scholarly journals LIF/STAT3/SOCS3 Signaling Pathway in Murine Bone Marrow Stromal Cells Suppresses Osteoblast Differentiation

2014 ◽  
Vol 115 (7) ◽  
pp. 1262-1268 ◽  
Author(s):  
Kenta Matsushita ◽  
Shousaku Itoh ◽  
Shun Ikeda ◽  
Yumiko Yamamoto ◽  
Yukako Yamauchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Murine Bone Marrow

scholarly journals BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells

PLoS ONE ◽  
2016 ◽  
Vol 11 (6) ◽  
pp. e0158481 ◽  
Author(s):  
Su-Hyang Yoo ◽  
Jae Goo Kim ◽  
Beom-Su Kim ◽  
Jun Lee ◽  
Sung-Hee Pi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells

scholarly journals Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation

Metabolomics ◽  
2021 ◽  
Vol 17 (10) ◽  
Author(s):  
Biswapriya B. Misra ◽  
Shobana Jayapalan ◽  
Alison K. Richards ◽  
Ron C. M. Helderman ◽  
Elizabeth Rendina-Ruedy
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Untargeted Metabolomics ◽  
Coupled Processes ◽  
Intracellular Metabolite ◽  
Mineral Density ◽  
Murine Bone Marrow

Abstract Introduction Skeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation. Objective The current project sought to rigorously characterize the distinct metabolic processes and intracellular metabolite profiles in stromal cells throughout osteoblast differentiation using untargeted metabolomics. Methods Primary, murine bone marrow stromal cells (BMSCs) were characterized throughout osteoblast differentiation using standard staining protocols, Seahorse XFe metabolic flux analyses, and untargeted metabolomics. Results We demonstrate here that the metabolic footprint of stromal cells undergoing osteoblast differentiation are distinct, and while oxidative phosphorylation drives adenosine triphosphate (ATP) generation early in the differentiation process, mature osteoblasts depend on glycolysis. Importantly, the intracellular metabolite profile supports these findings while also suggesting additional pathways critical for proper osteoblast function. Conclusion These data are the first of their kind to characterize these metabolites in conjunction with the bioenergetic profile in primary, murine stromal cells throughout osteoblast differentiation and provide provocative targets for future investigation.

scholarly journals Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential

2005 ◽  
Vol 25 (12) ◽  
pp. 5183-5195 ◽  
Author(s):  
Taisuke Mori ◽  
Tohru Kiyono ◽  
Hideaki Imabayashi ◽  
Yukiji Takeda ◽  
Kohei Tsuchiya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Life Span ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Murine Bone Marrow ◽  
Differentiated Cells ◽  
E6 And E7 ◽  
Old Donor

ABSTRACT Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.

The Transcription Repressor Gfi1 Directly Interacts With and Is Phosphorylated By Aurora A Kinase, Which Abrogates Myeloma-Induced Gfi1 Repression Of The Runx2 Promoter In Pre-Osteoblasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1844-1844
Author(s):  
Jixin Ding ◽  
Fengming Wang ◽  
ShunQian Jin ◽  
Judy Anderson ◽  
Deborah L. Galson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Disease ◽  
Stromal Cells ◽  
Protein Turnover ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Aurora A Kinase

Abstract Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Hyperactive Non-Canonical TGF-β Pathway Signaling in Fanconi Anemia Bone Marrow Stromal Cells Contributes to Growth Suppression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1039-1039
Author(s):  
Kevin O'Connor ◽  
Sofia Vidal-Cardenas ◽  
Haojian Zhang ◽  
Alfredo Rodriguez ◽  
Lisa Moreau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Bone Marrow Failure ◽  
Genotoxic Stress ◽  
Growth Suppression ◽  
Marrow Stromal Cells ◽  
Growth Defect ◽  
High Level

Abstract Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem cells (HSCs). FA is caused by autosomal recessive or X-linked mutations in one of nineteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to genotoxic DNA cross-linking agents. Although its mechanism is unknown, bone marrow failure in FA may be the result, directly or indirectly, of hyperactivation of cell-autonomous or microenvironmental growth-suppressive pathways induced, in part, due to genotoxic stress. We have recently identified canonical transforming growth factor-β (TGF-β) pathway-mediated growth suppression of HSCs as a cause of bone marrow failure in FA (Zhang H et al, Cell Stem Cell, 2016). We have shown that TGF-β pathway inhibition rescues genotoxic stress, proliferation defects and engraftment defects of FA-deficient HSCs, and ameliorates bone marrow failure in FA mice. Previous studies have suggested that bone marrow stromal fibroblasts from human FA patients and FA pathway-deficient mouse models, like HSCs, are hypersensitive to genotoxic stress and have impaired growth. Here, we therefore investigated the possible suppressive function of the TGF-β pathway in bone marrow stromal cells derived from FA mice and patients with FA. Methods: We established primary stromal cell lines from bone marrow of FA-deficient mice (Fancd2-/- mice) or wild-type sibling control mice. Primary bone marrow stromal cultures were also established from FA patients or normal healthy donors. The stromal cells were characterized and evaluated for growth kinetics, mitomycin C (MMC) sensitivity, chromosome breakage, inflammatory signals and response to the TGF-β inhibitors. CRISPR/Cas-9 technology was used to knockdown specific genes in stromal cells. Results: As expected,the primary bone marrow stromal cells from Fancd2-/- mice exhibited classical FA phenotypes, including hypersensitivity to a DNA cross-linking agent, MMC, and increased MMC-induced chromosomal radials. Fancd2-/- stromal cells also demonstrated a growth defect characterized by an enrichment of cells in G1 and elevated p21 expression. Interestingly, the FA stromal cells derived from FA patients or from Fancd2-/- mice expressed constitutively elevated levels of phosphorylated (activated) ERK1/2 (pERK) compared to control cells. In order to determine whether the factor responsible for inducing ERK1/2 phosphorylation in the murine FA stromal cells was cell-intrinsic or cell-extrinsic, we examined the conditioned media from the stromal cells. Indeed the FA stromal cells secreted a high level of TGF-β cytokine responsible for increased pERK levels, and expressed a high level of secreted TGF-β mRNA. The high level of pERK indicated that the TGF-β non-canonical pathway was hyperactive in the FA stromal cells. Interestingly, CRISPR/Cas9-mediated knockdown of Tgfbr1 or inhibition of the TGF-β pathway by a treatment with a small molecule inhibitor of TGFβR1 or a neutralizing antibody against TGF-β in these cells reduced pERK levels, promoted DNA repair and rescued MMC sensitivity. In addition, a MEK inhibitor also significantly improved the clonogenic growth of Fancd2-/- stromal cells. However, CRISPR/Cas9-mediated knockdown of Smad3, a downstream target of the canonical TGF-β pathway, did not rescue the growth inhibition of FA stromal cells in MMC, further indicating that hyperactivation of the canonical pathway is less relevant to their growth defect. Collectively, these results demonstrated that the hyperactive TGF-β pathway increases phosphorylation of ERK1/2 in FA stromal cells through the non-canonical signaling pathway and impairs their growth after genotoxic stress. Conclusions: The primary FA bone marrow stromal cells exhibit hyperactive non-canonical TGF-β pathway signaling and blocking this pathway improves their growth under genotoxic stress. The TGF-β signaling pathway-mediated growth suppression in bone marrow stromal cells may account, at least in part, for defective microenvironment, impaired HSC function and bone marrow failure in FA. This work suggests that the TGF-β signaling pathway may be a potential therapeutic target for the treatment of bone marrow failure in FA. Disclosures Shimamura: TransCellular Therapeutics: Other: Husband is founder. No revenue to date.; Novartis: Other: In discussion regarding possible clinical trial for aplastic anemia; Glaxo Smith Kline: Honoraria.

Sign in / Sign up

Export Citation Format

Share Document