Clathrin Heavy Chain Knockdown Impacts CXCR4 Signaling and Post-translational Modification

Recent research has implicated endocytic pathways as important regulators of receptor signaling. However, the role of endocytosis in regulating chemokine CXC receptor 4 (CXCR4) signaling remains largely unknown. In the present work we systematically investigate the impact of clathrin knockdown on CXCR4 internalization, signaling, and receptor post-translational modification. Inhibition of clathrin-mediated endocytosis (CME) significantly reduced CXCR4 internalization. In contrast to other receptors, clathrin knockdown increased CXCL12-dependent ERK1/2 signaling. Simultaneous inhibition of CME and lipid raft disruption abrogated this increase in ERK1/2 phosphorylation suggesting that endocytic pathway compensation can influence signaling outcomes. Interestingly, using an antibody sensitive to CXCR4 post-translational modification, we also found that our ability to detect CXCR4 was drastically reduced upon clathrin knockdown. We hypothesize that this effect was due to differences in receptor post-translational modification as total CXCR4 protein and mRNA levels were unchanged. Lastly, we show that clathrin knockdown reduced CXCL12-dependent cell migration irrespective of an observed increase in ERK1/2 phosphorylation. Altogether, this work supports a complex model by which modulation of endocytosis affects not only receptor signaling and internalization but also receptor post-translational modification

Vapd In Xylella Fastidiosa Is A Thermostable Protein With Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.10e014576

Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade.This work was funded by the National Institutes of Health (NIH/NCI) P50-CA97248 (University of Michigan Head and Neck SPORE)

PTEN Mediates Activation of Core Clock Protein BMAL1 and Accumulation of Epidermal Stem Cells

Tissue integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. In the skin, hair follicle stem cells (HFSCs) that reside within the bulge maintain tissue homeostasis in response to activating cues that occur with each new hair cycle or upon injury. We found that PTEN, a major regulator of the PI3K-AKT pathway, controlled HFSC number and size in the bulge and maintained genomically stable pluripotent cells. This regulatory function is central for HFSC quiescence, where PTEN-deficiency phenotype is in part regulated by BMAL1. Furthermore, PTEN ablation led to downregulation of BMI-1, a critical regulator of adult stem cell self-renewal, and elevated senescence, suggesting the presence of a protective system that prevents transformation. We found that short- and long-term PTEN depletion followed by activated BMAL1, a core clock protein, contributed to accumulation of HFSC

Periostin-driven cellular migration requires mTOR signaling.

<p>(<b>A</b>) Representative pictures of the NOK-SI cell scratch assay following treatment with vehicle, recombinant Periostin (50 ng/ml), and rapamycin (50 nM). Scale bars represent 50 μm. (<b>B</b>) Quantitative analysis of open-wounded area over time (n=4; mean ± S.E.M.). Note that rapamycin abrogates the Periostin migratory activity of epithelial cells (***p<0.001). (<b>C</b>) Proliferation assay using keratinocytes treated with rapamycin and/or Periostin. Note that Periostin alone induced significant cellular proliferation at 50 ng/ml (*p<0.05). Treatment with rapamycin blocked periostin-induced cell proliferation (ns p>0.05). (<b>D</b>) Representative immunoblot depicting knockdown of Raptor and Rictor after siRNA treatment. Scramble siRNA oligonucleotides sequences were used as controls. GAPDH was used as loading controls. (<b>E</b>) Graphic shows the quantitative analyses of open-wounded areas using NOK-SI cells over time (n=4; mean ± S.E.M.). Note that siRNA targeting Raptor abrogates Periostin-induced cellular migratory resulting on complete wound closure by 48 hours (**p<0.05). siRNA targeting Rictor did not change the Periostin induced accelerated cellular migration resulting on wound closure by 24 hours (ns p>0.05). (<b>F</b>) Proliferation assay using NOK-SI cells treated with siRNA for Raptor, Rictor, or siRNA scramble, and/or Periostin. Note that Periostin induced significant cellular proliferation (*p<0.05). Treatment with siRNA for Raptor or Rictor resulted in disruption of Periostin induced cellular proliferation (***p<0.001).</p

Co-expression of Periostin and mTOR during cellular migration and mechanical stress induced by tension.

<p>(<b>A</b>) A representative wound healing scratch assay shows keratinocytes stained for Periostin (TRITC-red), pS6 (FITC-green) and DNA (Hoechst-blue). Note colocalization of Periostin and pS6 staining (on merge and insert) in the migratory area. Scale bars represent 50 μm. (<b>B</b>) Quantification of positive cells co-expressing Periostin and pS6 are depicted. Note increased number of positive cells co-expressing Periostin and pS6. Most of these cells are in the migratory area (***p<0.001). (<b>C</b>) NOK-SI cells were subjected to biomechanical stimulation (load of 14% stretching at 6 cycles/min) at the indicated time points. (<b>D</b>) Western blot analysis for Periostin and pS6 expression in NOK-SI subjected to load assay. Non-stimulated cells (no load force) served as a control, and GAPDH was used as protein loading control.</p

Periostin-driven migration and proliferation.

<p>(<b>A</b>) Total cell lysates and conditioned medium (cond. medium) from NOK-SI and hPDL cells were blotted for Periostin. New cell culture medium supplemented with 10% FBS was used as a negative control (control). Intracellular Periostin is detected in epithelial cell lysate. However, conditioned medium from NOK-SI shows that keratinocytes did not secrete Periostin, as the same band was observed in the negative control media. hPDL cells have low levels of the intracellular Periostin isoform as observed in the cell lysate. Increased levels of secreted Periostin were found in the conditioned medium from hPDL. (<b>B</b>) hPDL conditioned medium induces keratinocyte proliferation compared to vehicle alone (***p<0.001), which is reduced upon administration of anti-Periostin antibody (*p<0.05). (<b>C</b>) Representative pictures of NOK-SI migration following treatment with recombinant Periostin (50 ng/ml), EGF (100 ng/ml) as the positive control, or vehicle. Scale bars represent 50 μm. (<b>D</b>) Graphic represents the quantification of the wound areas at indicated times (n=4; mean ± S.E.M). (<b>E</b>) Periostin enhances proliferation of keratinocytes compared to vehicle treated cells (***p<0.001). EGF treatment was used as positive control (*p<0.05) (n=6; mean ± S.E.M).</p

Expression of Periostin and CK6 during cutaneous wound healing.

<p><b>H</b>&<b>E</b>: Representative histological sections of cutaneous incisional wounds. (<b>A</b>) Morphology of the wounded site shows a thin edge of epithelial cells migrating across the wound bed, termed the epithelial tongue and (<b>B</b>) intact and normal skin adjacent to the wounded site were stained with Hematoxylin and Eosin (H&E). (<b>C</b>) Epithelial cells at the epithelial tongue express intracellular Periostin. (<b>D</b>) In normal adjacent skin, Periostin is in the connective tissue at the basal lamina, which is juxtaposed to the epithelial basal layer. (<b>E</b>) Note that basal and parabasal layers of the epithelial tongue have a large number of proliferating BrDU positive cells. (<b>F</b>) As expected, the epithelial basal layer of adjacent skin has few proliferating cells. (<b>G</b>) Upregulation of the epithelia stress/tension marker CK6 is depicted in the epithelial tongue compared to normal adjacent skin observed in (<b>H</b>). Scale bars represent 50 μm.</p

F-actin polarization and PI3K/mTOR signaling activation by Periostin-induced epithelial cell migration.

<p>(<b>A</b>) Phalloidin detection shows cells with polarized F-actin (white arrow) following treatment with recombinant Periostin compared to vehicle control. Scale bars represent 10 μm. (<b>B</b>) Graphic represents percentage of cells with stress fiber formation (polarized F-actin) after periostin or vehicle stimuli. Results were determined by measuring fields using independent triplicates (**p<0.01) (<b>C</b>) Activation of PI3K and mTOR signaling is triggered by Periostin treatment in a dose-dependent manner, as detected by phosphorylated AKT at Threonine 308 (pAKT^Thr308) and Serine 473 (pAKT^Ser473) and phosphorylated S6 (pS6). Note that 50 ng/ml of Periostin is the optimal concentration for PI3K activation. GAPDH was used as a loading control. </p

Functional And Small-angle X-ray Scattering Studies Of A New Stationary Phase Survival Protein E (sure) From Xylella Fastidiosa--evidence Of Allosteric Behaviour.

The genome data of bacterium Xylella fastidiosa strain 9a5c has identified several orfs related to its phytopathogenic adaptation and survival. Among these genes, the surE codifies a survival protein E (XfSurE) whose function is not so well understood, but functional assays in Escherichia coli revealed nucleotidase and exopolyphosphate activity. In the present study, we report the XfSurE protein overexpression in E. coli and its purification. The overall secondary structure was analyzed by CD. Small-angle X-ray scattering and gel filtration techniques demonstrated that the oligomeric state of the protein in solution is a tetramer. In addition, functional kinetics experiments were carried out with several monophosphate nucleoside substrates and revealed a highly positive cooperativity. An allosteric mechanism involving torsion movements in solution is proposed to explain the cooperative behaviour of XfSurE. This is the first characterization of a SurE enzyme from a phytopathogen organism and, to our knowledge, the first solution structure of a SurE protein to be described.2766751-6