In the pathogenesis of diabetic retinopathy, H-Ras (a small molecular weight G-protein) and matrix metalloproteinase-9 (MMP9) act as pro-apoptotic, accelerating the apoptosis of retinal capillary cells, a phenomenon that predicts its development and the activation of MMP9 is under the control of H-Ras. The goal of this study is to elucidate the cellular mechanism by which H-Ras activates MMP9 culminating in the development of diabetic retinopathy. Using isolated retinal endothelial cells, the effect of regulation of H-Ras downstream signaling cascade, Raf-1, MEK, and ERK, was investigated on glucose-induced activation of MMP9. In vitro results were confirmed in the retina obtained from diabetic mice manipulated for MMP9 gene, and also in the retinal microvasculature obtained from human donors with diabetic retinopathy. Regulation of Raf-1/MEK/ERK by their specific siRNAs and pharmacologic inhibitors prevented glucose-induced activation of MMP9 in retinal endothelial cells. In MMP9-KO mice, diabetes had no effect on retinal MMP9 activation, and H-Ras/Raf-1/MEK signaling cascade remained normal. Similarly, donors with diabetic retinopathy had increased MMP9 activity in their retinal microvessels, the site of histopathology associated with diabetic retinopathy, and this was accompanied by activated H-Ras signaling pathway (Raf-1/ERK). Collectively, these results suggest that Ras/Raf-1/MEK/ERK cascade has an important role in the activation of retinal MMP9 resulting in the apoptosis of its capillary cells. Understanding the upstream mechanism responsible for the activation of MMP9 should help identify novel molecular targets for future pharmacological interventions to inhibit the development/progression of diabetic retinopathy.
Human Pancreatic Cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras; Pdx1-Cre (KC) and K-Ras; Pdx1-Cre; INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b and miR-155, and down-regulation of miR-216a, miR-216b, miR-217 and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.
Ink4a; K-Ras; miRNAs; EGFR; RInk-l
The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS-4, and ADAMTS-5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1β) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7-promoted proteoglycan synthesis as assessed by 35S-sulfate incorporation. Protein-DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases.
articular cartilage; cartilage degeneration; regeneration; osteoarthritis; chondrosarcoma; resveratrol; matrix metalloprotease; MMP13; proteoglycan; cell survival; cancer
Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE-cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca2+-free medium, and VE-cadherin blocking antibody to alter VE-cadherin extracellular interactions to investigate the role of VE-cadherin outside-in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost-induced Rac activity at the sites of newly formed cell-cell junctions. In contrast, disruption of VE-cadherin extracellular trans-interactions suppressed iloprost-activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac-specific guanine nucleotide exchange factors (GEF) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE-cadherin domain further promoted iloprost-induced Rac signaling. In the model of thrombin-induced EC barrier recovery, blocking of VE-cadherin trans-interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE-cadherin outside-in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery.
endothelium; permeability; adherens junctions; cytoskeleton
Progressive multifocal leukoencephalopathy (PML) is a severe disease of the central nervous system (CNS), caused by infection with the Polyomavirus JC virus (JCV). Because there are no known treatments or prognostic factors, we performed a long-term study focusing mainly on cerebrospinal fluid (CSF) samples from PML patients to describe the virological features akin to the different forms of the disease. Twenty-eight PML patients were enrolled: 10 HIV-1+ patients with classical PML (CPML), 9 HIV-1+ patients with slowly progressing or stable neurological symptoms (benign PML), 3 HIV-1+ asymptomatic patients and 6 HIV-1-negative patients. CSF, urine and blood samples were collected at the enrollment (baseline) and every six months afterwards when possible. The JCV DNA and HIV-1 RNA loads were determined, and the JCV strains were characterized.
At baseline , the mean CSF JCV load was log 6.0 ± 1.2 copies/ml for CPML patients, log 4.0 ± 1.0 copies/ml for benign PML patients, log 4.2 ± 0.5 copies/ml for asymptomatic PML patients and log 5.8 ± 1.3 copies/ml for HIV-1-negative PML patients (CPML versus benign: p<0.01; CPML versus asymptomatic: p<0.05; HIV-1 negative versus benign: p<0.01). Organization of the JCV transcriptional control region (TCR) showed unusual archetype structures in 2 long-term survival patients; the NF1 sequence was found most commonly, whereas the Sp1 binding site was the most common for both CPML patients and HIV-1 negative patients.
Our results suggest that the JCV load in the CSF and the organization of the TCR should be considered as indicators of PML clinical outcome.
JC Virus; Cerebrospinal Fluid; Transcriptional Cellular Factors; Molecular organization
Reactive oxygen species (ROS), generated as a result of various reactions, control an array of cellular processes. The role of ROS during megakaryocyte (MK) development has been a subject of interest and research. The bone marrow niche is the major site of MK differentiation and maturation. In this environment, a gradient of oxygen tension, from normoxia to hypoxia results in different levels of ROS, impacting cellular physiology. This article provides an overview of major sources of ROS, their implication in different signaling pathways, and their effect on cellular physiology, with a focus on megakaryopoiesis. The importance of ROS-generating oxidases in MK biology and pathology, including myelofibrosis, is also described.
megakaryocytes; oxidases; reactive oxygen species; bone marrow
Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which flag-tagged Runx2 was driven by a cell-specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (postnatal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti-apoptotic genes Bcl-2, Opn, and Sox9 in transgenic mice by real-time RT-PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9 etc.). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification.
Runx2; Col10a1; transgenic mice; chondrocyte maturation; endochondral ossification
Epidemiological studies have shown that a diet rich in fruits and cruciferous vegetables is associated with a lower risk of prostate cancer. Indole-3-carbinol (I3C) and its dimeric product 3,3′-diindolylmethane (DIM) have been shown to exhibit anti-tumor activity both in vitro and in vivo. Recently, we have reported that a formulated DIM (B-DIM) induced apoptosis and inhibited growth, angiogenesis, and invasion of prostate cancer cells by regulating Akt, NF-κB, VEGF and the androgen receptor (AR) signaling pathway. However, the precise molecular mechanism(s) by which B-DIM inhibits prostate cancer cell growth and induces apoptosis have not been fully elucidated. Most importantly, it is not known how B-DIM affects cell cycle regulators and proteasome activity, which are critically involved in cell growth and apoptosis. In this study, we investigated the effects of B-DIM on proteasome activity and AR transactivation with respect to B-DIM-mediated cell cycle regulation and induction of apoptosis in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. We believe that our results show for the first time the cell cycle-dependent effects of B-DIM on proliferation and apoptosis of synchronized prostate cancer cells progressing from G1 to S phase. B-DIM inhibited this progression by induction of p27Kip1 and down-regulation of AR. We also show for the first time that B-DIM inhibits proteasome activity in S phase, leading to the inactivation of NF-κB signaling and induction of apoptosis in LNCaP and C4-2B cells. These results suggest that B-DIM could be a potent agent for the prevention and/or treatment of both hormone sensitive as well as hormone-refractory prostate cancer.
The discovery of JAK2 mutations in Philadelphia-negative myeloproliferative neoplasms has prompted investigators to evaluate mutation-targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1low mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27Kip 1 activity and is treatable by Aplidin®, a cyclic depsipeptide that activates p27 kip 1 in several cancer cells. Aplidin® restored expression of Gata1 and p27Kip 1 in Gata1 low hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF-β/VEGF levels released in the microenvironment by immature Gata1 low megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin®-treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1low progenitor cells remained low. These results indicate that Aplidin® effectively alters the natural history of myelofibrosis in Gata1low mice and suggest this drug as candidate for clinical evaluation in PMF.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer-related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF-κB play a critical role in the development of PDAC in the compound K-RasG12D and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K-Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real-time RT-PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K-RasG12D expressing mice led to high expression of PDGF-D signaling pathway in the tumor and tumor-derived cell line (RInk-1 cells). Furthermore, PDGF-D knock-down in RInk-1 cells resulted in the inhibition of pancreatosphere formation and down-regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial–mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF-D, signaling.
Mouse embryonic stem (mES) cells are pluripotent cells that can be propagated in vitro with leukemia inhibitory factor (LIF) and serum. Intracellular signaling by LIF is principally mediated by activation of STAT-3, although additional pathways for self-renewal have been described. Here, we identified a novel role for Insulin receptor substrate-1 (IRS-1) as a critical factor in mES cells self-renewal and differentiation. IRS-1 is expressed and tyrosyl phosphorylated during mES cells self-renewal. Differentiation of mES cells, by LIF withdrawal, is associated with a marked reduction in IRS-1 expression. Targeting of IRS-1 by si-IRS-1 results in a severe reduction of Oct-4 protein expression and alkaline phosphatase activity, markers of undifferentiated mES cells. IRS-1 targeting does not interfere with LIF-induced STAT-3 phosphorylation, but negatively affects protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3β) phosphorylation, which are downstream effectors of the LIF-mediated PI3K signaling cascade. Targeting of IRS-1 also results in a marked down regulation of Id-1 and Id-2 proteins expression, which are important components for self-renewal of ES cells. Conversely, over expression of IRS-1 inhibits mES cell differentiation. Taken together, these results suggest that expression and activity of IRS-1 are critical to the maintenance of the self-renewal program in mES cells.
Inhibitor of differentiation-1 (Id-1) is a member of helix–loop–helix (HLH) family of proteins that regulate gene transcription through their inhibitory binding to basic-HLH transcription factors. Similarly to other members of this family, Id-1 is involved in the repression of cell differentiation and activation of cell growth. The dual function of Id-1, inhibition of differentiation, and stimulation of cell proliferation, might be interdependent, as cell differentiation is generally coupled with the exit from the cell cycle. Fibroblast growth factor-2 (FGF-2) has been reported to play multiple roles in different biological processes during development of the central nervous system (CNS). In addition, FGF-2 has been described to induce “neuronal-like” differentiation and trigger apoptosis in neuroblastoma SK-N-MC cells. Although regulation of Id-1 protein by several mitogenic factors is well-established, little is known about the role of FGF-2 in the regulation of Id-1. Using human neuroblastoma cell line, SK-N-MC, we found that treatment of these cells with FGF-2 resulted in early induction of both Id-1 mRNA and protein. The induction occurs within 1 h from FGF-2 treatment and is mediated by ERK1/2 pathway, which in turn stimulates expression of the early growth response-1 (Egr-1) transcription factor. We also demonstrate direct interaction of Egr-1 with Id-1 promoter in vitro and in cell culture. Finally, inhibition of Id-1 expression results in G2/M accumulation of FGF-2-treated cells and delayed cell death.
Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.
Unsaturated fatty acids; Mesenchymal stem cells; Bone marrow; Linoleic acid; Oleic acid; Cell proliferation; Gene expression; Angiogenesis
The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic acute lung injury in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP-1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type-II cells (AT-II) and THP-1 co-cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase-1, IL-1β cleavage and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short hairpin RNA silencing of inflammasome components abrogated ceramide-induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide induced alveolar epithelial permeability in AT-II. Collectively, our results demonstrate for the first time that ceramide-induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation.
Macrophage; Inflammasome; ceramide; reactive oxygen species; acute lung injury; IL-1β
Diet and energy metabolism affect gene expression, which influences human health and disease. Here, we discuss the role of epigenetics as a mechanistic link between energy metabolism and control of gene expression. A number of key energy metabolites including SAM, acetyl-CoA, NAD+, and ATP serve as essential co-factors for many, perhaps most, epigenetic enzymes that regulate DNA methylation, posttranslational histone modifications, and nucleosome position. The relative abundance of these energy metabolites allows a cell to sense its energetic state. And as co-factors, energy metabolites act as rheostats to modulate the activity of epigenetic enzymes and upregulate/downregulate transcription as appropriate to maintain homeostasis.
Energy Metabolism; Epigenetics; SAM; CpG Methylation; Acetyl-CoA; Histone Modifications; NAD+
AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.
Ack1; TNK2; ACK; AKT; SRC; TBK1; IKBKE; DNAPKcs; PI3K; PTEN; Tyrosine phosphorylation; Cancer; EGFR; HER2; Kinase inhibitor
Adenosine A1 receptor (A1R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β1-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β1-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A1R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β1-adrenergic stimulation on PKCε translocation elicited by A1R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A1R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β1-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A1R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A1R. This increase was attenuated by the A1R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A1R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A1R, it is apparent that the interactive effects of adenosine and β1-adrenergic agonists on function are complex in the heart.
adrenergic; antiadrenergic; heart; RACK2; PKA; PKCε
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.
Lactoferricin; Interleukin-1; Lipopolysaccharide; Intervertebral disc degeneration; Anti-Inflammation; Anti-catabolic effect
Ribonuclease P is an ancient enzyme that cleaves pre-tRNAs to generate mature 5′ ends. It contains an essential RNA subunit in Bacteria, Archaea, and Eukarya, but the degree to which the RNA subunit relies on proteins to supplement catalysis is highly variable. The eukaryotic nuclear holoenzyme has recently been found to contain almost twenty times the protein content of the bacterial enzymes, in addition to having split into at least two related enzymes with distinct substrate specificity. In this review, recent progress in understanding the molecular architecture and functions of nuclear forms of RNase P will be considered.
An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.
JCV agnoprotein; chemokine; CXCL5/LIX; neuron; apoptosis
Deletion of the highly conserved gene for the major Ca2+ efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P<0.001) ATP levels, significantly (P<0.001) greater cytosolic Ca2+ concentration ([Ca2+]c) and ~10-fold higher mitochondrial sequestration, indicating Ca2+ overload. Investigating the mechanism involved, we used coimmunoprecipitation studies to show that CASK (Ca2+/calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b’s enzymatic activity, consequent Ca2+ accumulation, and a ~6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca2+ homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.
hyperactivated motility; flagellum’s principal piece; calcium efflux; electron dense mitochondria
Organisms frequently encounter a wide variety of proteotoxic stressors. The heat-shock response, an ancient cytoprotective mechanism, has evolved to augment organismal survival and longevity in the face of proteotoxic stress from without and within. These broadly recognized beneficial effects, ironically, contrast sharply with its emerging role as a culprit in the pathogenesis of cancers. Here, we present an overview of the normal biology of the heat-shock response and highlight its implications in oncogenic processes, including the proteotoxic stress phenotype of cancer; the function of this stress response in helping cancer survive and adapt to proteotoxic stress; and perturbation of proteome homeostasis in cancer as a potential therapeutic avenue.
Oncogenesis; Proteotoxic stress; Heat-shock response; HSF1
The family of insulin receptor substrates (IRS) consists of four proteins (IRS-1 - IRS-4), which were initially characterized as typical cytosolic adaptor proteins involved in insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) signaling. The first cloned and characterized member of the IRS family, IRS-1, has predicted molecular weight of 132 kDa, however, as a result of its extensive serine phosphorylation it separates on a SDS gel as a band of approximately 160–185 kDa. In addition to its metabolic and growth-promoting functions, IRS-1 is also suspected to play a role in malignant transformation. The mechanism by which IRS-1 supports tumor growth is not fully understood, and the argument that IRS-1 merely amplifies the signal from the IGF-1R and/or IR requires further investigation. Almost a decade ago, we reported the presence of nuclear IRS-1 in medulloblastoma clinical samples, which express viral oncoprotein, large T-antigen of human polyomavirus JC (JCV T-antigen). This first demonstration of nuclear IRS-1 was confirmed in several other laboratories. The nuclear IRS-1 was also detected by cells expressing the SV40 T-antigen, v-Src, in immortalized fibroblasts stimulated with IGF-I, in hepatocytes, 32D cells, and in an osteosarcoma cell line. More recently, nuclear IRS-1 was detected in breast cancer cells in association with estrogen receptor alpha (ERα), and in JC virus negative medulloblastoma cells expressing ERβ, further implicating nuclear IRS-1 in cellular transformation. Here, we discuss how nuclear IRS-1 acting on DNA repair fidelity, transcriptional activity, and cell growth can support tumor development and progression.
Biosynthesis of selenium-containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3′-untranslated region of the mRNA, as well as several SECIS-binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS-binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross-linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co-precipitated with NSEP1 in both human and rat cell lines. Co-immunoprecipitation of an epitope-tagged GPX1 construct depended upon an intact SECIS element in its 3′-untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS-dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co-transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells.