Increased nuclear translocation of catalytically active PKC-ζ during mouse colonocyte hyperproliferation

2000 ◽  
Vol 279 (1) ◽  
pp. G223-G237 ◽  
Author(s):  
Shahid Umar ◽  
Joseph H. Sellin ◽  
Andrew P. Morris
Keyword(s):  
Kinase Activity ◽  
Nuclear Translocation ◽  
Physiological Role ◽  
Subcellular Fractionation ◽  
Nuclear Antigen ◽  
Nuclear Staining ◽  
Catalytically Active ◽  
Pkc Ζ

Protein kinase (PK) C-ζ is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-ζ, 3.2-fold), 48-kDa catalytic subunit (PKM-ζ, 3- to 9-fold), and 24-kDa membrane-bound fragment (Mf-ζ, >10-fold) expression. Both PKC-ζ and PKM-ζ exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-ι/PKM-ι expression occurred. The subcellular distribution of immunoreactive PKC-ζ changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-ζ and PKM-ζ expression and activity within nuclei, which preferentially accumulated PKM-ζ. These results suggest separate cellular and nuclear roles, respectively, for PKC-ζ in quiescent and mitotically active colonocytes. PKM-ζ may specifically act as a modulator of proliferation during TMCH.

scholarly journals Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets

2017 ◽  
Vol 37 (5) ◽  
pp. 823-835 ◽  
Author(s):  
Christopher W. Smith ◽  
Steven G. Thomas ◽  
Zaher Raslan ◽  
Pushpa Patel ◽  
Maxwell Byrne ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Thrombus Formation ◽  
Physiological Role ◽  
Src Family Kinase ◽  
Glycoprotein Vi ◽  
Collagen Receptor ◽  
Deficient Mice

Objective— Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif–containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif–containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1–deficient mice. Approach and Results— Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1–deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI–specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI–FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Conclusions— Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein–coupled receptors.

238 EFFECTS OF LEPTIN SUPPLEMENTATION ON NUCLEAR AND CYTOPLASMIC IN VITRO MATURATION OF RABBIT OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 198 ◽  
Author(s):  
M. Arias-Alvarez ◽  
R. M. Garcia-Garcia ◽  
L. Revuelta ◽  
P. G. Rebollar ◽  
P. L. Lorenzo
Keyword(s):  
Nutritional Status ◽  
Laser Scanning ◽  
Physiological Role ◽  
White Female ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Nuclear Staining ◽  
Cytoplasmic Maturation

Reproductive function is affected substantially by nutritional status. Leptin is a peptide secreted mainly by adipocytes that reflects the amount of body fat and acts as a modulator of oocyte quality. The aim of this study was to analyze, for the first time in the rabbit, the influence of leptin on meiotic and cytoplasmic maturation (cortical granule (CG) migration) of rabbit oocytes in vitro (IVM). Cumulus–oocyte complexes (COCs) were collected from 25 young New Zealand white female rabbits (<3 parturitions) in 3 replicates. COCs were aspirated from ovarian follicles >1 mm in size and were matured in TCM-199 medium, containing sodium pyruvate, sodium bicarbonate, BSA, and 10 ng mL–1 epidermal growth factor (EGF), and supplemented with 0, 10, or 100 ng mL–1 leptin. A total of 163 COCs were treated progressively with hyaluronidase (2 mm), 0.5% pronase, 4% paraformaldehyde, 0.02% Triton X-100, and 7.5% BSA after the maturation period. Oocytes were incubated with 100 mg mL–1 fluorescein isothiocyanate (FITC)-conjugated Lens culinaris agglutinin (LCA) for CG staining and with 4′,6-diamino-2-phenylindole (DAPI) for nuclear staining, and observed under a confocal laser-scanning microscope. In addition, 17 ovulated oocytes recovered from oviducts at 20 h post- GnRH were used as in vivo-maturated controls for CG distribution. Most of the ovulated oocytes at metaphase II (MII, 100%) presented CGs located in the cortex beneath the plasma membrane (61.1 ± 11.8%). Addition of 10 ng mL–1 leptin to IVM medium significantly increased the rate of oocytes reaching MII, compared to the 0 and 100 ng mL–1 leptin concentrations (P < 0.05). The percentage of oocytes showing CG migration to the cortex was significantly increased in the 10 ng mL–1 leptin treatment group (Table 1) compared to that in the 100 ng mL–1 leptin group (P < 0.05) and tended to be higher than that in the 0 ng mL–1 leptin group (P < 0.08). The rest of the oocytes showed homogeneous CG distribution, as they were not cytoplasmic maturated. Moreover, both in vivo- and in vitro-matured oocytes had a GC-free domain overlying the MII spindle. In conclusion, addition of leptin to IVM medium at physiological dose (10 ng mL–1) improves both meiotic and cytoplasmic maturation of rabbit oocytes, whereas an excessive leptin concentration does not exert a beneficial effect. These findings suggest a physiological role for leptin in the relationship between nutritional status and regulation of oocyte maturation. Table 1. Nuclear maturation and CG distribution of oocytes after IVM This research was supported by AGL05-196 and UCM-CM research program (920249). MAA received a grant from CM and FSE. RMGG was supported by the Juan de la Cierva-MEC Program.

scholarly journals TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Mohammad B. Hossain ◽  
Rehnuma Shifat ◽  
Jingyi Li ◽  
Xuemei Luo ◽  
Kenneth R. Hess ◽  
...  
Keyword(s):  
Dna Repair ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Subcellular Fractionation ◽  
Tyrosine Kinase Receptor ◽  
Histone H4 ◽  
Small Interfering Rnas ◽  
Caveolin 1

ABSTRACT DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies—chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo. This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

scholarly journals Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo.

1989 ◽  
Vol 37 (2) ◽  
pp. 241-247 ◽  
Author(s):  
K Schilling ◽  
C Duvernoy ◽  
S Keck ◽  
C Pilgrim
Keyword(s):  
Nuclear Antigen ◽  
Computer Search ◽  
Neural Cells ◽  
Nuclear Staining ◽  
Developmentally Regulated ◽  
Western Blots ◽  
Molecular Weights ◽  
Nuclear Antigens

We report that a monoclonal antibody directed against phosphorylated neurofilaments (SMI 31) recognizes nuclear antigens present in embryonic but not in adult neural cells. On Western blots, the antibody reacts with four proteins of apparent MW 35, 37, 52/54, and 250 KD which are found exclusively in developing brain tissue. These nuclear antigens are expressed by glial and neuronal cells. Both nuclear staining and immunoreactive proteins decrease with ongoing in vitro differentiation. A computer search for proteins that share the epitope recognized by antibody SMI 31 did not yield any proteins of known nuclear localization that exhibit the same molecular weights and solubility characteristics as the above immunoreactive proteins. We conclude that antibody SMI 31 recognizes hitherto unknown nuclear proteins which, in neural cells, are developmentally regulated.

PGE2 induces angiogenesis via MT1-MMP–mediated activation of the TGFβ/Alk5 signaling pathway

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1120-1128 ◽  
Author(s):  
Arántzazu Alfranca ◽  
Juan Manuel López-Oliva ◽  
Laura Genís ◽  
Dolores López-Maderuelo ◽  
Isabel Mirones ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Kinase Activity ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Transforming Growth Factor Β ◽  
Tgfβ Signaling ◽  
In Vivo Models ◽  
Latent Form

Abstract The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E2 (PGE2). Here, we provide evidence for a novel signaling route through which PGE2 activates the Alk5-Smad3 pathway in endothelial cells. PGE2 induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFβ from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE2. MT1-MMP–dependent transforming growth factor β (TGFβ) signaling through Alk5 is also required for PGE2-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE2-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE2 and TGFβ, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.

scholarly journals Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

2021 ◽  
Vol 22 (1) ◽  
pp. 434
Author(s):  
Yuria Jang ◽  
Hong Moon Sohn ◽  
Young Jong Ko ◽  
Hoon Hyun ◽  
Wonbong Lim
Keyword(s):  
Nuclear Translocation ◽  
Critical Role ◽  
Osteoclast Differentiation ◽  
Point Mutations ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mice Model ◽  
Gsk 3Β ◽  
Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

scholarly journals Phaseolin Attenuates Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells and Zebrafish

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 420
Author(s):  
Su-Jung Hwang ◽  
Ye-Seul Song ◽  
Hyo-Jong Lee
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Raw 264.7 Cells ◽  
Network Pharmacology ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Bioactive Constituents

Kushen (Radix Sophorae flavescentis) is used to treat ulcerative colitis, tumors, and pruritus. Recently, phaseolin, formononetin, matrine, luteolin, and quercetin, through a network pharmacology approach, were tentatively identified as five bioactive constituents responsible for the anti-inflammatory effects of S. flavescentis. However, the role of phaseolin (one of the primary components of S. flavescentis) in the direct regulation of inflammation and inflammatory processes is not well known. In this study, the beneficial role of phaseolin against inflammation was explored in lipopolysaccharide (LPS)-induced inflammation models of RAW 264.7 macrophages and zebrafish larvae. Phaseolin inhibited LPS-mediated production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), without affecting cell viability. In addition, phaseolin suppressed pro-inflammatory mediators such as cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in a dose-dependent manner. Furthermore, phaseolin reduced matrix metalloproteinase (MMP) activity as well as macrophage adhesion in vitro and the recruitment of leukocytes in vivo by downregulating Ninjurin 1 (Ninj1), an adhesion molecule. Finally, phaseolin inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB). In view of the above, our results suggest that phaseolin could be a potential therapeutic candidate for the management of inflammation.

scholarly journals Plant Physiology, 2021 Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

2021 ◽  
Author(s):  
Jianghao Wu ◽  
Liwei Rong ◽  
Weijun Lin ◽  
Lingxi Kong ◽  
Dengjie Wei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Disulfide Bonds ◽  
Kinase Activity ◽  
Light Harvesting ◽  
Photosynthetic Electron Transport ◽  
State Transitions ◽  
Light Harvesting Complex ◽  
Light Harvesting Complex Ii

Abstract In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

scholarly journals LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽  
2021 ◽  
Author(s):  
Jiaying Zhu ◽  
Zhu Zhu ◽  
Yipin Ren ◽  
Yukang Dong ◽  
Yaqi Li ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Mice Model ◽  
Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

scholarly journals RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Ruizhao Li ◽  
Xingchen Zhao ◽  
Shu Zhang ◽  
Wei Dong ◽  
Li Zhang ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Transcription Factor ◽  
Nuclear Translocation ◽  
Kidney Injury ◽  
Septic Acute Kidney Injury ◽  
Transcription Factor Eb ◽  
Autophagic Degradation ◽  
Lps Treatment

AbstractAutophagy is an important renal-protective mechanism in septic acute kidney injury (AKI). Receptor interacting protein kinase 3 (RIP3) has been implicated in the renal tubular injury and renal dysfunction during septic AKI. Here we investigated the role and mechanism of RIP3 on autophagy in septic AKI. We showed an activation of RIP3, accompanied by an accumulation of the autophagosome marker LC3II and the autophagic substrate p62, in the kidneys of lipopolysaccharide (LPS)-induced septic AKI mice and LPS-treated cultured renal proximal tubular epithelial cells (PTECs). The lysosome inhibitor did not further increase the levels of LCII or p62 in LPS-treated PTECs. Moreover, inhibition of RIP3 attenuated the aberrant accumulation of LC3II and p62 under LPS treatment in vivo and in vitro. By utilizing mCherry-GFP-LC3 autophagy reporter mice in vivo and PTECs overexpression mRFP-GFP-LC3 in vitro, we observed that inhibition of RIP3 restored the formation of autolysosomes and eliminated the accumulated autophagosomes under LPS treatment. These results indicated that RIP3 impaired autophagic degradation, contributing to the accumulation of autophagosomes. Mechanistically, the nuclear translocation of transcription factor EB (TFEB), a master regulator of the lysosome and autophagy pathway, was inhibited in LPS-induced mice and LPS-treated PTECs. Inhibition of RIP3 restored the nuclear translocation of TFEB in vivo and in vitro. Co-immunoprecipitation further showed an interaction of RIP3 and TFEB in LPS-treated PTECs. Also, the expression of LAMP1 and cathepsin B, two potential target genes of TFEB involved in lysosome function, were decreased under LPS treatment in vivo and in vitro, and this decrease was rescued by inhibiting RIP3. Finally, overexpression of TFEB restored the autophagic degradation in LPS-treated PTECs. Together, the present study has identified a pivotal role of RIP3 in suppressing autophagic degradation through impeding the TFEB-lysosome pathway in septic AKI, providing potential therapeutic targets for the prevention and treatment of septic AKI.

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