Publications by authors named "Sudarshana M Sharma"

27 Publications

  • Page 1 of 1

An Emerging Role of miRNAs in Neurodegenerative Diseases: Mechanisms and Perspectives on .

Antioxid Redox Signal 2021 Feb 15. Epub 2021 Feb 15.

Darby Children's Research Institute, Medical University of South Carolina, Charleston, South Carolina, USA.

Advancements in and access to health care have led to unprecedented improvements in the quality of life and increased lifespan of human beings in the past century. However, aging is a significant risk factor for neurodegenerative diseases (NDs). Hence, improved life expectancy has led to an increased incidence of NDs. Despite intense research, effective treatments for NDs remain elusive. The future of neurotherapeutics development depends on effective disease modification strategies centered on carefully scrutinized targets. As a promising new direction, recent evidence has demonstrated that epigenetic processes modify diverse biochemical pathways, including those related to NDs. Small non-coding RNAs, known as microRNAs (miRNAs), are components of the epigenetic system that alter the expression of target genes at the post-transcriptional level. miRNAs are expressed abundantly in the central nervous system and are critical for the normal functioning and survival of neurons. Here, we review recent advances in elucidating miRNAs' roles in NDs and discuss their potential as therapeutic targets. In particular, neuroinflammation is a major pathological hallmark of NDs and is a crucial regulator of inflammation. Finally, we explore the possibilities of developing as a potential biomarker and therapeutic target where additional research may help facilitate the detection and amelioration of neuroinflammation in NDs.
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http://dx.doi.org/10.1089/ars.2020.8256DOI Listing
February 2021

Nanofiber-expanded human CD34 cells heal cutaneous wounds in streptozotocin-induced diabetic mice.

Sci Rep 2019 06 10;9(1):8415. Epub 2019 Jun 10.

Department of Internal Medicine, Wexner Medical Center at The Ohio State University, Columbus, Ohio, USA.

Despite advances in diabetic wound care, the significant number of amputations that occur every year demands more effective therapeutics. Herein, we offer an aminated polyethersulfone nanofiber-expanded human umbilical cord blood-derived CD34 cells (henceforth CD34 cells) effective therapy, tested in cutaneous wounds developed in streptozotocin-induced diabetic NOD/SCID mice. We show that systemic administration of CD34 cells homed to the wound site and significantly accelerated wound closure. Wound closure was associated with improved re-epithelialization and increased neovascularization; and with decreased sustained pro-inflammatory activity of NF-κB and its downstream effector molecules TNF-α, IL-1β, and IL-6 at the wound bed. This finding was further supported by the observation of a decreased number of myeloperoxidase positive neutrophils, and concomitantly increased levels of IL-10. In addition, improved granulation tissue formation was observed along with higher collagen deposition and myofibroblasts and decreased expressions of MMP-1. Mechanistically, CD34 cells reduced the level of MMP-1 expression by inhibiting recruitment of NF-κB to the MMP-1 promoter site in dermal fibroblasts. In summary, we provide evidence of a novel nanofiber-expanded CD34 stem cell therapeutic development for treating diabetic wounds by defining their cellular and molecular mechanisms.
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http://dx.doi.org/10.1038/s41598-019-44932-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557810PMC
June 2019

Eomes partners with PU.1 and MITF to Regulate Transcription Factors Critical for osteoclast differentiation.

iScience 2019 Jan 27;11:238-245. Epub 2018 Dec 27.

Department of Cancer Biology and Genetics and Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA; Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Hollings Cancer Center, 86 Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:

Bone-resorbing osteoclasts (OCs) are derived from myeloid precursors (MPs). Several transcription factors are implicated in OC differentiation and function; however, their hierarchical architecture and interplay are not well known. Analysis for enriched motifs in PU.1 and MITF chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) data from differentiating OCs identified eomesodermin (EOMES) as a potential novel binding partner of PU.1 and MITF at genes critical for OC differentiation and function. We were able to demonstrate using co-immunoprecipitation and sequential ChIP analysis that PU.1, MITF, and EOMES are in the same complex and present as a complex at OC genomic loci. Furthermore, EOMES knockdown in MPs led to osteopetrosis associated with decreased OC differentiation and function both in vitro and in vivo. Although EOMES is associated with embryonic development and other hematopoietic lineages, this is the first study demonstrating the requirement of EOMES in the myeloid compartment.
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http://dx.doi.org/10.1016/j.isci.2018.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327072PMC
January 2019

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

Nat Commun 2018 08 24;9(1):3431. Epub 2018 Aug 24.

Department of Cancer Biology and Genetics, Columbus, OH, 43210, USA.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.
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http://dx.doi.org/10.1038/s41467-018-05910-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109146PMC
August 2018

Enhancer variants reveal a conserved transcription factor network governed by PU.1 during osteoclast differentiation.

Bone Res 2018 28;6. Epub 2018 Mar 28.

1Department of Cancer Biology and Genetics and Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA.

Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states.
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http://dx.doi.org/10.1038/s41413-018-0011-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874256PMC
March 2018

Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia.

Neoplasia 2016 09;18(9):541-52

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; Department of Cancer Biology & Genetics, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.
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http://dx.doi.org/10.1016/j.neo.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031867PMC
September 2016

Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant.

J Cell Physiol 2016 Mar;231(3):630-40

Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, Ohio.

The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation.
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http://dx.doi.org/10.1002/jcp.25108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664053PMC
March 2016

The multifunctional protein fused in sarcoma (FUS) is a coactivator of microphthalmia-associated transcription factor (MITF).

J Biol Chem 2014 Jan 20;289(1):326-34. Epub 2013 Nov 20.

From the Department of Molecular and Cellular Biochemistry, Comprehensive Cancer Center, and.

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.
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http://dx.doi.org/10.1074/jbc.M113.493874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879555PMC
January 2014

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

PLoS One 2013 16;8(8):e71533. Epub 2013 Aug 16.

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America.

Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745457PMC
April 2014

TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in duchenne muscular dystrophy.

PLoS One 2010 Aug 30;5(8):e12479. Epub 2010 Aug 30.

Human Cancer Genetics and Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, United States of America.

Background: Classical NF-kappaB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFalpha on skeletal muscle differentiation are mediated in part through sustained NF-kappaB activity. In dystrophic muscles, NF-kappaB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFalpha that is also under IKKbeta and NF-kappaB control.

Methodology/principal Findings: Based on these findings we speculated that in DMD, TNFalpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFalpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappaB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFalpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2.

Conclusions/significance: We propose that in dystrophic muscles, elevated levels of TNFalpha and NF-kappaB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012479PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930001PMC
August 2010

An ets2-driven transcriptional program in tumor-associated macrophages promotes tumor metastasis.

Cancer Res 2010 Feb 9;70(4):1323-33. Epub 2010 Feb 9.

Department of Molecular and Cellular Biochemistry, Center for Biostatistics, College of Public Health, and Tumor Microenvironment Program, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.

Tumor-associated macrophages (TAM) are implicated in breast cancer metastasis, but relatively little is known about the underlying genes and pathways that are involved. The transcription factor Ets2 is a direct target of signaling pathways involved in regulating macrophage functions during inflammation. We conditionally deleted Ets in TAMs to determine its function at this level on mouse mammary tumor growth and metastasis. Ets2 deletion in TAMs decreased the frequency and size of lung metastases in three different mouse models of breast cancer metastasis. Expression profiling and chromatin immunoprecipitation assays in isolated TAMs established that Ets2 repressed a gene program that included several well-characterized inhibitors of angiogenesis. Consistent with these results, Ets2 ablation in TAMs led to decreased angiogenesis and decreased growth of tumors. An Ets2-TAM expression signature consisting of 133 genes was identified within human breast cancer expression data which could retrospectively predict overall survival of patients with breast cancer in two independent data sets. In summary, we identified Ets2 as a central driver of a transcriptional program in TAMs that acts to promote lung metastasis of breast tumors.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-1474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822898PMC
February 2010

Pten in stromal fibroblasts suppresses mammary epithelial tumours.

Nature 2009 Oct;461(7267):1084-91

Department of Molecular Genetics, College of Biological Sciences, The Ohio State University, Columbus, Ohio 43210, USA.

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.
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http://dx.doi.org/10.1038/nature08486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2767301PMC
October 2009

Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells.

Science 2009 Aug 20;325(5944):1142-6. Epub 2009 Aug 20.

Immunology and Hematopoiesis Division, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.
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http://dx.doi.org/10.1126/science.1176077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859703PMC
August 2009

Ets1 and Ets2 are required for endothelial cell survival during embryonic angiogenesis.

Blood 2009 Jul 1;114(5):1123-30. Epub 2009 May 1.

Department of Molecular and Cellular Biochemistry, The OhioState University, Columbus, OH 43210, USA.

The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.
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http://dx.doi.org/10.1182/blood-2009-03-211391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2721789PMC
July 2009

Defective co-activator recruitment in osteoclasts from microphthalmia-oak ridge mutant mice.

J Cell Physiol 2009 Jul;220(1):230-7

Department of Molecular and Cellular Biochemistry, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.
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http://dx.doi.org/10.1002/jcp.21755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852098PMC
July 2009

Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K.

Circ Res 2009 Feb 2;104(4):455-65. Epub 2009 Jan 2.

Department of Medicine, Columbia University, New York, NY 10032, USA.

The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.
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http://dx.doi.org/10.1161/CIRCRESAHA.108.182568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680702PMC
February 2009

NFATc1 in mice represses osteoprotegerin during osteoclastogenesis and dissociates systemic osteopenia from inflammation in cherubism.

J Clin Invest 2008 Nov 9;118(11):3775-89. Epub 2008 Oct 9.

Department of Infectious Diseases and Immunology, Harvard School of Public Health, Boston, Massachusetts, USA.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.
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http://dx.doi.org/10.1172/JCI35711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2564610PMC
November 2008

The Ewing sarcoma protein (EWS) binds directly to the proximal elements of the macrophage-specific promoter of the CSF-1 receptor (csf1r) gene.

J Immunol 2008 May;180(10):6733-42

Australian Research Council Special Research Centre for Functional and Applied Genomics and the Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland, Australia.

Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.
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http://dx.doi.org/10.4049/jimmunol.180.10.6733DOI Listing
May 2008

PU.1 and NFATc1 mediate osteoclastic induction of the mouse beta3 integrin promoter.

J Cell Physiol 2008 Jun;215(3):636-44

The New England Baptist Bone and Joint Institute, Department of Rheumatology, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02115, USA.

Expression of the alpha(v)beta(3) integrin is required for normal osteoclast function. We previously showed that an evolutionary conserved NFATc1 binding site is required for RANKL induction and NFATc1 transactivation of the human beta(3) promoter. The mechanism conferring specificity for RANKL induction and NFATc1 transduction of the beta(3) gene in osteoclast differentiation is unclear since NFATc1 is expressed and activated in numerous cell types that do not express the beta(3) gene. PU.1 is an ETS family transcription factor in myeloid cells associated with expression of various osteoclast genes. The present study investigates the role of NFATc1 in concert with PU.1 in osteoclast-specific transcription of the mouse beta(3) integrin gene. The mouse beta(3) promoter was transactivated by NFATc1 in RAW264.7 cells and deletion or mutation of either of the conserved NFAT and PU.1 binding sites abrogated transactivation. NFATc1 transactivation of the mouse beta(3) promoter was specifically dependent on co-transfected PU.1 in HEK293 cells, to the exclusion of other ETS family members. Direct binding of NFATc1 and PU.1 to their cognate sequences was demonstrated by EMSA and NFATc1 and PU.1 occupy their cognate sites in RANKL-treated mouse marrow precursors in chromatin immuno-precipitation (ChIP) assays. TAT-mediated transduction with dominant-negative NFATc1 dose-dependently blocked endogenous expression of the mouse beta(3) integrin and the formation of TRAP positive multinucleated cells in RANKL-treated mouse macrophages. These data provide evidence that NFATc1, in concert with PU.1, are involved in regulation of beta(3) integrin expression during osteoclast differentiation and suggest that PU.1 confers specificity to the NFATc1 response to macrophage lineage cells.
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http://dx.doi.org/10.1002/jcp.21344DOI Listing
June 2008

Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors.

Mol Cell Biol 2007 Jun 2;27(11):4018-27. Epub 2007 Apr 2.

Department of Molecular and Cellular Biochemistry and Comprehensive Cancer Center, 370A Tzagournis Medical Research Facility, Ohio State University, 420 West 12th Avenue, Columbus, OH 43210, USA.

Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.
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http://dx.doi.org/10.1128/MCB.01839-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1900027PMC
June 2007

MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation.

J Biol Chem 2007 May 2;282(21):15921-9. Epub 2007 Apr 2.

Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.

Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-kappaB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells.
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http://dx.doi.org/10.1074/jbc.M609723200DOI Listing
May 2007

The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor.

J Biol Chem 2007 Jan 14;282(3):1891-904. Epub 2006 Nov 14.

Institute for Molecular Biosciences, the University of Queensland, St. Lucia, Queensland 4072, Australia.

Microphthalmia transcription factor (MITF) regulates osteoclast function by controling the expression of genes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to receptor activator of nuclear factor-kappaB ligand (RANKL)-induced signaling. To identify novel MITF target genes, we have overexpressed MITF in the murine macrophage cell line RAW264.7 subclone 4 (RAW/C4) and examined the gene expression profile after sRANKL-stimulated osteoclastogenesis. Microarray analysis identified a set of genes superinduced by MITF overexpression, including Clcn7 (chloride channel 7) and Ostm1 (osteopetrosis-associated transmembrane protein 1). Using electrophoretic mobility shift assays, we identified two MITF-binding sites (M-boxes) in the Clcn7 promoter and a single M-box in the Ostm1 promoter. An anti-MITF antibody supershifted DNA-protein complexes for promoter sites in both genes, whereas MITF binding was abolished by mutation of these sites. The Clcn7 promoter was transactivated by coexpression of MITF in reporter gene assays. Mutation of one Clcn7 M-box prevented MITF transactivation, but mutation of the second MITF-binding site only reduced basal activity. Chromatin immunoprecipitation assays confirmed that the two Clcn7 MITF binding and responsive regions in vitro bind MITF in genomic DNA. The expression of Clcn7 is repressed in the dominant negative mutant Mitf mouse, mi/mi, indicating that the dysregulated bone resorption seen in these mice can be attributed in part to transcriptional repression of Clcn7. MITF regulation of the TRAP, cathepsin K, Clcn7, and Ostm1 genes, which are critical for osteoclast resorption, suggests that the role of MITF is more significant than previously perceived and that MITF may be a master regulator of osteoclast function and bone resorption.
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http://dx.doi.org/10.1074/jbc.M608572200DOI Listing
January 2007

Genetics and genomics of osteoclast differentiation: integrating cell signaling pathways and gene networks.

Crit Rev Eukaryot Gene Expr 2006 ;16(3):253-77

Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, OH 43210, USA.

The regulation of osteoclast differentiation in the bone microenvironment is critical for normal bone remodeling, as well as for various human bone diseases. Over the last decade, our knowledge of how osteoclast differentiation occurs has progressed rapidly. We highlight some of the major advances in understanding how cell signaling and transcription are integrated to direct the differentiation of this cell type. These studies used genetic, molecular, and biochemical approaches. Additionally, we summarize data obtained from studies of osteoclast differentiation that used the functional genomic approach of global gene profiling applied to osteoclast differentiation. This genomic data confirms results from studies using the classical experimental approaches and also may suggest new modes by which osteoclast differentiation and function can be modulated. Two conclusions that emerge are that osteoclast differentiation depends on a combination of fairly ubiquitously expressed transcription factors rather than unique osteoclast factors, and that the overlay of cell signaling pathways on this set of transcription factors provides a powerful mechanism to fine tune the differentiation program in response to the local bone microenvironment.
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http://dx.doi.org/10.1615/critreveukargeneexpr.v16.i3.40DOI Listing
December 2006

Microphthalmia-associated transcription factor interactions with 14-3-3 modulate differentiation of committed myeloid precursors.

Mol Biol Cell 2006 Sep 5;17(9):3897-906. Epub 2006 Jul 5.

Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, OH 43210, USA.

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a target for signaling pathways engaged by colony stimulating factor (CSF)-1 and receptor-activator of nuclear factor-kappaB ligand (RANKL). Work presented here demonstrates that MITF can shuttle from cytoplasm to nucleus dependent upon RANKL/CSF-1 action. 14-3-3 was identified as a binding partner of MITF in osteoclast precursors, and overexpression of 14-3-3 in a transgenic model resulted in increased cytosolic localization of MITF and decreased expression of MITF target genes. MITF/14-3-3 interaction was phosphorylation dependent, and Ser173 residue, within the minimal interaction region of amino acid residues 141-191, was required. The Cdc25C-associated kinase (C-TAK)1 interacted with an overlapping region of MITF. C-TAK1 increased MITF/14-3-3 complex formation and thus promoted cytoplasmic localization of MITF. C-TAK1 interaction was disrupted by RANKL/CSF-1 treatment. The results indicate that 14-3-3 regulates MITF activity by promoting the cytosolic localization of MITF in the absence of signals required for osteoclast differentiation. This work identifies a mechanism that regulates MITF activity in monocytic precursors that are capable of undergoing different terminal differentiation programs, and it provides a mechanism that allows committed precursors to rapidly respond to signals in the bone microenvironment to promote specifically osteoclast differentiation.
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http://dx.doi.org/10.1091/mbc.e06-05-0470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1593166PMC
September 2006

Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression.

J Biol Chem 2004 Feb 10;279(8):6783-93. Epub 2003 Nov 10.

Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA.

The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (-9, -102, -347 with respect to the +1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (+152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylation-mediated inhibition of ribosomal RNA gene expression.
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http://dx.doi.org/10.1074/jbc.M309393200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242730PMC
February 2004

Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1.

J Cell Biochem 2003 Apr;88(5):855-64

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.
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http://dx.doi.org/10.1002/jcb.10457DOI Listing
April 2003

Nitric oxide: an antioxidant and neuroprotector.

Ann N Y Acad Sci 2002 May;962:389-401

Laboratory of Experimental and Clinical Neuroscience, Indian Institute of Chemical Biology, Calcutta, India.

Indirect evidence, including neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-neurotoxicity by nitric oxide synthase (NOS) inhibitors and resistance of transgenic animals deficient in NOS, is controversial. We have reviewed evidence in favor of oxidative stress during the development of MPTP-neurotoxicity and the influence of antioxidants, including nitric oxide (NO) and NO donors, on MPTP-induced dopaminergic neurotoxicity. Systemic administration of MPTP causes dose-dependent generation of hydroxyl radicals (OH) in vivo in the striatum in mice; OH scavengers protect dopaminergic neurons from this insult. On the other hand the role of NO in MPTP-neurotoxicity is controversial. Hitherto, no direct evidence for the involvement of NO in MPTP neurotoxicity has been available. MPTP does not affect inducible-NOS mRNA level or its expression in SN or the striatum. Nitroglycerine, a NO donor, can attenuate MPTP-induced dopamine depletion in the striatum by virtue of its OH scavenging action. Several other NO donors have also been shown to scavenge the OH generated, following Fenton chemistry in vitro, and to protect against in vivo dopaminergic neurotoxicity by small mass iron complex formation. This evidence suggests that NO renders protection against MPTP-induced OH-mediated nigrostriatal lesions, acting as an antioxidant.
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http://dx.doi.org/10.1111/j.1749-6632.2002.tb04083.xDOI Listing
May 2002