66 research outputs found
Analysis of transcription factor interactions in osteoblasts using competitive chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) is a widely used technique for quantifying proteināDNA interactions in living cells. This method commonly uses fixed (crosslinked) chromatin that is fragmented by sonication (X-ChIP). We developed a simple new ChIP procedure for the immunoprecipitation of sonicated chromatin isolated from osteoblasts in the absence of crosslinking (N-ChIP). The use of noncrosslinked chromatin allowed development of a new modification of the ChIP assay: the combination of N-ChIP and competition with double-stranded oligonucleotides containing specific binding sites for individual transcription factors (Competitive N-ChIP). Using this approach, we were able to discriminate between individual binding sites for the Runx2 transcription factor in the osteocalcin and bone sialoprotein genes that cannot be resolved by traditional X-ChIP. N-ChIP assays were also able to detect several other types of chromatin interactions including those with Dlx homeodomain factors and nuclear proteins such as Sin3a that lack an intrinsic DNA-binding motif and, therefore, bind to chromatin via interactions with other proteins
Transcriptional Regulation of Osteoblasts
The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. Among these are RUNX2, Osterix (OSX), ATF4, and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations well before the branching of chondrogenic and osteogenic lineages. Given that RUNX2 is expressed so early and participates in several stages of bone formation, it is not surprising that it is subject to a variety of controls. These include regulation by nuclear accessory factors and posttranslational modification, especially phosphorylation. Specific examples of RUNX2 regulation include interactions with DLX proteins and ATF4 and phosphorylation by the ERK/MAP kinase pathway. RUNX2 is regulated via phosphorylation of critical serine residues in the P/S/T domain. MAPK activation of RUNX2 was also found to occur in vivo . Transgenic expression of constitutively active MEK1 in osteoblasts accelerated skeletal development while a dominant-negative MEK1 retarded development in a RUNX2-dependent manner. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation in response to extracellular stimuli including ECM interactions, mechanical loads, and hormonal stimulation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75356/1/annals.1402.081.pd
Chemical transfection of dyeāconjugated microRNA precursors for microRNA functional analysis of M2 macrophages
MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumorāassociated macrophages (TAMs). To aid the functional analyses of miRNAs in an ināvitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNAāconstruct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescenceāactivated cell sorting. The chemical transfection of these dyeāconjugated miRNA precursors yield an efficiency of 37.5āĀ±ā0.6% and a cell viability of 74āĀ±ā1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dyeāconjugated miRNA precursors was determined to be the bestāsuited approach for the functional analysis of M2 macrophages. J. Cell. Biochem. 113: 1714ā1723, 2012. Ā© 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90531/1/24041_ftp.pd
Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages
Apoptosis occurs at an extraordinary rate in the human body and the effective clearance of dead cells (efferocytosis) is necessary to maintain homeostasis and promote healing, yet the contribution and impact of this process in bone is unclear. Bone formation requires that bone marrow stromal cells (BMSCs) differentiate into osteoblasts which direct matrix formation and either become osteocytes, bone lining cells, or undergo apoptosis. A series of experiments were performed to identify the regulators and consequences of macrophage efferocytosis of apoptotic BMSCs (apBMSCs). Bone marrow derived macrophages treated with the antiāinflammatory cytokine interleukinā10 (ILā10) exhibited increased efferocytosis of apBMSCs compared to vehicle treated macrophages. Additionally, ILā10 increased antiāinflammatory M2ālike macrophages (CD206+), and further enhanced efferocytosis within the CD206+ population. Stattic, an inhibitor of STAT3 phosphorylation, reduced the ILā10āmediated shift in M2 macrophage polarization and diminished ILā10ādirected efferocytosis of apBMSCs by macrophages implicating the STAT3 signaling pathway. Cell culture supernatants and RNA from macrophages coācultured with apoptotic bone cells showed increased secretion of monocyte chemotactic protein 1/chemokine (CāC motif) ligand 2 (MCPā1/CCL2) and transforming growth factor beta 1 (TGFāĪ²1) and increased ccl2 gene expression. In conclusion, ILā10 increases M2 macrophage polarization and enhances macrophageāmediated engulfment of apBMSCs in a STAT3 phosphorylationādependent manner. After engulfment of apoptotic bone cells, macrophages secrete TGFāĪ²1 and MCPā1/CCL2, factors which fuel the remodeling process. A better understanding of the role of macrophage efferocytosis as it relates to normal and abnormal bone turnover will provide vital information for future therapeutic approaches to treat bone related diseases. J. Cell. Biochem. 117: 2697ā2706, 2016. Ā© 2016 Wiley Periodicals, Inc.The process of efferocytosis (clearance of apoptotic cells) has been characterized in various tissues but the role of efferocytosis in the bone microenvironment is unclear. Bone marrow macrophage efferocytosis of apoptotic osteoblastic cells was enhanced by interleukinā10 in a STATā3 dependent manner and resulted in increased production of TGFāĪ²1 and CCLā2. The process of efferocytosis is likely important in bone remodeling and osseous wound healing.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/1/jcb25567.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/2/jcb25567_am.pd
Immune mediators in the tumor microenvironment of prostate cancer
Prostate cancer tissue is composed of both cancer cells and host cells. The milieu of host components that compose the tumor is termed the tumor microenvironment (TME). Host cells can be those derived from the tissue in which the tumor originates (e.g., fibroblasts and endothelial cells) or those recruited, through chemotactic or other factors, to the tumor (e.g., circulating immune cells). Some immune cells are key players in the TME and represent a large proportion of nonātumor cells found within the tumor. Immune cells can have both antiātumor and proātumor activity. In addition, crosstalk between prostate cancer cells and immune cells affects immune cell functions. In this review, we focus on immune cells and cytokines that contribute to tumor progression. We discuss Tāregulatory and T helper 17 cells and macrophages as key modulators in prostate cancer progression. In addition, we discuss the roles of interleukinā6 and receptor activator of nuclear factor kappaāB ligand in modulating prostate cancer progression. This review highlights the concept that immune cells and cytokines offer a potentially promising target for prostate cancer therapy.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152631/1/cac2s4088001701983.pd
Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.
We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy
A bioinformatics approach reveals novel interactions of the OVOL transcription factors in the regulation of epithelial ā mesenchymal cell reprogramming and cancer progression
Abstract
Background
Mesenchymal to Epithelial Transition (MET) plasticity is critical to cancer progression, and we recently showed that the OVOL transcription factors (TFs) are critical regulators of MET. Results of that work also posed the hypothesis that the OVOLs impact MET in a range of cancers. We now test this hypothesis by developing a model, OVOL Induced MET (OI-MET), and sub-model (OI-MET-TF), to characterize differential gene expression in MET common to prostate cancer (PC) and breast cancer (BC).
Results
In the OI-MET model, we identified 739 genes differentially expressed in both the PC and BC models. For this gene set, we found significant enrichment of annotation for BC, PC, cancer, and MET, as well as regulation of gene expression by AP1, STAT1, STAT3, and NFKB1. Focusing on the target genes for these four TFs plus the OVOLs, we produced the OI-MET-TF sub-model, which shows even greater enrichment for these annotations, plus significant evidence of cooperation among these five TFs. Based on known gene/drug interactions, we prioritized targets in the OI-MET-TF network for follow-on analysis, emphasizing the clinical relevance of this work. Reflecting these results back to the OI-MET model, we found that binding motifs for the TF pair AP1/MYC are more frequent than expected and that the AP1/MYC pair is significantly enriched in binding in cancer models, relative to non-cancer models, in these promoters. This effect is seen in both MET models (solid tumors) and in non-MET models (leukemia). These results are consistent with our hypothesis that the OVOLs impact cancer susceptibility by regulating MET, and extend the hypothesis to include mechanisms not specific to MET.
Conclusions
We find significant evidence of the OVOL, AP1, STAT1, STAT3, and NFKB1 TFs having important roles in MET, and more broadly in cancer. We prioritize known gene/drug targets for follow-up in the clinic, and we show that the AP1/MYC TF pair is a strong candidate for intervention.http://deepblue.lib.umich.edu/bitstream/2027.42/109509/1/12918_2013_Article_1293.pd
ILā4 induces proliferation in prostate cancer PC3 cells under nutrientādepletion stress through the activation of the JNKāpathway and survivin upāregulation
Interleukin (IL)ā4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of ILā4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrientādepleted environment, ILā4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrientādepletion stress, ILā4 activates mitogenāactivated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAPāsignalingāspecific inhibitors, it was shown that ILā4āinduced proliferation is mediated by JNK activation. In fact, JNKāinhibitorāV (JNKiāV) stunted ILā4āmediated cell proliferation. Furthermore, it was found that ILā4 induces survivin upāregulation in nutrientādepleted cancer cells. Using survivināshortāhairpināRNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of ILā4āmediated proliferation. In addition, the significance of survivin upāregulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL ā4 could induce proliferation in cancer cells from multiple origins: MDAāMBā231 (breast), A253 (head and neck), and SKOVā3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, ILā4 triggers a simultaneous activation of the JNKāpathway and the upāregulation of survivin turning on a cancer proliferation mechanism. J. Cell. Biochem. 113: 1569ā1580, 2012. Ā© 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90542/1/24025_ftp.pd
Inflammatory bone loss associated with MFGāE8 deficiency is rescued by teriparatide
Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/1/fsb2fj201701238r-sup-0002.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/2/fsb2fj201701238r.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/3/fsb2fj201701238r-sup-0001.pd
- ā¦