Loss of p53 in mesenchymal stem cells promotes alteration of bone remodeling through negative regulation of osteoprotegerin

Abstractp53 plays a pivotal role in controlling the differentiation of mesenchymal stem cells (MSCs) by regulating genes involved in cell cycle and early steps of differentiation process. In the context of osteogenic differentiation of MSCs and bone homeostasis, the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) axis is a critical signaling pathway. The absence or loss of function of p53 has been implicated in aberrant osteogenic differentiation of MSCs that results in higher bone formation versus erosion, leading to an unbalanced bone remodeling. Here, we show by microCT that mice with p53 deletion systemically or specifically in mesenchymal cells possess significantly higher bone density than their respective littermate controls. There is a negative correlation between p53 and OPG both in vivo by analysis of serum from p53+/+, p53+/−, and p53−/− mice and in vitro by p53 knockdown and ChIP assay in MSCs. Notably, high expression of Opg or its combination with low level of p53 are prominent features in clinical cancer lesion of osteosarcoma and prostate cancer respectively, which correlate with poor survival. Intra-bone marrow injection of prostate cancer cells, together with androgen can suppress p53 expression and enhance local Opg expression, leading to an enhancement of bone density. Our results support the notion that MSCs, as osteoblast progenitor cells and one major component of bone microenvironment, represent a cellular source of OPG, whose amount is regulated by the p53 status. It also highlights a key role for the p53-OPG axis in regulating the cancer associated bone remodeling.

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KAT6A regulates stemness of aging bone marrow-derived mesenchymal stem cells through Nrf2/ARE signaling pathway

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02164-5 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Dongdong Fei ◽

Yazheng Wang ◽

Qiming Zhai ◽

Xige Zhang ◽

Yang Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Colony Formation ◽

Cell Sheet ◽

Loss Of Function ◽

Acetylation Level ◽

Ros Accumulation

Abstract Background This study aimed to explore the effect of KAT6A on the decreased stemness of aging bone marrow-derived mesenchymal stem cells (BMSCs) and its potential mechanism. Methods The acetylation level and KAT6A expression of BMSCs from the young (YBMSCs) and the old (OBMSCs) were examined. Gain- and loss-of-function experiments were performed to determine the effect of KAT6A on BMSC proliferation, colony formation, and osteogenic differentiation. The effect of KAT6A on Nrf2/ARE signaling pathway was investigated after KAT6A inhibition in YBMSCs or overexpression in OBMSCs, and the role of Nrf2/ARE signaling pathway on stemness was examined by investigating proliferation, colony formation, and osteogenic differentiation. Further in vivo study was performed to explore osteogenesis ability of OBMSCs after modulation of KAT6A and Nrf2/ARE pathway through cell sheet technology. Results The acetylation level and KAT6A expression of OBMSCs were decreased, and KAT6A downregulation resulted in decreased proliferation, colony formation, and osteogenic differentiation of OBMSCs. Mechanically, KAT6A was found to regulate Nrf2/ARE signaling pathway and inhibit ROS accumulation in OBMSCs, thus promoting proliferation, colony formation, and osteogenic differentiation of OBMSCs. Further study demonstrated that KAT6A could promote osteogenesis of OBMSCs by regulating Nrf2/ARE signaling pathway. Conclusions Downregulation of KAT6A resulted in the decreased stemness of OBMSCs by inhibiting the Nrf2/ARE signaling pathway. Graphical abstract KAT6A was downregulated in aging bone marrow-derived mesenchymal stem cells (BMSCs), and downregulation of KAT6A resulted in Nrf2/ARE signaling pathway inhibition and ROS accumulation, thus leading to decreased stemness of aging BMSCs.

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Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

International Journal of Molecular Sciences ◽

10.3390/ijms23020856 ◽

2022 ◽

Vol 23 (2) ◽

pp. 856

Author(s):

Kamini Kaushal ◽

Apoorvi Tyagi ◽

Janardhan Keshav Karapurkar ◽

Eun-Jung Kim ◽

Parthasaradhireddy Tanguturi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Protein Degradation ◽

Osteogenic Differentiation ◽

Soft Tissues ◽

Bone Mineralization ◽

Loss Of Function ◽

Differentiation Potential ◽

Specific Protease ◽

Ubiquitin Specific Protease

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.

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Chikungunya virus infection impairs osteogenic differentiation of bone marrow-derived mesenchymal stem cells

10.1101/780791 ◽

2019 ◽

Author(s):

Enakshi Roy ◽

Wen Shi ◽

Bin Duan ◽

St Patrick Reid

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Chikungunya Virus ◽

Bone Homeostasis ◽

Bone Pathology ◽

Chikv Infection ◽

Osteogenic Cells ◽

First Time

AbstractChikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus, belonging to the genus alphavirus in the family Togaviridae. The virus is spread by the Aedes species (sp.) mosquitoes in tropical and subtropical regions of the world. CHIKV causes Chikungunya fever (CHIKF), where the acute stage of infection is characterized by high fever, headache, rash, and polyarthralgia. In 30-40% of cases, patients develop a chronic stage with debilitating joint pain persisting for months to years imposing a burden on the population in terms of disability adjusted life years (DALY). Presently, no vaccines or treatment options are available for this infection. Prior investigations reveal that CHIKV infection is associated with bone pathology; however, the molecular mechanism underlying CHIKV-induced bone pathology remains poorly defined. Studies show that disruption of osteogenic differentiation and function of bone marrow-derived mesenchymal stem cells (BMMSCs) can lead to bone pathologies. However, to date pathogenesis of CHIKV infection in this context has not been studied. In the current study, we investigated the susceptibility of BMMSCs to CHIKV and studied the effect of infection on BMMSCs-derived osteogenic cells. To our knowledge, for the first time we report that CHIKV can productively infect BMMSCs. We observed a decrease in the intracellular and extracellular alkaline phosphatase (ALP) activity and reduction in calcium phosphate deposition in infected cells compared to mock-infected control. Thus, we conclude that CHIKV infects BMMSCs and disrupts function of osteogenic cells.ImportanceAlthough studies have shown association of bone pathology and CHIKV infection, the pathogenesis of infection causing altered bone homeostasis is not fully understood. Here, we demonstrate for the first time that BMMSCs are susceptible to CHIKV infection. Furthermore, we observe that infection causes disruption in the function of BMMSC- derived osteogenic cells. Impaired function of these osteogenic cells will likely lead to a disruption in bone homeostasis and in part, provides a mechanism for the observed bone pathology associated with CHIKV pathogenesis.

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Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Cellular Physiology and Biochemistry ◽

10.1159/000374069 ◽

2015 ◽

Vol 36 (1) ◽

pp. 259-273 ◽

Cited By ~ 4

Author(s):

Caterina Morabito ◽

Iolanda D'Alimonte ◽

Laura Pierdomenico ◽

Caterina Pipino ◽

Simone Guarnieri ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Amniotic Fluid ◽

Osteogenic Differentiation ◽

Physiological Role ◽

Bone Homeostasis ◽

Calcitonin Receptor ◽

Enzymatic Assays ◽

Cell Therapies

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules.

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YAP as a key regulator of adipo-osteogenic differentiation in human MSCs

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1494-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 15

Author(s):

Chanchao Lorthongpanich ◽

Kanjana Thumanu ◽

Kantpitchar Tangkiettrakul ◽

Nittaya Jiamvoraphong ◽

Chuti Laowtammathron ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Bone Cells ◽

Effector Protein ◽

Human Mscs ◽

Hippo Signaling ◽

Bone Homeostasis ◽

Loss Of Function ◽

Ftir Microspectroscopy ◽

Msc Differentiation

Abstract Background Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. As a common progenitor, MSC differentiation has to be tightly regulated to maintain the balance of their differentiation commitment. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Several factors have been suggested as critical factors that affect adipo-osteogenic decision, including melatonin and smad4. Yes-associated protein (YAP) is an important effector protein in the Hippo signaling pathway that acts as a transcriptional regulator by activating the transcription of the genes involved in cell proliferation and anti-apoptosis. The non-canonical role of YAP in regulating bone homeostasis by promoting osteogenesis and suppressing adipogenesis was recently demonstrated in a mouse model. However, it is unclear whether YAP is also crucial for modulating human MSC differentiation to fat and bone. Methods The expression level of YAP during MSC differentiation was modulated using pharmaceutical molecule and genetic experiments through gain- and loss-of-function approaches. Results We demonstrated for the first time that YAP has a non-canonical role in regulating the balance of adipo-osteogenic differentiation of human MSCs. The result from synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy showed unique metabolic fingerprints generated from YAP-targeted differentiated cells that were clearly distinguished from non-manipulated control. Conclusions These results, thus, identify YAP as an important effector protein that regulates human MSC differentiation to fat and bone and suggests the use of FTIR microspectroscopy as a promising technique in stem cell research.

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Distal Low Homeobox 3 Promotes Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation and Bone Synthesis and Ameliorates Osteoporosis by Activating Typical Wnt Signaling

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2209 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1776-1782

Author(s):

Yongyi Xu ◽

Lei Chen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Bone Density ◽

Osteogenic Differentiation ◽

Caspase 3 ◽

Type I Collagen ◽

Type I ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

The distal low homeobox 3 (DLX3) regulates the bone marrow mesenchymal stem cells (BMSC) osteogenic differentiation. However, whether DLX3 affects osteoporosis (OP) remains unclear. An OVX-induced OP rat model was constructed and DLX3 plasmid was injected followed by analysis of bone mineral density and ALP activity. Rat BMSCs were isolated and divided into control group, OP group and DLX3 group (transfected with DLX3 plasmid) followed by analysis of chondrocytes survival rate by MTT assay, Caspase 3 activity, type I collagen and Osterix expression by Real time PCR and -catenin level by Western blot. DLX3 expression was significantly down-regulated in OP rats with deceased bone density and ALP activity compared to sham group (P < 0 05). When DLX3 was transfected into OP rats, DLX3 expression was significantly up-regulated with increased bone density and ALP activity compared with OP group (P < 0 05). BMSCs survival was significantly decreased in OP group and Caspase 3 activity was significantly increased with downregulated type I collagen, Osterix and -catenin (P < 0 05). However, transfection of DLX3 plasmid into OP group BMSCs cells can significantly reverse the above changes, compared to OP group (P < 0 05). DLX3 expression is reduced in osteoporosis. Up-regulation of DLX3 can promote osteogenic differentiation of BMSCs by regulating typical Wnt signaling, promote differentiation into osteoblasts, increase bone density increase, and then ameliorate osteoporosis.

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Ionomycin Ameliorates Hypophosphatasia via Rescuing Alkaline Phosphatase Deficiency-mediated L-type Ca2+ Channel Internalization in Mesenchymal Stem Cells

10.1101/545418 ◽

2019 ◽

Author(s):

Bei Li ◽

Xiaoning He ◽

Zhiwei Dong ◽

Kun Xuan ◽

Wei Sun ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Cell Lineage ◽

Bone Homeostasis ◽

Loss Of Function ◽

Channel Trafficking ◽

Lineage Differentiation ◽

Promising Therapeutic Approach ◽

Intracellular Ca

AbstractLoss-of-function mutations in ALPL result in hypophosphatasia (HPP), an inborn error of metabolism that causes skeletal mineralization defect. In adults, main clinical involvement includes early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused reduction of intracellular Ca2+ influx resulting in osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse BMSCs. To elevate intracellular level of calcium in bone marrow mesenchymal stem cells (BMSCs) by ionomycin treatment rescues the osteoporotic phenotype in alpl+/- mice and BMSC-specific (Prrx1-alpl-/-) conditional alpl knockout mice. Mechanistically, ALPL is required to maintain intracellular Ca2+ influx by regulating L-type Ca2+ channel trafficking via binding to the α2δ subunits, which regulates the internalization of L-type Ca2+ channel. Decreased Ca2+ flux inactivates Akt/GSK3β/β-catenin signaling pathway that regulates lineage differentiation of BMSCs. This study identifies a previous unknown role of ectoenzyme ALPL in maintenance of calcium channel trafficking to keep stem cell lineage differentiation and bone homeostasis. Accelerating Ca2+ flux through L-type Ca2+ channel by ionomycin treatment may be a promising therapeutic approach for adult HPP patients.One Sentence SummaryALP regulates internalization of L-Type Ca2+ Channel of BMSCs in Hypophosphatasia.

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Periostin: A Downstream Mediator of EphB4-Induced Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Stem Cells International ◽

10.1155/2016/7241829 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Fei Zhang ◽

Zehua Zhang ◽

Dong Sun ◽

Shiwu Dong ◽

Jianzhong Xu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Pathway ◽

Glycogen Synthase ◽

Underlying Mechanism ◽

Bone Homeostasis ◽

Osteogenic Markers

Erythropoietin-producing hepatocyte B4 (EphB4) has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN) within bone marrow-derived mesenchymal stem cells (MSCs) and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3β-serine 9 (p-GSK-3β-Ser9) andβ-catenin, as well as the osteogenic markers Runx2 and COL1. The results showed that GSK-3βactivation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrinαvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis.

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