scholarly journals Four and a Half LIM Domains Protein 2 Mediates Bortezomib-Induced Osteogenic Differentiation of Mesenchymal Stem Cells in Multiple Myeloma Through p53 Signaling and β-Catenin Nuclear Enrichment

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhenqing Xie ◽  
Yan Xu ◽  
Xiaojing Wei ◽  
Gang An ◽  
Mu Hao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Bone Disease ◽  
Marker Genes ◽  
Human Mscs ◽  
P53 Signaling ◽  
Lim Domains

Myeloma bone disease (MBD), caused by the inhibition of osteoblast activity and the activation of osteoclast in the bone marrow environment, is the most frequent and life-threatening complication in multiple myeloma (MM) patients. Bortezomib (Bzb) was shown to promote MM-derived mesenchymal stem cells (MM-MSCs) differentiation to osteoblast in vitro and in animal models, promoting the bone formation and regeneration, may be mediated via β-catenin/T-cell factor (TCF) pathway. Further defining molecular mechanism of Bzb-enhanced bone formation in MM will be beneficial for the treatment of myeloma patients. The present study has identified for the first time four and a half LIM domains protein 2 (FHL2), a tissue-specific coregulator that interacts with many osteogenic marker molecules, as a therapeutic target to ameliorate MM bone disease. First, increased messenger RNA (mRNA) and protein levels of FHL2, and the mRNA level of main osteoblast markers (including Runx2, ALP, and Col1A1), were found in MM-patients-derived MSCs after Bzb treatment. FHL2 KD with short hairpin RNA (shRNA) reduced the expression of osteoblast marker genes and blocked the osteogenic differentiation of MM-MSCs regardless of the presence or absence of Bzb, implying that FHL2 is an important activator of the osteogenic differentiation of human MSCs under a proteasome inhibition condition. Molecular analysis showed that the enhanced expression of FHL2 was associated with the Bzb-induced upregulation of p53. No significant change at protein level of total β-catenin was observed with or without Bzb treatment. However, it was mostly enriched to nuclei in MSCs after Bzb treatment. Moreover, β-catenin was restricted to the perinuclear region in FHL2 KD cells. These data provide evidence that FHL2 is essential for promoting β-catenin nuclear enrichment in MM-MSCs. In conclusion, FHL2 is critical for Bzb-induced osteoblast differentiation of MM-MSCs and promotes the osteogenesis, through p53 signaling and β-catenin activation. Targeting FHL2 in MM may provide a new therapeutic strategy for treating MBD.

scholarly journals Knockdown of POSTN Inhibits Osteogenic Differentiation of Mesenchymal Stem Cells From Patients With Steroid-Induced Osteonecrosis

2020 ◽  
Vol 8 ◽  
Author(s):  
Lizhi Han ◽  
Song Gong ◽  
Ruoyu Wang ◽  
Shaokai Liu ◽  
Bo Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Control Group ◽  
Nodule Formation ◽  
Matricellular Protein ◽  
Specific Gene ◽  
Endogenous Expression

Steroid-induced osteonecrosis of femoral head (SONFH) is a common and serious complication caused by long-term and/or excessive use of glucocorticoids (GCs). The decreased activity and abnormal differentiation of bone marrow mesenchymal stem cells (BMSCs) are considered to be one of the major reasons for the onset and progression of this disease. Periostin (POSTN) is a matricellular protein which plays an important role in regulating osteoblast function and bone formation. Sclerostin (SOST) is a secreted antagonist of Wnt signaling that is mainly expressed in osteocytes to inhibit bone formation. However, the exact role of POSTN and SOST in SONFH has not been reported yet. Therefore, we detected the differential expression of POSTN and SOST in BMSCs of SONFH Group patients, and Control Group was patients with traumatic ONFH (TONFH) and developmental dysplasia of the hip (DDH). Furthermore, we used lentiviral transfection to knockdown POSTN expression in BMSCs of patients with SONFH to study the effect of POSTN knockdown on the SOST expression and osteogenic differentiation of BMSCs. The results indicated that the endogenous expression of POSTN and SOST in BMSCs of SONFH Group was upregulated, compared with Control Group. POSTN was upregulated gradually while SOST was downregulated gradually at days 0, 3, and 7 of osteogenic differentiation of BMSCs in Control Group. Contrarily, POSTN was gradually downregulated while SOST was gradually upregulated during osteogenic differentiation of BMSCs in SONFH Group. This could be due to increased expression of SOST in BMSCs, which was caused by excessive GCs. In turn, the increased expression of POSTN in BMSCs may play a role in antagonizing the continuous rising of SOST during the osteogenic differentiation of BMSCs in patients with SONFH. POSTN knockdown significantly attenuated osteo-specific gene expression, alkaline phosphatase activity, and calcium nodule formation in vitro; thus inhibiting the osteogenic differentiation of BMSCs in patients with SONFH. Besides, POSTN knockdown upregulated SOST expression, increased GSK-3β activity, and downregulated β-catenin. These findings suggest that POSTN have an essential role in regulating the expression of SOST and osteogenic differentiation of BMSCs in patients with SONFH, and POSTN knockdown suppresses osteogenic differentiation by upregulating SOST and partially inactivating Wnt/β-catenin signaling pathway. Therefore, targeting POSTN and SOST may serve as a promising therapeutic target for the prevention and treatment of SONFH.

Effect of Vitamin D Pretreatment of Human Mesenchymal Stem Cells on Ectopic Bone Formation

10.1563/760.1 ◽  
2006 ◽  
Vol 32 (3) ◽  
pp. 103-109 ◽  
Author(s):  
I. J. De Kok ◽  
K. C. Hicok ◽  
R. J. Padilla ◽  
R. G. Young ◽  
L. F. Cooper
Keyword(s):  
Stem Cells ◽  
Vitamin D ◽  
Ascorbic Acid ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Scid Mice ◽  
Osteogenic Potential ◽  
Human Mscs ◽  
Adult Mesenchymal Stem Cells ◽  
Undifferentiated Cells

Abstract Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow–derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.

Systemically Injected Mesenchymal Stem Cells Traffic to Myelomatous Bone and Inhibit Bone Disease, and Intralesionally Promotes Bone Formation and Delays Myeloma Progression During the Disease Active Stage or Remission

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 980-980
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Sharmin Khan ◽  
Yuping Wang ◽  
Angela Pennisi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Tumor Growth ◽  
Cell Line ◽  
Bone Disease ◽  
Active Stage ◽  
Myeloma Cells ◽  
Intravenous Injections ◽  
Bone Injection

Abstract Abstract 980 Mesenchymal stem cells (MSCs) cytotherapy has been clinically tested in various applications including bone regeneration, autoimmune diseases, and cancer. The aims of the study were to test the effect of MSCs cytotherapy on myeloma (MM) bone disease and tumor growth, and determine their ability to traffic into myelomatous bone during active disease stage and induction of remission by melphalan. We exploited the SCID-rab model for MM and a novel human myeloma cell line, Hg, established through our previously reported procedure (Xin et al., BJH 2007). Hg myeloma cells do not grow in culture but are capable of sequential passaging in SCID-rab mice. Microarray analysis revealed similar gene expression profiling between the Hg cell line and the original patient's myeloma plasma cells, and that these cells are classified in the MMSET subgroup and express DKK1, indicating their authenticity and clinical relevancy. The human fetal MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector for in vivo tracking. In Hg-bearing hosts (5 mice/group), intra-bone injection of MSCs (1×106 cells/mouse) increased bone mineral density (BMD) by 21±4% while in control, diluent injected bones it was reduced by 14±5% (p<0.001). Increased bone formation by MSCs was associated with reduced tumor growth by 50% (p<0.01). Bioluminescence analysis revealed disappearance of the majority of the intralesionally injected MSCs within 4 weeks, suggesting that MSCs exert their effects on bone remodeling as a bystander cells (trophic effect). To test effect on relapse, remission was induced by treating Hg-bearing hosts with a total of 4 subcutaneous injections of melphalan (10 mg/kg/4 days), followed by intra-bone injection of diluent or MSCs (10 mice/group). Three weeks post-cytotherapy BMD was increased by 23±5% in bones injected with MSCs and reduced by 23±3% in bones injected with diluent (p<0.001). Eleven weeks post-cytotherapy, BMD was reduced by 33±6% and 9±7% in bones injected with diluent and MSCs, respectively (p<0.03). Following melphalan treatment circulating immunoglobulin (Ig) level (MM burden) was undetected while at 2 weeks after cytotherapy it was detected in 80% and 30% of hosts injected with diluent and MSCs, respectively. At experiment's end, Ig levels were significantly lower by 6 folds in hosts treated with MSCs than diluent (p<0.01). To further validate clinical relevancy, MSCs or diluent were intravenously injected into Hg-bearing hosts. In contrast to a single injection, 4 weekly, intravenous injections of MSCs (10 mice/group) prevented reduction of the BMD of the myelomatous bone while in control hosts the BMD was reduced by 14±3% (p<0.006 vs. pretreatment). MM growth was not affected by single or multiple intravenous injections of MSCs. Ex vivo imaging of tissues from hosts with active MM or treated with melphalan detected MSCs in implanted bones and murine lungs indicating that myeloma cells or conditions induced by MM or melphalan attract MSCs to myelomatous bones. We conclude that intra-bone injection of MSCs effectively promotes bone formation and delays MM progression during the disease active stage or remission. We also conclude that exogenous MSCs are capable of trafficking to myelomatous bone and that systemic, weekly injections of MSCs inhibit MM bone disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

Open Biology ◽  
2016 ◽  
Vol 6 (3) ◽  
pp. 150258 ◽  
Author(s):  
Nan Wang ◽  
Zubin Zhou ◽  
Tianyi Wu ◽  
Wei Liu ◽  
Peipei Yin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Bone Cells ◽  
Bone Diseases ◽  
Cytokine Activation ◽  
Tnf Α ◽  
Marrow Mesenchymal Stem Cells ◽  
Underlying Mechanisms

Although systemic or local inflammation, commonly featured by cytokine activation, is implicated in patients with bone loss, the underlying mechanisms are still elusive. As microRNAs (miR), a class of small non-coding RNAs involved in essential physiological processes, have been found in bone cells, we aimed to investigate the role of miR for modulating osteogenesis in inflammatory milieu using human bone marrow mesenchymal stem cells (hBM-MSCs). Induced by proinflammatory cytokine TNF-α, miR-150-3p was identified as a key player in suppressing osteogenic differentiation through downregulating β-catenin, a transcriptional co-activator promoting bone formation. TNF-α treatment increased the levels of miR-150-3p, which directly targeted the 3′-UTR of β-catenin mRNA and in turn repressed its expression. In addition, we observed that miR-150-3p expression was increased by TNF-α via IKK-dependent NF-κB signalling. There are three putative NF-κB binding sites in the promoter region of miR-150, and we identified −686 region as the major NF-κB binding site for stimulation of miR-150 expression by TNF-α. Finally, the osteogenic differentiation of hBM-MSCs was inhibited by either miR-150-3p overexpression or TNF-α treatment, which was prevented by anti-miR-150-3p oligonucleotides. Taken together, our data suggested that miR-150-3p integrated inflammation signalling and osteogenic differentiation and may contribute to the inhibition effects of inflammation on bone formation, thus expanding the pathophysiological functions of microRNAs in bone diseases.

scholarly journals Effect of 20(S)-Hydroxycholesterol on Multilineage Differentiation of Mesenchymal Stem Cells Isolated from Compact Bones in Chicken

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1360
Author(s):  
Roshan Adhikari ◽  
Chongxiao Chen ◽  
Woo Kyun Kim
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Health ◽  
Hedgehog Signaling ◽  
Myogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Bioactive Molecules ◽  
Marker Genes ◽  
Human Mscs ◽  
Differentiation Potential

Bone health and body weight gain have significant economic and welfare importance in the poultry industry. Mesenchymal stem cells (MSCs) are common progenitors of different cell lineages such as osteoblasts, adipocytes, and myocytes. Specific oxysterols have shown to be pro-osteogenic and anti-adipogenic in mouse and human MSCs. To determine the effect of 20(S)-hydroxycholesterol (20S) on osteogenic, adipogenic, and myogenic differentiation in chicken, mesenchymal stem cells isolated from compact bones of broiler chickens (cBMSCs) were subjected to various doses of 20S, and markers of lineage-specific mRNA were analyzed using real-time PCR and cell cytochemistry. Further studies were conducted to evaluate the molecular mechanisms involved in lineage-specific differentiation pathways. Like human and mouse MSCs, 20S oxysterol expressed pro-osteogenic, pro-myogenic, and anti-adipogenic differentiation potential in cBMSCs. Moreover, 20(S)-Hydroxycholesterol induced markers of osteogenic genes and myogenic regulatory factors when exposed to cBMSCs treated with their specific medium. In contrast, 20S oxysterol suppressed expression of adipogenic marker genes when exposed to cBMSCs treated with OA, an adipogenic precursor of cBMSCs. To elucidate the molecular mechanism by which 20S exerts its differentiation potential in all three lineages, we focused on the hedgehog signaling pathway. The hedgehog inhibitor, cyclopamine, completely reversed the effect of 20S induced expression of osteogenic and anti-adipogenic mRNA. However, there was no change in the mRNA expression of myogenic genes. The results showed that 20S oxysterol promotes osteogenic and myogenic differentiation and decreases adipocyte differentiation of cBMSCs. This study also showed that the induction of osteogenesis and adipogenesis inhibition in cBMSCs by 20S is mediated through the hedgehog signaling mechanism. The results indicated that 20(S) could play an important role in the differentiation of chicken-derived MSCs and provided the theory basis on developing an intervention strategy to regulate skeletal, myogenic, and adipogenic differentiation in chicken, which will contribute to improving chicken bone health and meat quality. The current results provide the rationale for the further study of regulatory mechanisms of bioactive molecules on the differentiation of MSCs in chicken, which can help to address skeletal health problems in poultry.

A Transgenic Model Of Multiple Myeloma Bone Disease Shows Profound Mesenchymal Stem Cell Impairment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 130-130 ◽  
Author(s):  
Monica Gomez-Palou ◽  
Huang Fang ◽  
Richard Kremer ◽  
Michael Sebag
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Mesenchymal Stem Cells ◽  
Mouse Model ◽  
Transgenic Mice ◽  
Bone Disease ◽  
Ex Vivo ◽  
Wild Type ◽  
Lytic Lesions ◽  
Msc Differentiation

Abstract Bone disease affects 70% of Multiple Myeloma (MM) patients during the course of their illness. While new treatments for the MM itself have prolonged their survival, patients are living longer with their bone disease. Bisphophonates have been shown to reduce skeletal related events in MM, but they neither eliminate these events nor reverse skeletal damage. The transgenic mouse model of MM (vk*myc) is the only mouse model that has been shown to faithfully recapitulate the clinical disease, including its bony abnormalities. The original publication of the model showed principally a decrease in bone mineral density of affected animals but scant lytic lesions. We have cross bred this model with a substrain of C56/BL6 mice, KaLwRiJ, that is the basis of a competing model of MM and its bone disease the 5T33 model. This new transgenic substrain shows significant number bony lesions on x-ray and microCT analyses. Immunohistochemistry of the femurs and spines of these animals show an increased number of osteoclast and a decreased number of osteoblasts as compared to non-transgenic wild type mice of the same substrain. Dynamic labeling of bone shows a decreased mineral apposition rate (1.21mm/day ±0.03 vs 2.05mm/day ±0.04) in mice with MM vs wild type mice. In addition to the demineralization associated with MM bone disease and unique to this model, we show a dramatic decrease in the formation of osteoblasts from cultured mesenchymal stem cells (MSCs). These are grown ex-vivo from affected transgenic mice and induced to differentiate into osteoblasts in culture. We demonstrate a close to 90% reduction in observed and quantified mineralized colonies indicating a dramatic impairment in MSC differentiation in these mice, similar to what is seen in human MM. Gene expression profiling analyses using mRNA from ex-vivo mesenchymal stem cells derived from transgenic and wild type mice reveal a number of pathways and genes that can potentially play a role in the inhibition of MSC differentiation in this model. These include the wnt signaling pathway as well as genes involved in histone acetyltransferase activity. The latter suggests that MSCs are ‘permanently’ affected by the presence of MM cells in vivo and that this inhibition does not improve even when they are separated from MM cells for a prolonged period of time. Bortezomib has been shown in MM patients to improve the appearance of MM lytic lesions radiographically as well as to improve serum markers of osteoblastic activity. This improvement is thought to be due to a reversal of MSC differentiation impairment. Wild type and transgenic mice with MM and bone disease were treated with bortezomib (0.5mg/Kg twice per week) for two weeks. CT analyses of mice pre and post bortezomib treatment showed a 28% improvement in the treated transgenic mice. We also show a concomitant improvement in the ex-vivo ability of MSCs to differentiate into osteoblasts. In summary, we present a unique animal model to study MM bone disease that shows profound MSC impairment. This model responds to a treatment strategy that has been shown to work in the human disease and opens itself to further study and use for future developments targeting MM bone disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Alpha-5 Integrin Mediates Simvastatin-Induced Osteogenesis of Bone Marrow Mesenchymal Stem Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 506 ◽  
Author(s):  
Pei-Lin Shao ◽  
Shun-Cheng Wu ◽  
Zih-Yin Lin ◽  
Mei-Ling Ho ◽  
Chung-Hwan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Calcium Deposition ◽  
Marker Genes ◽  
Expression Levels ◽  
Gene Expression Levels

Simvastatin (SVS) promotes the osteogenic differentiation of mesenchymal stem cells (MSCs) and has been studied for MSC-based bone regeneration. However, the mechanism underlying SVS-induced osteogenesis is not well understood. We hypothesize that α5 integrin mediates SVS-induced osteogenic differentiation. Bone marrow MSCs (BMSCs) derived from BALB/C mice, referred to as D1 cells, were used. Alizarin red S (calcium deposition) and alkaline phosphatase (ALP) staining were used to evaluate SVS-induced osteogenesis of D1 cells. The mRNA expression levels of α5 integrin and osteogenic marker genes (bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen type I, ALP and osteocalcin (OC)) were detected using quantitative real-time PCR. Surface-expressed α5 integrin was detected using flow cytometry analysis. Protein expression levels of α5 integrin and phosphorylated focal adhesion kinase (p-FAK), which is downstream of α5 integrin, were detected using Western blotting. siRNA was used to deplete the expression of α5 integrin in D1 cells. The results showed that SVS dose-dependently enhanced the gene expression levels of osteogenic marker genes as well as subsequent ALP activity and calcium deposition in D1 cells. Upregulated p-FAK was accompanied by an increased protein expression level of α5 integrin after SVS treatment. Surface-expressed α5 integrin was also upregulated after SVS treatment. Depletion of α5 integrin expression significantly suppressed SVS-induced osteogenic gene expression levels, ALP activity, and calcium deposition in D1 cells. These results identify a critical role of α5 integrin in SVS-induced osteogenic differentiation of BMSCs, which may suggest a therapeutic strategy to modulate α5 integrin/FAK signaling to promote MSC-based bone regeneration.

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