteogenic differentiation and matrix mineralization in vivo. Mechanistically, we found that ICAM-1 enhances MSC proliferation but causes stem cell marker loss. Furthermore, overexpression of ICAM-1 stably activated the MAPK and NF-?B pathways but suppressed the PI3K/AKT pathway in MSCs. More importantly, specific inhibition of the ERK/MAPK and NF-?B pathways or activation of the PI3K/AKT pathway partially rescued osteogenic differentiation, while inhibition of the p38/MAPK and PI3K/AKT pathway caused more serious osteogenic suppression. In summary, our findings reveal a novel function of ICAM-1 in osteogenesis and suggest a new molecular target to improve bone regeneration and repair in inflammatory microenvironments.
Project description:Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of differentiating into osteoblasts, chondrocytes, and adipocytes. Skewed differentiation of BM-MSCs contributes to the pathogenesis of osteoporosis. Yet how BM-MSC lineage commitment is regulated remains unclear. We show that ablation of p38? in Prx1+ BM-MSCs produced osteoporotic phenotypes, growth plate defects, and increased bone marrow fat, secondary to biased BM-MSC differentiation from osteoblast/chondrocyte to adipocyte and increased osteoclastogenesis and bone resorption. p38? regulates BM-MSC osteogenic commitment through TAK1-NF-?B signaling and osteoclastogenesis through osteoprotegerin (OPG) production by BM-MSCs. Estrogen activates p38? to maintain OPG expression in BM-MSCs to preserve the bone. Ablation of p38? in BM-MSCs positive for Dermo1, a later BM-MSC marker, only affected osteogenic differentiation. Thus, p38? mitogen-activated protein kinase (MAPK) in Prx1+ BM-MSCs acts to preserve the bone by promoting osteogenic lineage commitment and sustaining OPG production. This study thus unravels previously unidentified roles for p38? MAPK in skeletal development and bone remodeling.
Project description:BACKGROUND:Stem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs. METHODS:SCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates. RESULTS:Pretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin. CONCLUSIONS:Our findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.
Project description:Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1? production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.
Project description:Mesenchymal stem cells (MSCs) are multipotent stem cells that have a strong osteogenic differentiation capacity. However, the molecular mechanism underlying the osteogenic differentiation of MSCs remains largely unknown and thus hinders further development of MSC-based cell therapies for bone repair in the clinic. RSP5, also called NEDD4L (NEDD4-like E3 ubiquitin protein ligase), belongs to the HECT (homologous to E6-AP carboxyl terminus) domain-containing E3 ligase family. Nevertheless, although many studies have been conducted to elucidate the role of RSP5 in various biological processes, its effect on osteogenesis remains elusive. In this study, we demonstrated that the expression of RSP5 was elevated during the osteogenesis of MSCs and positively regulated the osteogenic capacity of MSCs by inducing K63-linked polyubiquitination and activation of the Akt pathway. Taken together, our findings suggest that RSP5 may be a promising target to improve therapeutic efficiency by using MSCs for bone regeneration and repair.
Project description:Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated I?B kinase (IKK)-NF-?B and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK-NF-?B significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK-NF-?B activation promoted ?-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-?B in inflammation and infection, our results suggest that targeting IKK-NF-?B may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.
Project description:Oleanolic acid (OA), a pentacyclic triterpenoid, has been shown to modulate multiple signaling pathways in a variety of cell linages. But the mechanisms underlying OA-mediated mesenchymal stromal cell (MSC) osteogenic differentiation are not known. In this study, we examined effects of OA on cell viability, osteogenic differentiation in MSCs, and the involvement of Notch and BMP signaling. OA induced bone marrow derived MSC differentiation towards osteoprogenitor cells and inhibited Notch signaling in a dose dependent manner. Constitutive activation of Notch signaling fully blocked OA induced MSC osteogenic differentiation. The expression level of early osteogenic marker genes, ALP, Runx2, and type I collagen, which play a critical role in MSC to osteoblast transition and servers as a downstream target of BMP signaling, was significantly induced by OA. Furthermore, BMP2 mediated MSC osteogenic differentiation was significantly enhance by OA treatment, indicating a synergistic effect between BMP2 and OA. Our results suggest that OA is a promising bioactive agent for bone tissue regeneration, and inhibition of Notch signaling is required for its osteogenic effects on MSCs.
Project description:Myeloma bone disease (MBD) is one of the clinical features of multiple myeloma, which contributes to the attenuation of osteoblast function. Bone marrow mesenchymal stem cells exhibit a high potential for differentiation into osteoblasts. A number of studies have reported that microRNAs (miRs) serve a vital role in mesenchymal stem cell (MSC) osteogenesis; however, the role of miR-221-5p in the osteogenic differentiation of MBD-MSCs remains unclear. The present study revealed that the osteogenic differentiation capacity of MBD-MSCs was reduced compared with that of normal (N)-MSCs. Further experiments demonstrated that miR-221-5p expression was downregulated in N-MSCs following osteoblast induction while no obvious alterations in expression levels were observed in MBD-MSCs. The inhibition of miR-221-5p promoted the osteogenic differentiation of MBD-MSCs. Bioinformatics, luciferase reporter assays, reverse transcription-quantitative PCR and western blotting assays indicated that smad family member 3 (smad3) was a direct target of miR-221-5p in MBD-MSCs. A negative association was identified between the expression levels of smad3 and miR-221-5p. Investigations of the molecular mechanism indicated that suppressed miR-221-5p could regulate the osteogenic differentiation of MBD-MSCs by upregulating smad3 expression. It was also identified that the PI3K/AKT/mTOR signaling pathway was activated following miR-221-5p inhibition, and this increased the osteogenic differentiation capacity of MBD-MSCs. The present study may improve the understanding regarding the role of miR-221-5p in the regulation of osteogenic differentiation, and may contribute to the development of a novel therapy for MBD.
Project description:Mesenchymal stem cells (MSCs) play a crucial role in tissue repair by secretion of tissue nutrient factors such as hepatocyte growth factor (HGF). However, studies examining the effects of HGF on the proliferation and differentiation of MSCs used different concentrations of HGF and reported conflicting conclusions. This study aimed to determine the mechanisms by which different concentrations of HGF regulate MSC proliferation and osteogenic differentiation, and validate the mechanism in an animal model of early stage avascular necrosis of femoral head (ANFH). Our results demonstrate that a low concentration of HGF (20 ng/ml) preferentially promotes MSC osteogenic differentiation through increased c-Met expression and phosphorylation, Akt pathway activation, and increased expression of p27, Runx2 and Osterix. In contrast, a high concentration of HGF (100 ng/ml) strongly induced proliferation by inducing strong activation of the ERK1/2 signalling pathway. As validated by animal experiments, high localized expression of HGF achieved by transplantation of HGF transgenic MSCs into ANFH rabbits increased the number of MSCs. Subsequently, 2 weeks after transplantation, HGF levels decreased and MSCs differentiated into osteoblasts and resulted in efficient tissue repair. Our results demonstrate that sequential concentration changes in HGF control the proliferation and osteogenic differentiation of MSCs in vivo. This phenomenon can be exploited therapeutically to induce bone regeneration and, in turn, improve the efficacy of pharmacological intervention for ANFH treatment.
Project description:Mesenchymal stem cells (MSCs) are a reliable resource for bone regeneration and tissue engineering, but the molecular mechanisms of differentiation remain unclear. The tumor antigen 15-leucine-rich repeat containing membrane protein (LRRC15) is a transmembrane protein demonstrated to play important roles in cancer. However, little is known about its role in osteogenesis. This study was to evaluate the functions of LRRC15 in osteogenic differentiation of MSCs.Osteogenic-induction treatment and the ovariectomized (OVX) model were performed to investigate the potential relationship between LRRC15 and MSC osteogenesis. A loss-of-function study was used to explore the functions of LRRC15 in osteogenic differentiation of MSCs in vitro and in vivo. NF-?B pathway inhibitor BAY117082, siRNA, nucleocytoplasmic separation, and ChIP assays were performed to clarify the molecular mechanism of LRRC15 in bone regulation.Our results first demonstrated that LRRC15 expression was upregulated upon osteogenic induction, and the level of LRRC15 was significantly decreased in OVX mice. Both in-vitro and in-vivo experiments detected that LRRC15 was required for osteogenesis of MSCs. Mechanistically, LRRC15 inhibited transcription factor NF-?B signaling by affecting the subcellular localization of p65. Further studies indicated that LRRC15 regulated osteogenic differentiation in a p65-dependent manner.Taken together, our findings reveal that LRRC15 is an essential regulator for osteogenesis of MSCs through modulating p65 cytoplasmic/nuclear translocation, and give a novel hint for MSC-mediated bone regeneration.
Project description:Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin ?4?1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.