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2.
Free Radic Biol Med ; 118: 1-12, 2018 04.
Article in English | MEDLINE | ID: mdl-29452246

ABSTRACT

During endochondral ossification, cartilage template is eventually replaced by bone. This process involves several well characterized, stereotypic, molecular and cellular changes in the cartilage primordia. These steps involve transition from resting to proliferative and then pre-hypertrophic to finally hypertrophic cartilage. BMP signaling is necessary and sufficient for osteogenesis. However, the specific step(s) of endochondral ossification in which BMP signaling plays an essential role is not yet known. In this study we have identified Prdx1, a known scavenger of ROS, to be expressed in pre-hypertrophic chondrocytes in a BMP signaling-dependent manner. We demonstrate that BMP signaling inhibition increases ROS levels in osteogenic cells. Further, Prdx1 regulates osteogenesis in vivo by helping maintenance of Ihh expressing pre-hypertrophic cells, in turn regulating these cells' transition into hypertrophy. Therefore, our data suggests that one of the key roles of BMP signaling in endochondral ossification is to maintain pre-hypertrophic state.


Subject(s)
Chondrocytes/metabolism , Osteogenesis/physiology , Peroxiredoxins/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Cell Enlargement , Chick Embryo , Mice , Rats , Signal Transduction/physiology
3.
Biochim Biophys Acta ; 1863(4): 510-9, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26778333

ABSTRACT

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.


Subject(s)
Core Binding Factor Alpha 1 Subunit/metabolism , Osteogenesis/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , S-Phase Kinase-Associated Proteins/physiology , Animals , Cells, Cultured , Down-Regulation/drug effects , Down-Regulation/genetics , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Osteogenesis/drug effects , RNA, Small Interfering/pharmacology , Rats , Rats, Sprague-Dawley , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , Ubiquitin/metabolism
4.
J Biol Chem ; 290(52): 30975-87, 2015 Dec 25.
Article in English | MEDLINE | ID: mdl-26542806

ABSTRACT

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3ß are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3ß was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3ß was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3ß-dependent manner and thus provides a plausible explanation for GSK3ß-mediated bone loss as described before.


Subject(s)
Cell Differentiation , Core Binding Factor Alpha 1 Subunit/metabolism , F-Box Proteins/metabolism , Osteoblasts/metabolism , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Animals , Bone Resorption/genetics , Bone Resorption/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Female , Glycogen Synthase Kinase 3/biosynthesis , Glycogen Synthase Kinase 3 beta , HEK293 Cells , Humans , Mice , Osteogenesis/genetics , Rats , Rats, Sprague-Dawley , Transcriptional Activation , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics
5.
Biochim Biophys Acta ; 1833(12): 2639-2652, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23820376

ABSTRACT

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3ß), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3ß are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3ß negatively regulates G-CSFR expression and its downstream signaling.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Differentiation , F-Box Proteins/metabolism , Granulocytes/cytology , Granulocytes/metabolism , Proteolysis , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , F-Box-WD Repeat-Containing Protein 7 , Gene Knockdown Techniques , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Kinetics , Mice , Mutant Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , STAT3 Transcription Factor/metabolism , Ubiquitin/metabolism
6.
PLoS One ; 8(6): e65330, 2013.
Article in English | MEDLINE | ID: mdl-23762344

ABSTRACT

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.


Subject(s)
Adipogenesis , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Down-Regulation , Ubiquitin-Protein Ligases/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/metabolism , Animals , Biocatalysis , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , CCAAT-Enhancer-Binding Protein-alpha/genetics , Cell Differentiation , Cell Separation , Gene Knockdown Techniques , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Models, Biological , Mutant Proteins/metabolism , RNA, Small Interfering/metabolism , Transcriptional Activation/genetics , Up-Regulation
7.
Proteomics ; 13(14): 2100-12, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23576398

ABSTRACT

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.


Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Profilins/chemistry , Profilins/metabolism , Proteome/analysis , Amino Acid Motifs , Amino Acid Sequence , Breast Neoplasms/drug therapy , Female , Humans , MCF-7 Cells , Models, Molecular , Molecular Sequence Data , Protein Binding , Proteomics/methods , Tamoxifen/pharmacology
8.
Expert Opin Drug Discov ; 7(12): 1165-75, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22971110

ABSTRACT

INTRODUCTION: Global protein expression profiling between healthy vs diseased states helps identifying differential expression and post-translational modifications of proteins, thereby providing better insights into the molecular changes of disease diagnosis and prognosis. In addition, analytical separation and identification of proteins from complex mixtures can provide insight into targeted drug therapy and prediction of response to different therapeutics. AREAS COVERED: In the present review the authors summarize the readily available quantitative proteomics tools for the analytical separation and identification of target proteins in myeloid leukemia, AML in particular, and its future perspectives in its diagnostics and therapeutics. Within, the authors highlight some of the proteomics approaches such as gel-based quantitation strategies (2D, 2D-DIGE); MS-based quantitative proteomics tools (metabolic labeling (SILAC), chemical labeling (ITRAQ, ICAT)); MS techniques (MALDI-MS/MS). In addition, some of the target proteins identified using proteomics approaches in myeloid leukemia are also discussed that may encourage cancer biology investigators to undertake proteomics as a vital tool in their study. EXPERT OPINION: With suitable, selective application of diverse set of quantitative proteomics strategies integrated with bioinformatics software and precise statistical analysis in myeloid leukemia holds tremendous promise in deciphering cancer proteome, understanding tumor pathophysiology and development of personalized molecular medicine and therapy.


Subject(s)
Chemistry Techniques, Analytical/methods , Drug Discovery/methods , Leukemia, Myeloid/drug therapy , Neoplasm Proteins/metabolism , Proteome/metabolism , Proteomics/methods , Antineoplastic Agents/therapeutic use , Chromatography, Liquid/methods , Electrophoresis, Gel, Two-Dimensional/methods , Humans , Leukemia, Myeloid/metabolism , Mass Spectrometry/methods
9.
Electrophoresis ; 32(3-4): 357-67, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21254132

ABSTRACT

After human genome is decoded, the characterization of the proteins is the next challenging task. The study of the complete protein complement of the genome, the 'proteome' referred to as proteomics, is an important tool for the identification of new therapeutic targets. Research efforts are underway to develop the technology necessary to compare the specific protein profiles of diseased versus healthy states. These technologies provide a wealth of information by rapidly generating large quantities of data. These data can be useful for predictive mathematical descriptions of biological systems for rapid identification of novel therapeutic targets and identification of biomarkers in metabolic disorders. In recent years, using proteomics, we and others have identified various interacting as well as target proteins, PTMs and protein markers in myeloid leukemia. This review summarizes the usage of proteomics in recent years as an important technique in defining the proteome of myeloid leukemia, which has helped in elaborate understanding of the disease and has provided new avenues for developing better therapeutics.


Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/analysis , Genetic Markers , Leukemia, Myeloid/diagnosis , Proteomics/instrumentation , Proteomics/methods , CCAAT-Enhancer-Binding Protein-alpha/genetics , Diagnostic Techniques and Procedures , Drug Design , Humans , Leukemia, Myeloid/blood , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/genetics , Models, Molecular , Mutation/genetics , Proteins , Proteome/analysis , Proteome/metabolism
10.
Eur J Med Chem ; 45(6): 2265-76, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20207053

ABSTRACT

A series of tetrahydro-beta-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC50 value of 740 nM, also arrests cell cycle in G1 phase and induces apoptosis in MCF7 and MDA MB231 cell lines.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carbolines/chemistry , Triazines/chemistry , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Cell Line, Tumor , DNA Fragmentation/drug effects , G1 Phase/drug effects , Humans , Mitosis/drug effects
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