scholarly journals Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation

2019 ◽  
Vol 294 (20) ◽  
pp. 8184-8196 ◽  
Author(s):  
William N. Addison ◽  
Martin Pellicelli ◽  
René St-Arnaud
Keyword(s):  
Transcription Factor ◽  
Osteoblast Differentiation ◽  
Transcriptional Activity ◽  
Affinity Purification ◽  
Catalytic Subunit ◽  
Luciferase Reporter ◽  
Matrix Mineralization ◽  
Regulatory Subunits ◽  
Matrix Metallopeptidase ◽  
And Function

The transcriptional cofactor nascent polypeptide-associated complex and co-regulator α (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA's activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified protein phosphatase 1 catalytic subunit α (PP1A) as a NACA-associated Ser/Thr phosphatase. NACA interacted with multiple components of the PP1A holoenzyme complex: the PPP1CA catalytic subunit and the regulatory subunits PPP1R9B, PPP1R12A and PPP1R18. MS analysis revealed that NACA co-expression with PPP1CA causes dephosphorylation of NACA at Thr-89, Ser-151, and Thr-174. NACA Ser/Thr-to-alanine variants displayed increased nuclear localization, and NACA dephosphorylation was associated with specific recruitment of novel NACA interactants, such as basic transcription factor 3 (BTF3) and its homolog BTF3L4. NACA and PP1A cooperatively potentiated cJUN transcriptional activity of the AP-1–responsive MMP9-luciferase reporter, which was abolished when Thr-89, Ser-151, or Thr-174 were substituted with phosphomimetic aspartate residues. We confirmed the NACA–PP1A interaction in MC3T3-E1 osteoblastic cells and observed that NACA phosphorylation status at PP1A-sensitive sites is important for the regulation of AP-1 pathway genes and for osteogenic differentiation and matrix mineralization. These results suggest that PP1A dephosphorylates NACA at specific residues, impacting cJUN transcriptional activity and osteoblast differentiation and function.

Canine H(+)-K(+)-ATPase alpha-subunit gene promoter: studies with canine parietal cells in primary culture

1996 ◽  
Vol 271 (6) ◽  
pp. G1104-G1113 ◽  
Author(s):  
A. Muraoka ◽  
M. Kaise ◽  
Y. J. Guo ◽  
J. Yamada ◽  
I. Song ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Primary Culture ◽  
Transcriptional Activity ◽  
Parietal Cells ◽  
Luciferase Reporter ◽  
Cell Type ◽  
High Concentration ◽  
Specific Expression ◽  
Atpase Gene ◽  
Cell Type Specific

H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) is the principal enzyme responsible for the process of gastric acid secretion. This enzyme is expressed in a cell-type-specific manner in gastric parietal cells. To explore the mechanisms regulating its expression, we transfected differentiated canine parietal cells in primary culture with H(+)-K(+)-ATPase-luciferase reporter genes and assessed transcriptional activities. Deletional analysis of the 5'-flanking region of this gene demonstrated a remarkable increment in transcriptional activity associated with a segment between bases -54 to -45 (5' GCTCCGCCTC 3') relative to the transcriptional initiation site. Gel shift assays with competition and supershift analysis demonstrated that this segment is specifically bound by the transcription factor Sp1. A point mutation, eliminating Sp1 binding, diminished basal transcriptional activity by 80%, indicating that this Sp1 binding site is important for constitutive transcriptional activity. Although these studies indicate that Sp1 is required to maintain a high concentration of the H(+)-K(+)-ATPase gene in the parietal cell, its cell-type-specific expression must rely on other elements because Sp1 is a ubiquitously expressed transcription factor.

scholarly journals MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1

2016 ◽  
Vol 38 (2) ◽  
pp. 809-820 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xiaojian Cao ◽  
Yingbin Ge ◽  
Hongtao Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Postmenopausal Osteoporosis ◽  
Osteoblast Differentiation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Matrix Mineralization ◽  
Direct Target

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

scholarly journals Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor

2020 ◽  
Vol 21 (24) ◽  
pp. 9579
Author(s):  
Kyung-Ran Park ◽  
SooHyun Kim ◽  
MyoungLae Cho ◽  
Sang Wook Kang ◽  
Hyung-Mun Yun
Keyword(s):  
Transcription Factor ◽  
Cell Migration ◽  
Protein Level ◽  
Osteoblast Differentiation ◽  
Signaling Molecules ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Related Transcription Factor ◽  
Dose Dependent

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

scholarly journals The PERK-EIF2α-ATF4 signaling branch regulates osteoblast differentiation and proliferation by PTH

2019 ◽  
Vol 316 (4) ◽  
pp. E590-E604 ◽  
Author(s):  
Kefan Zhang ◽  
Miaomiao Wang ◽  
Yingjiang Li ◽  
Chunping Li ◽  
Shaidi Tang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Er Stress ◽  
Signaling Pathway ◽  
Osteoblast Differentiation ◽  
Type I ◽  
Matrix Mineralization ◽  
Related Peptide ◽  
Differentiation And Proliferation

Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1–34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG’44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.

rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs

1990 ◽  
Vol 10 (7) ◽  
pp. 3810-3812
Author(s):  
C S Pikaard ◽  
S D Smith ◽  
R H Reeder ◽  
L Rothblum
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Affinity Purification ◽  
Binding Specificity ◽  
Rna Polymerase I ◽  
Dna Binding Specificity ◽  
Apparent Homogeneity ◽  
And Function ◽  
Polymerase I

Rat cells contain a DNA-binding polymerase I transcription factor, rUBF, with properties similar to UBF homologs that have been purified from both human (hUBF) and frog (xUBF) cells. In this note we report the affinity purification of rUBF to apparent homogeneity and show that UBFs from both rat and frog have identical footprinting characteristics on templates from either species. Furthermore, xUBF was able to stimulate transcription from rat RNA polymerase I promoters in a partially fractionated rat extract that was UBF dependent. These results strengthen the conclusion that all vertebrate cells contain a UBF homolog whose DNA-binding specificity and function have been strongly conserved.

Loss Of Tumor Suppressor BTG1 Enhances ATF4 Function and Promotes Cell Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3796-3796
Author(s):  
Laurens T. van der Meer ◽  
Laurensia Yuniati ◽  
Esther J.H. Tijchon ◽  
Liesbeth van Emst ◽  
Manon Alkema ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Er Stress ◽  
B Cell ◽  
Cell Survival ◽  
Transcriptional Activity ◽  
Cellular Stress ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Leukemia Development ◽  
Error Bars

Abstract We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth. We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells.Figure 1BTG1 knockout cells are resistant to Asparaginase treatment.Figure 1. BTG1 knockout cells are resistant to Asparaginase treatment. As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2).Figure 2BTG1 attenuates ATF4 transcriptional activity.Figure 2. BTG1 attenuates ATF4 transcriptional activity. Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells. Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones. Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean. A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CCAAT/Enhancer-binding Protein β Promotes Osteoblast Differentiation by Enhancing Runx2 Activity with ATF4

2008 ◽  
Vol 19 (12) ◽  
pp. 5373-5386 ◽  
Author(s):  
Hiroyuki Tominaga ◽  
Shingo Maeda ◽  
Makoto Hayashi ◽  
Shu Takeda ◽  
Shizuo Akira ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Specific Element ◽  
Chondrocyte Maturation ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Osteoblast Maturation

Although CCAAT/enhancer-binding protein β (C/EBPβ) is involved in osteocalcin gene expression in osteoblast in vitro, the physiological importance of and molecular mechanisms governing C/EBPβ in bone formation remain to be elucidated. In particular, it remains unclear whether C/EBPβ acts as a homodimer or a heterodimer with other proteins during osteoblast differentiation. Here, deletion of the C/EBPβ gene from mice resulted in delayed bone formation with concurrent suppression of chondrocyte maturation and osteoblast differentiation. The expression of type X collagen as well as chondrocyte hypertrophy were suppressed in mutant bone, providing new insight into the possible roles of C/EBPβ in chondrocyte maturation. In osteoblasts, luciferase reporter, gel shift, DNAP, and ChIP assays demonstrated that C/EBPβ heterodimerized with activating transcription factor 4 (ATF4), another basic leucine zipper transcription factor crucial for osteoblast maturation. This complex interacted and transactivated osteocalcin-specific element 1 (OSE1) of the osteocalcin promoter. C/EBPβ also enhanced the synergistic effect of ATF4 and Runx2 on osteocalcin promoter transactivation by enhancing their interaction. Thus, our results provide evidence that C/EBPβ is a crucial cofactor in the promotion of osteoblast maturation by Runx2 and ATF4.

scholarly journals miRNA-340 inhibits osteoclast differentiation via repression of MITF

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Hongying Zhao ◽  
Jun Zhang ◽  
Haiyu Shao ◽  
Jianwen Liu ◽  
Mengran Jin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Tartrate Resistant Acid Phosphatase ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Macrophage Colony ◽  
And Function

Many miRNAs play critical roles in modulating various biological processes of osteoclast differentiation and function. Microphthalmia-associated transcription factor (MITF), a target of miR-340, served as pivotal transcription factor involved in osteoclast differentiation. However, the role of miR-340 and MITF during osteoclast differentiation has not yet been clearly established. Tartrate-resistant acid phosphatase (TRAP) staining assay was performed to identify osteoclasts differentiated from bone marrow-derived macrophages (BMMs). Quantitative reverse transcription PCR (qRT-PCR) or Western blotting was undertaken to examine the mRNA or protein expression respectively. Luciferase reporter assay was performed to investigate the interaction between miR-340 and MITF. MITF was knocked down and miR-340 was overexpressed and transfected into BMMs to detect their effects on osteoclast differentiation. Firstly, qRT-PCR analysis showed that miR-340 was down-regulated during osteoclast differentiation stimulated by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB (RANK) ligand (RANKL). Besides, we found that overexpression of miRNA-340 inhibited osteoclast differentiation and suppressed both the mRNA and protein level of MITF. Finally, Western blot and qRT-PCR analysis revealed that silencing MITF inhibited TRAP, calcitonin receptor, V-ATPase d2, and cathepsin K. miR-340 suppresses osteoclast differentiation by inhibiting MITF. Our findings may provide promising therapeutic targets for osteoclast-associated diseases.

scholarly journals High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1

2013 ◽  
Vol 305 (3) ◽  
pp. F362-F369 ◽  
Author(s):  
Xiaoming Zhou ◽  
Hong Wang ◽  
Maurice B. Burg ◽  
Joan D. Ferraris
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
Transcriptional Activity ◽  
Protein Targeting ◽  
Inhibitory Effect ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Protein Abundance ◽  
Negative Effect ◽  
Sirna Screening

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.

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