scholarly journals 3152 The role of myostatin in diabetic bone disease

2019 ◽  
Vol 3 (s1) ◽  
pp. 24-25
Author(s):  
Evangelia Kalaitzoglou ◽  
Callie Knuckles ◽  
John Fowlkes
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Bone Disease ◽  
Osteoblast Differentiation ◽  
Bone Cells ◽  
Negative Regulator ◽  
Negative Effects ◽  
Osteoblastic Cell Line ◽  
Primary Osteoblasts

OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine the mechanism of action of myostatin on osteoblasts by measuring markers of osteoblast differentiation. With these experiments we will evaluate the effects of myostatin on an osteoblastic cell line (MC3T3 cells) and primary murine osteoblasts during baseline and hyperglycemic conditions and assess whether these effects are altered in the presence of a hyperglycemic environment. METHODS/STUDY POPULATION: Primary osteoblasts from calvaria of WT mice will be isolated and cultured per previously published protocol. MC3T3 cells (murine pre-osteoblast cell line) and primary osteoblasts will be plated in 6-well plates until they reach confluency. They will subsequently be stimulated with or without myostatin at various concentrations under control and hyperglycemic conditions. Additional experiments will assess myostatin stimulation during cell differentiation/maturation in the presence of osteogenic induction medium. Subsequently, cells will be lysed and processed for gene analysis with qPCR. Genes of interest (e.g., myostatin, RUNX2, osteocalcin etc.) will be assessed. Additionally, cells will be collected and processed for protein quantification with western blot to assess myostatin-related pathways, such as Smad2/3 and MAPK signaling. RESULTS/ANTICIPATED RESULTS: We have demonstrated that the receptor for myostatin (Activin receptor 2b, AcvR2b) is present in MC3T3 cells and we have evidence of Smad2 phosphorylation in MC3T3 cells as a result of myostatin stimulation, confirming that myostatin can exert intracellular signaling events in bone cells (Fig 1). We anticipate to observe negative effects of myostatin on differentiation of primary osteoblasts and MC3T3 cells. Specifically, we anticipate suppression of Runt-related transcription factor 2 (RUNX-2), a transcription factor known as the “ master regulator” of osteogenic gene expression and programming, as a result of signaling downstream of Smad 2/3. Additionally we anticipate downregulation of osterix and osteocalcin, two essential genes for osteoblast differentiation and activity. We anticipate that hyperglycemia will potentiate the negative effects of myostatin on osteoblastogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: We have demonstrated that myostatin can directly act on osteoblastic cells. As myostatin is a negative regulator or bone mass, its direct effects on bone cells can be detrimental to the bone health of patients with elevated myostatin levels and/or activity. There is evidence suggesting that myostatin is elevated in Type 1 diabetes, and its effects might be potentiated in a hyperglycemic environments. Future experiments will be evaluating the role of myostatin on a diabetic animal model and in humans. Our experiments provide an additional mechanism by which muscle-bone interactions could be contributing to the development of diabetic bone disease.

scholarly journals Type 2 Iodothyronine Selenodeiodinase Is Expressed throughout the Mouse Skeleton and in the MC3T3-E1 Mouse Osteoblastic Cell Line during Differentiation

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Cecilia H. A. Gouveia ◽  
Marcelo A. Christoffolete ◽  
Clarissa R. Zaitune ◽  
José Miguel Dora ◽  
John W. Harney ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Cell Line ◽  
Bone Cells ◽  
Bone Development ◽  
Cell Types ◽  
Osteoblastic Cell ◽  
Dependent Manner ◽  
Osteoblastic Cell Line ◽  
Serum T3

Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nm, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5–7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30–40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFα, TGFβ, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.

scholarly journals miR-539 mediates osteoblast mineralization by regulating Distal-less genes 2 in MC3T3-E1 cell line

2017 ◽  
Vol 12 (1) ◽  
pp. 294-299 ◽  
Author(s):  
Jianguo Han ◽  
Li Su ◽  
Chunyang Zhang ◽  
Rongcai Jiang
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Cell Line ◽  
Osteoblast Differentiation ◽  
Marker Gene ◽  
Inhibitory Effect ◽  
Negative Regulator ◽  
Expression Level ◽  
Matrix Mineralization ◽  
Marker Gene Expression

AbstractmicroRNAs (miRNAs) play an important role in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still needs to be further cleared. Distal-less genes 2 (Dlx2) plays an important role in osteoblast differentiation. We have found that miR-539 was significantly downregulated and Dlx2 was found to be inversely correlated with miR-539 in MC3T3-E1 cell line during osteoblast mineralization. The overexpression of miR-539 significantly decreased the expression level of Dlx2 and suppressed the osteogenic marker gene expression level, alkaline phosphatase activity and matrix mineralization. Our study showed that miR-539 was a negative regulator in osteoblast mineralization and that the targeting of Dlx2 gene partly contributes to this inhibitory effect exerted by miR-539.

scholarly journals A CCAAT/Enhancer Binding Protein β Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

2005 ◽  
Vol 25 (5) ◽  
pp. 1971-1979 ◽  
Author(s):  
Kenji Hata ◽  
Riko Nishimura ◽  
Mio Ueda ◽  
Fumiyo Ikeda ◽  
Takuma Matsubara ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Osteoblast Differentiation ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Mesenchymal Cells ◽  
Dominant Negative ◽  
Inhibitory Protein ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein β (C/EBPβ), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPβ, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPβ, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPβ isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2267-2267
Author(s):  
Eva Sahakian ◽  
John Powers ◽  
Jie Chen ◽  
Allison Distler ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Hematopoietic Cells ◽  
Negative Regulator ◽  
Over Expression

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Expanding Role of MT1-MMP in Cancer Progression

2019 ◽  
Vol 12 (2) ◽  
pp. 77 ◽  
Author(s):  
Anna M. Knapinska ◽  
Gregg B. Fields
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Cancer Progression ◽  
Negative Regulator ◽  
Fibrillar Collagen ◽  
Biological Functions ◽  
Complete Mapping ◽  
Membrane Type

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array of MT1-MMP substrates, and MT1-MMP selective inhibitors have allowed for a more complete mapping of MT1-MMP biological functions. MT1-MMP has extensive sheddase activities, is both a positive and negative regulator of angiogenesis, can act intracellularly and as a transcription factor, and modulates immune responses. We presently examine the multi-faceted role of MT1-MMP in cancer, with a consideration of how the diversity of MT1-MMP behaviors impacts the application of MT1-MMP inhibitors.

scholarly journals Impaired cyclic AMP-dependent phosphorylation renders CREB a repressor of C/EBP-induced transcription of the somatostatin gene in an insulinoma cell line.

1995 ◽  
Vol 15 (1) ◽  
pp. 415-424 ◽  
Author(s):  
M Vallejo ◽  
M E Gosse ◽  
W Beckman ◽  
J F Habener
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Cell Line ◽  
Target Genes ◽  
Gene Promoter ◽  
Chloramphenicol Acetyltransferase ◽  
Negative Regulator ◽  
Insulinoma Cell ◽  
Insulinoma Cell Line

Transcription factor CREB regulates cyclic AMP (cAMP)-dependent gene expression by binding to and activating transcription from cAMP response elements (CREs) in the promoters of target genes. The transcriptional transactivation functions of CREB are activated by its phosphorylation by cAMP-dependent protein kinase A (PKA). In studies of many different phenotypically distinct cells, the CRE of the somatostatin gene promoter is a prototype of a highly cAMP-responsive element regulated by CREB. We now report on a somatostatin-producing rat insulinoma cell line, RIN-1027-B2, in which transcription from the somatostatin gene promoter is paradoxically repressed by CREB. We find that CREB fails to transactivate a CRE-containing somatostatin-chloramphenicol acetyltransferase reporter even when coexpressed with the catalytic subunit of PKA. CAAT box/enhancer-binding protein beta (C/EBP beta) and C/EBP-related activating transcription factor bind to the CRE in the promoter of the somatostatin gene and transactivate transcription. CREB binds competitively with C/EBP beta to the somatostatin CRE in vitro and represses C/EBP beta-induced transcription of the CRE-containing somatostatin-chloramphenicol acetyltransferase reporter. The lack of CREB-mediated transcriptional stimulation is due to the presence of a heat-stable inhibitor of PKA that prevents activation of PKA and subsequent CREB phosphorylation in the nucleus. These findings indicate that dephosphorylated CREB is a negative regulator of C/EBP-activated transcription of the somatostatin gene promoter in RIN-1027-B2 cells.

scholarly journals A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides

2015 ◽  
Vol 28 (2) ◽  
pp. 154-166 ◽  
Author(s):  
Dana Ment ◽  
Noam Alkan ◽  
Neta Luria ◽  
Fang-Cheng Bi ◽  
Eli Reuveni ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Colletotrichum Gloeosporioides ◽  
Gene Expression Regulation ◽  
Tomato Fruit ◽  
Negative Regulator ◽  
Sequencing Data ◽  
Fungal Pathogenicity ◽  
Sequencing Data Analysis

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

scholarly journals A novel role of MNT as a negative regulator of REL and the NF-κB pathway

Oncogenesis ◽  
2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Judit Liaño-Pons ◽  
M. Carmen Lafita-Navarro ◽  
Lorena García-Gaipo ◽  
Carlota Colomer ◽  
Javier Rodríguez ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Transcriptional Regulator ◽  
Negative Regulator ◽  
Biological Processes ◽  
Helix Loop Helix ◽  
Bona Fide ◽  
E Boxes ◽  
Independent Protein

AbstractMNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix–loop–helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and REL, the oncogenic member of the NF-κB family. REL participates in important biological processes and it is altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with IκB and translocates to the nucleus when the NF-κB pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-κB pathway. Meanwhile, MNT overexpression results in the repression of IκBα, a bona fide REL target. Both MNT and REL bind to the IκBα gene on the first exon, suggesting its regulation as an MNT–REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-κB pathway by two mechanisms: (1) retention of REL in the cytoplasm by MNT interaction, and (2) MNT-driven repression of REL-target genes through an MNT–REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and NF-κB pathways, two of the most prominent pathways in cancer.

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