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1.  [No title available] 
PMCID: PMC3865079  PMID: 24038283
2.  [No title available] 
PMCID: PMC3865101  PMID: 23966233
3.  [No title available] 
PMCID: PMC4088938  PMID: 24038340
4.  Bone Morphogenetic Protein-4 Enhances Vascular Endothelial Growth Factor Secretion by Human Retinal Pigment Epithelial Cells 
Journal of cellular biochemistry  2006;98(5):1196-1202.
Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.
doi:10.1002/jcb.20831
PMCID: PMC4306351  PMID: 16514669
retinal pigment epithelium; ARPE-19; bone morphogenetic protein; vascular endothelial growth factor; angiogenesis; diabetic retinopathy
5.  Microtubule-Associated Protein Tau In Human Prostate Cancer Cells: Isoforms, Phosphorylation, And Interactions 
Journal of cellular biochemistry  2009;108(3):555-564.
SUMMARY
Tau is a microtubule-associated protein whose function has been investigated primarily in neurons. Recently, tau expression has been correlated with increased drug resistance in various cancers of non-neuronal tissues. In this report, we investigate the tau expressed in cancerous prostate lines ALVA-31, DU 145, and PC-3. Prostate cancer tau is heat-stable and highly phosphorylated, containing many of the modifications identified in Alzheimer’s Disease brain tau. RT-PCR and phosphatase treatment indicated that all six alternatively spliced adult brain tau isoforms are expressed in ALVA-31 cells and isoforms containing exon 6 as well as high molecular weight tau isoforms containing either exon 4A or a larger splice variant of exon 4A are also present. Consistent with its hyperphosphorylated state, a large proportion of ALVA-31 tau does not bind to microtubules, as detected by confocal microscopy and biochemical tests. Finally, endogenous ALVA-31 tau can interact with the p85 subunit of phosphatidylinositol 3-kinase, as demonstrated by co-immunoprecipitations and in vitro protein binding assays. Our results suggest that tau in prostate cancer cells does not resemble that from normal adult brain and support the hypothesis that tau is a multifunctional protein.
doi:10.1002/jcb.22287
PMCID: PMC4299866  PMID: 19681044
tau; prostate cancer cells; microtubule binding; phosphorylation; phosphatidylinositol 3-kinase
6.  [No title available] 
PMCID: PMC4293017  PMID: 19259948
7.  HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses 
Journal of cellular biochemistry  2008;105(5):1183-1193.
HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29’s ability to modulate actions of HS-binding growth factors (HBGFs) in angiogenesis. Between 1–2.5 μg/ml (ca. 60–150 nM), HIP/RPL29 inhibited HBGF-stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 μg/ml). At this concentration, HIP/RPL29 had no effect on HBGF- stimulated MAPK phosphorylation or VEGF-stimulated receptor-2 phosphorylation at site Y-996. Partial inhibition occured at VEGF receptor-2 site Y951, associated with cell migration. HBGF displacement from HS-bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 μg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE-dependent release of HS-bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 μg/ml and by 77% at 40 μg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 μg/ml (ca 2.5 mM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors.
doi:10.1002/jcb.21899
PMCID: PMC4287213  PMID: 18980226
HIP/RPL29; heparan sulfate; VEGF; FGF2; heparanase
8.  Nuclear Structure as a Source of Cancer Specific Biomarkers 
Journal of cellular biochemistry  2008;104(6):1988-1993.
There are few biomarkers that have been developed which have proven clinical utility for the detection and prognosis of cancer. Cancer is diagnosed today, in large part, by examining cells under the microscope and determining the shape and texture of the nucleus. The molecular underpinnings of this hallmark of cancer are the components of the nuclear matrix. Utilizing proteomics focused on this subset of proteins, biomarkers have been identified that are specific for cancer types including prostate, colon and bladder cancer. These cancer biomarkers now serve as the basis of assays which can specifically identify individuals with cancer by sampling their blood and/or urine. In addition, these may serve as potential therapeutic targeting or imaging approaches.
doi:10.1002/jcb.21363
PMCID: PMC4281772  PMID: 17455233
nuclear matrix; nuclear structure; tumor markers; biomarkers
9.  Malignant Mesothelioma: Development to Therapy 
Journal of cellular biochemistry  2014;115(1):10.1002/jcb.24642.
Malignant mesothelioma (MM) is an aggressive cancer of the mesothelium caused by asbestos. Asbestos use has been reduced but not completely stopped. In addition, natural or man-made disasters will continue to dislodge asbestos from old buildings into the atmosphere and as long as respirable asbestos is available, MM will continue to be a threat. Due to the long latency period of MM development, it would still take decades to eradicate this disease if asbestos was completely removed from our lives today. Therefore, there is a need for researchers and clinicians to work together to understand this deadly disease and find a solution for early diagnosis and treatment. This article focuses on developmental mechanisms as well as current therapies available for MM.
doi:10.1002/jcb.24642
PMCID: PMC3856563  PMID: 23959774
malignant mesothelioma; asbestos; mesothelium; inflammation
10.  Eliminative signaling by Janus kinases: role in the down-regulation of associated receptors 
Journal of cellular biochemistry  2014;115(1):10.1002/jcb.24647.
Activation of cytokine receptor-associated Janus kinases (JAKs) mediates most, if not all, of the cellular responses to peptide hormones and cytokines. Consequently, JAKs play a paramount role in homeostasis and immunity. Members of this family of tyrosine kinases control the cytokine/hormone-induced alterations in cell gene expression program. This function is largely mediated through an ability to signal towards activation of the signal transducer and activator of transcription proteins (STAT) as well as towards some other pathways. Importantly, JAKs are also instrumental in tightly controlling the expression of associated cytokine and hormone receptors, and, accordingly, in regulating the cell sensitivity to these cytokines and hormones. This review highlights the enzymatic and non-enzymatic mechanisms of this regulation and discusses the importance of the ambidextrous nature of JAK as a key signaling node that integrates the combining the functions of forward signaling and eliminative signaling. Attention to the latter aspect of JAK function may contribute to emancipating our approaches to the pharmacologic modulation of JAKs.
doi:10.1002/jcb.24647
PMCID: PMC3856693  PMID: 23959845
JAK; kinase; cytokine; receptor; downregulation
11.  Roles of Parathyroid Hormone (PTH) Receptor and Reactive Oxygen Species in Hyperlipidemia-Induced PTH(1-34) Resistance in Preosteoblasts. 
Journal of cellular biochemistry  2014;115(1):179-188.
Bioactive lipids initiate inflammatory reactions leading to pathogenesis of atherosclerosis. Evidence shows that they also contribute to bone loss by inhibiting parathyroid hormone receptor (PTH1R) expression and differentiation of osteoblasts. We previously demonstrated that bone anabolic effects of PTH(1-34) are blunted in hyperlipidemic mice and that these PTH effects are restored by antioxidants. However, it is not clear which osteoblastic cell developmental stage is targeted by bioactive lipids. To investigate the effects of hyperlipidemia at the cellular level, hyperlipidemic Ldlr−/− mice were bred with Col3.6GFPtpz mice, in which preosteoblasts/osteoblasts carry a topaz fluorescent label, and with Col2.3GFPcyan mice, in which more mature osteoblasts/osteocytes carry a cyan fluorescent label. Histological analyses of trabecular bone surfaces in femoral as well as calvarial bones showed that intermittent PTH(1-34) increased fluorescence intensity in WT-Tpz mice, but not in Tpz-Ldlr−/− mice. In contrast, PTH(1-34) did not alter fluorescence intensity in femoral cortical envelopes of either WT-Cyan or Ldlr−/−-Cyan mice. To test the mechanism of PTH1R downregulation, preosteoblastic MC3T3-E1 cells were treated with bioactive lipids and the antioxidant Trolox. Results showed that inhibitory effects of PTH1R levels by bioactive lipids were rescued by pretreatment with Trolox. The inhibitory effects on expression of PTH1R as well as on PTH-induced osteoblastic genes were mimicked by xanthine/xanthine oxidase, a known generator of reactive oxygen species. These findings suggest an important role of preosteoblasts as the target development stage and downregulation of PTH receptor expression mediated by intracellular oxidant stress as a mechanism in hyperlipidemia-induced PTH resistance.
doi:10.1002/jcb.24648
PMCID: PMC3889484  PMID: 24038594
Hyperlipidemia; bioactive lipids; preosteoblasts; PTH receptor; reactive oxygen species
12.  Lithium chloride induces TNFα in mouse macrophages via MEK-ERK-dependent pathway 
Journal of cellular biochemistry  2014;115(1):71-80.
Lithium (Li) is one of the currently prescribed drugs for bipolar disorders and has many neuro-regulatory and immune-modulating properties. Because many neuro-pathological diseases including bipolar disorders have been associated with some level of inflammation, Li's effect on inflammation may have some crucial consequences. Even though Li has been shown to have proand anti-inflammatory activities in different cell models, mechanisms involved in these effects are not well understood. Moreover, Li's effect on inflammation in the presence of activators of Toll-like receptors (TLRs), especially TLR-2 (that activates MyD88-dependent pathway) and TLR-3 (that activates TRIF-dependent pathway) is not known. Here we tested the role of Li in the presence and absence of TLR2, and TLR3 on MAPK and NFκB pathways and the consequent production of tumor necrosis factor-α (TNFα) in Raw264.7 macrophages. Our results indicate that Li enhances TNFα production both in the absence and presence of TLR stimulation. Interestingly, Li differentially modulates MAPK and NFκB pathways in the absence and presence of TLR2/3 ligands. Our results further indicate that the effect of Li on TNFα occurs at the post-transcriptional level. Together, these studies demonstrate that Li induces TNFα production in macrophages and that it modulates signaling at different levels depending on the presence or absence of TLR2/3 stimulation.
doi:10.1002/jcb.24634
PMCID: PMC4030382  PMID: 23904208
13.  Glucose availability and glycolytic metabolism dictate glycosphingolipid levels† 
Journal of cellular biochemistry  2015;116(1):67-80.
Cancer therapeutics has seen an emergence and re-emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramide's essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre-clinical inhibitor ABT-263, this effect was augmented and production of pro-apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia.
doi:10.1002/jcb.24943
PMCID: PMC4229434  PMID: 25145677
sphingolipid; glucose; ceramide; glycosphingolipid; GSL; glucosylceramide; GlcCer; GluCer; glucosylceramide synthase; GCS; glycolysis; pentose phosphate pathway; PPP; metabolism; apoptosis; 2-deoxyglucose; 2-DG; 6-aminonicatinomide; 6-AN; ABT-263; ABT-737
14.  Gender Differences in Adiponectin Modulation of Cardiac Remodeling in Mice Deficient in Endothelial Nitric Oxide Synthase 
Journal of cellular biochemistry  2012;113(10):3276-3287.
Left ventricular hypertrophy (LVH) is a risk factor for cardiovascular disease, a leading cause of death. Alterations in endothelial nitric oxide synthase (eNOS), an enzyme involved in regulating vascular tone, and in adiponectin, an adipocyte-derived secretory factor, are associated with cardiac remodeling. Deficiency of eNOS is associated with hypertension and LVH. Adiponectin exhibits vaso-protective, anti-inflammatory, and anti-atherogenic properties. We hypothesized that increased levels of adiponectin would alleviate cardiac pathology resulting from eNOS deficiency, while decreased levels of adiponectin would exacerbate the pathology. Male and female mice, deficient in eNOS, and either lacking or over-expressing adiponectin, were fed high fat diet (HFD) or normal chow. Cardiac magnetic resonance imaging was performed to serially assess heart morphology and function up to 40 weeks of age. Thirty-two weeks of HFD feeding led to significantly greater LV mass in male mice deficient in eNOS and either lacking or over-expressing adiponectin. Heart function was significantly reduced when the mice were deficient in either eNOS, adiponectin or both eNOS and adiponectin; for female mice, heart function was only reduced when both eNOS and adiponectin were lacking. Thus, while over-expression of adiponectin in the eNOS deficient HFD fed male mice preserved function at the expense of significantly increased LV mass, female mice were protected from decreased function and increased LVH by over-expression of adiponectin. Our results demonstrate a sexual dimorphism in response of the heart to alterations in eNOS and adiponectin during high fat feeding and suggest that adiponectin might require eNOS for some of its metabolic effects.
doi:10.1002/jcb.24206
PMCID: PMC4268866  PMID: 22644792
ADIPONECTIN; ENOS; LEFT VENTRICULAR HYPERTROPHY; MRI; CARDIAC REMODELING; HYPERTENSION
15.  Acetylcholine Induces Mesenchymal Stem Cell Migration via Ca2+/PKC/ERK1/2 Signal Pathway 
Journal of cellular biochemistry  2012;113(8):2704-2713.
Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks Ach-mediated MSC migration. Interestingly, intracellular Ca2+ ATPase specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca2+ storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca2+, PKC and ERK1/2 signal pathways.
doi:10.1002/jcb.24148
PMCID: PMC4263956  PMID: 22441978
Mesenchymal stem cell; Acetylcholine; Migration; Protein kinase C; Ras mitogen-activated protein kinase; Calcium; Muscarinic receptors; ERK1/2
16.  Altered Signaling in the G1 Phase Deregulates Chondrocyte Growth in a Mouse Model With Proteoglycan Undersulfation 
Journal of Cellular Biochemistry  2014;115(10):1779-1786.
In several skeletal dysplasias defects in extracellular matrix molecules affect not only the structural and mechanical properties of cartilage, but also the complex network of signaling pathways involved in cell proliferation and differentiation. Sulfated proteoglycans, besides playing an important structural role in cartilage, are crucial in modulating the transport, diffusion, and interactions of growth factors with their specific targets, taking part in the regulation of signaling pathways involved in skeletal development and growth. In this work, we investigated by real time PCR and Western blots of the microdissected growth plate and by immunohistochemistry the molecular basis of reduced chondrocyte proliferation in the growth plate of the dtd mouse, a chondrodysplastic model with defective chondroitin sulfate proteoglycan sulfation of articular and growth plate cartilage. We detected activation of the Wnt pathway, leading to an increase in the non-phosphorylated form of nuclear β-catenin and subsequent up-regulation of cyclin D1 expression in the G1 phase of the cell cycle. β-Catenin was further stabilized by up-regulation of Smad3 expression through TGF-β pathway synergistic activation. We demonstrate that notwithstanding cyclin D1 expression increase, cell cycle progression is compromised in the G1 phase due to reduced phosphorylation of the pocket protein p130 leading to inhibition of transcription factors of the E2F family which are crucial for cell cycle progression and DNA replication. These data, together with altered Indian hedgehox signaling detected previously, explain at the molecular level the reduced chondrocyte proliferation rate of the dtd growth plate leading to reduced skeletal growth. J. Cell. Biochem. 115: 1779–1786, 2014.
doi:10.1002/jcb.24844
PMCID: PMC4262066  PMID: 24820054
GROWTH PLATE; CELL CYCLE; SKELETAL DYSPLASIA; PROTEOGLYCAN SULFATION; WNT PATHWAY
17.  Core Binding Factor β (CBFβ) and the Leukemogenic Fusion Protein CBFβ-Smooth Muscle Myosin Heavy Chain (SMMHC) Associate with Mitotic Chromosomes to Epigenetically Regulate Ribosomal Gene Expression* 
Journal of cellular biochemistry  2014;115(12):2155-2164.
Mitotic bookmarking is an epigenetic control mechanism that sustains gene expression in progeny cells; it is often found in genes related to the maintenance of cellular phenotype and growth control. RUNX transcription factors regulate a broad spectrum of RNA Polymerase (Pol II) transcribed genes important for lineage commitment but also regulate RNA Polymerase I (Pol I) driven ribosomal gene expression, thus coordinating control of cellular identity and proliferation. In this study, using fluorescence microscopy and biochemical approaches we show that the principal RUNX co-factor, CBFβ, associates with nucleolar organizing regions (NORs) during mitosis to negatively regulate RUNX-dependent ribosomal gene expression. Of clinical relevance, we establish for the first time that the leukemogenic fusion protein CBFβ-SMMHC (smooth muscle myosin heavy chain) also associates with ribosomal genes in interphase chromatin and mitotic chromosomes to promote and epigenetically sustain regulation of ribosomal genes through RUNX factor interactions. Our results demonstrate that CBFβ contributes to the transcriptional regulation of ribosomal gene expression and provide further understanding of the epigenetic role of CBFβ-SMMHC in proliferation and maintenance of the leukemic phenotype.
Background
Runt-related transcription factors (RUNX) bookmark genes important for phenotype, but the mitotic behavior of RUNX cofactor, Core Binding Factor β (CBFβ) is unknown.
Results
CBFβ and leukemogenic fusion protein CBFβ-SMMHC associate with chromosomes during mitosis and regulate ribosomal genes.
Conclusion
CBFβ and CBFβ-SMMHC contribute to epigenetic control of ribosomal genes.
Significance
CBFβ-SMMHC alters regulation linking phenotypic control with cell growth, thereby promoting cancer.
doi:10.1002/jcb.24892
PMCID: PMC4199869  PMID: 25079347
Leukemia; Epigenetics; Ribosomal RNA (rRNA); Molecular cell biology; Transcription coactivators; Core binding factor β; Mitotic bookmarking; Ribosomal gene expression
18.  Prospects on Strategies for Therapeutically Targeting Oncogenic Regulatory Factors by Small-Molecule Agents 
Journal of cellular biochemistry  2014;115(4):611-624.
Although the Human Genome Project has raised much hope for the identification of druggable genetic targets for cancer and other diseases, this genetic target-based approach has not improved productivity in drug discovery over the traditional approach. Analyses of known human target proteins of currently marketed drugs reveal that these drugs target only a limited number of proteins as compared to the whole proteome. In contrast to genome-based targets, mechanistic targets are derived from empirical research, at cellular or molecular levels, in disease models and/or in patients, thereby enabling the exploration of a greater number of druggable targets beyond the genome and epigenome. The paradigm shift has made a tremendous headway in developing new therapeutic agents targeting different clinically relevant mechanisms/pathways in cancer cells. In this Prospects article, we provide an overview of potential drug targets related to the following four emerging areas: (1) tumor metabolism (the Warburg effect), (2) dysregulated protein turnover (E3 ubiquitin ligases), (3) protein–protein interactions, and (4) unique DNA high-order structures and protein–DNA interactions. Nonetheless, considering the genetic and phenotypic heterogeneities that characterize cancer cells, the development of drug resistance in cancer cells by adapting signaling circuitry to take advantage of redundant pathways or feedback/crosstalk systems is possible. This “phenotypic adaptation” underlies the rationale of using therapeutic combinations of these targeted agents with cytotoxic drugs.
doi:10.1002/jcb.24704
PMCID: PMC4240004  PMID: 24166934
WARBURG EFFECT; E3 LIGASES; PROTEIN–PROTEIN INTERACTIONS; PROTEIN–DNA INTERACTIONS; DNA QUADRUPLEX
19.  Differences in Nuclear DNA Organization Between Lymphocytes, Hodgkin and Reed–Sternberg Cells Revealed by Structured Illumination Microscopy 
Journal of Cellular Biochemistry  2014;115(8):1441-1448.
Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that of DNA-free space in control lymphocytes, Hodgkin cells and Reed–Sternberg cells using 3D structured illumination microscopy (SIM). We have observed detail in these SIM images that was not observed in conventional widefield images. We have measured the size distribution of the DNA structure using granulometry and noted a significant, progressive increase in the amount of sub-micron structures from control lymphocytes to Hodgkin cells to Reed–Sternberg cells. The DNA-free space changes as well; “holes” in the DNA distribution start to appear in the malignant cells. We have studied whether these “holes” are nucleoli by staining for upstream binding factor (UBF), a protein associated with the nucleolus. We have found that the relative UBF content progressively and significantly decreases—or is absent—in the DNA-free space when measured as either the Pearson correlation coefficient with the DNA-free space or as the number of “holes” that contain UBF. Similar differences exist within the population of Reed–Sternberg cells between binucleated and multinucleated cells with four or more subnuclei. To our knowledge, this is the first study that investigates the changes of the nuclear DNA structure in any disease with superresolution light microscopy. J. Cell. Biochem. 115: 1441–1448, 2014. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
doi:10.1002/jcb.24800
PMCID: PMC4231252  PMID: 24590512
STRUCTURED ILLUMINATION MICROSCOPY; NUCLEAR ARCHITECTURE; QUANTITATIVE MICROSCOPY; HODGKIN’S LYMPHOMA
20.  An Intact Connexin43 is Required to Enhance Signaling and Gene Expression in Osteoblast-like Cells 
Journal of cellular biochemistry  2013;114(11):2542-2550.
The cytoplasmic C-terminus of connexin43 (Cx43) interacts with numerous signaling complexes. We hypothesize that signal complex docking to the Cx43 C-terminus (CT) is required to propagate the molecules being shared by gap junctions. We have previously shown that Cx43 impacts the responsiveness of osteoblasts to FGF2 in a PKCδ- and ERK-dependent manner, converging on Runx2 activity. Here, we mapped the interaction domain of Cx43 and PKCδ to amino acids 243–302 of the Cx43 CT by GST pulldown assay. Using Runx2-responsive luciferase reporter assays, a Cx43 deletion construct (Cx43 S244Stop), which lacks the C-terminus (amino acids 244 to 382), failed to support the Cx43-dependent potentiation of transcription following FGF2 treatment in MC3T3 osteoblast-like cells. Similarly, overexpression of Cx43 S244Stop could not mimic the ability of the full length Cx43 to stimulate expression of osteoblast genes. In contrast to full length Cx43, overexpression of just the Cx43 CT (amino acids 236 to 382) inhibited both transcription from a Runx2 reporter and signaling via PKCδ and ERK. Inhibition of signaling by the CT did not occur in HeLa cells, which lack endogenous Cx43. In summary, the data support a model in which an intact Cx43 is required for both signal propagation/permeability (i.e., channel function) and local recruitment of signaling complexes to the CT (i.e., docking function) in order to mediate its cellular effects. Further, while the CT alone has channel independent activity, it is opposing to the effect of overexpression of the full length Cx43 channel.
doi:10.1002/jcb.24603
PMCID: PMC3963279  PMID: 23744706
Gap Junction; Bone; Fibroblast Growth Factor 2; Protein Kinase C delta; Signal Transduction; Cell-to-Cell Communication
21.  Acquisition of Paclitaxel Resistance Is Associated With a More Aggressive and Invasive Phenotype in Prostate Cancer 
Journal of cellular biochemistry  2013;114(6):1286-1293.
Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration-resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen-insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel-resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel-resistant (DU145-TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145-TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial-to-mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans-differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients.
doi:10.1002/jcb.24464
PMCID: PMC4211414  PMID: 23192682
PROSTATE CANCER; PACLITAXEL; EPITHELIAL MESENCHYMAL TRANSITION; INVASION; CELL TRACTION FORCE; CYTOSKELETAL REMODELING
22.  Methylation of the PTPRO Gene in Human Hepatocellular Carcinoma and Identification of VCP as Its Substrate 
Journal of cellular biochemistry  2013;114(8):1810-1818.
We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy.
doi:10.1002/jcb.24525
PMCID: PMC4199230  PMID: 23533167
TYROSINE PHOSPHATASE; PTPRO; P97/VCP; HCC; METHYLATION
23.  An Autonomous BMP2 Regulatory Element in Mesenchymal Cells 
Journal of cellular biochemistry  2011;112(2):666-674.
BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements.
doi:10.1002/jcb.22975
PMCID: PMC4198144  PMID: 21268088
GENE REGULATION; POST-TRANSCRIPTIONAL; GROWTH FACTOR; PATHOLOGICAL CALCIFICATION; CORONARY VASCULATURE; AORTA
24.  Fyn Promotes Phosphorylation of Collapsin Response Mediator Protein 1 at Tyrosine 504, a Novel, Isoform-Specific Regulatory Site 
Journal of cellular biochemistry  2010;111(1):20-28.
In vertebrates the Collapsin Response Mediator Proteins (CRMPs) are encoded by five highly-related genes. CRMPs are cytosolic phosphoproteins abundantly expressed in developing and mature mammalian brains. CRMPs are best understood as effectors of Semaphorin 3A signaling regulating growth cone collapse in migratory neurons. Phosphorylation in the carboxyl-terminal regulatory domain of CRMPs by several serine/threonine kinases has been described. These phoshorylation events appear to function, at least in part, to disrupt the interaction of CRMPs with tubulin heterodimers. In a large-scale phosphoproteomic analysis of murine brain, we recently identified a number of in vivo tyrosine phosphorylation sites on CRMP isoforms. Using biochemical approaches and quantitative mass spectrometry we demonstrate that one of these sites, CRMP1 tyrosine 504 (Y504), is a primary target of the Src family of tyrosine kinases (SFKs), specifically Fyn. Y504 is adjacent to CDK5 and GSK-3β sites that regulate the interaction of CRMPs with tubulin. Although Y504 is highly conserved among vertebrate CRMP1 orthologs, a residue corresponding to Y504 is absent in CRMP isoforms 2-5. This suggests an isoform-specific regulatory role for CRMP1 Y504 phosphorylation and may help explain the observation that CRMP1-deficient mice exhibit neuronal migration defects not compensated for by CRMPs 2-5.
doi:10.1002/jcb.22659
PMCID: PMC4198339  PMID: 20506281
Collapsin Response Mediator Protein, CRMP; Fyn; Phosphorylation; Tyrosine Kinase; Quantitative mass spectrometry, quantitative proteomics; Neuronal positioning; Growth Cone; Absolute Quantification, AQUA; Stable Isotope Labeling with Amino Acids in Cell Culture, SILAC
25.  Heat and Radiofrequency Plasma Glow Discharge Pretreatment of a Titanium Alloy Promote Bone Formation and Osseointegration 
Journal of cellular biochemistry  2013;114(10):10.1002/jcb.24585.
Orthopedic and dental implants manifest increased failure rates when inserted into low density bone. We determined whether chemical pretreatments of a titanium alloy implant material stimulated new bone formation to increase osseointegration in vivo in trabecular bone using a rat model. Titanium alloy rods were untreated or pretreated with heat (600°C) or radiofrequency plasma glow discharge (RFGD). The rods were then coated with the extracellular matrix protein fibronectin (1 nM) or left uncoated and surgically implanted into the rat femoral medullary cavity. Animals were euthanized 3 or 6 weeks later, and femurs were removed for analysis. The number of trabeculae in contact with the implant surface, surface contact between trabeculae and the implant, and the length and area of bone attached to the implant were measured by histomorphometry. Implant shear strength was measured by a pull-out test. Both pretreatments and fibronectin enhanced the number of trabeculae bonding with the implant and trabeculae-to-implant surface contact, with greater effects of fibronectin observed with pretreated compared to untreated implants. RFGD pretreatment modestly increased implant shear strength, which was highly correlated (r2 = 0.87 – 0.99) with measures of trabecular bonding for untreated and RFGD-pretreated implants. In contrast, heat pretreatment increased shear strength 3 to 5-fold for both uncoated and fibronectin-coated implants at 3 and 6 weeks, suggesting a more rapid increase in implant-femur bonding compared to the other groups. In summary, our findings suggest that the heat and RFGD pretreatments can promote the osseointegration of a titanium alloy implant material.
doi:10.1002/jcb.24585
PMCID: PMC3786157  PMID: 23649564
Dental implant; fibronectin; osteoblast; cell differentiation; bone mineralization; osseointegration

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