Evelyn Png

Hyperosmolarity-mediated mitochondrial dysfunction requires Transglutaminase-2 in human Corneal epithelial cells

Hyperosmolar‐induced ocular surface cell death is a key mitochondria‐mediated event in inflammatory eye diseases. Transglutaminase (TGM)‐2, a cross‐linking enzyme, is purported to mediate cell death, but its link to mitochondria is unclear. In the cornea, the integrity of the epithelial cells is important for maintaining transparency of the cornea and therefore functional vision. We evaluated the role of TGM‐2 and its involvement in hyperosmolarity‐stimulated mitochondrial cell death in human corneal epithelial (HCE‐T) cells. HCE‐T cell lines stably expressing either shRNA targeting TGM‐2 (shTG) or scrambled shRNA (shRNA) were constructed. Hyperosmolar conditions reduced viability and increased mitochondrial depolarization in shRNA cells. However, hyperosmolarity failed to induce mitochondrial depolarization to the same extent in shTG cells. Transient overexpression of TGM‐2 resulted in very high levels of TGM‐2 expression in shTG and shRNA cells. In the case of shTG cells after overexpression of TGM‐2, hyperosmolarity induced the same extent of mitochondrial depolarization as similarly treated shRNA cells. Overexpression of TGM‐2 also elevated transamidase activity and reduced viability. It also induced mitochondrial depolarization, increased caspase‐3/7 and ‐9 activity, and these increases were partially suppressed by pan‐caspase inhibitor Z‐VAD‐FMK. Corneal epithelial apoptosis via mitochondrial dysfunction after hyperosmolar stimulation is partially dependent on TGM‐2. This TGM‐2‐dependent mechanism occurs in part via caspase‐3/7 and ‐9. Protection against mitochondrial stress in the ocular surface targeting TGM‐2 may have important implications in the survival of cells in hyperosmolar stress. J. Cell. Physiol. 226: 693–699, 2011.

Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing.

Transglutaminase-2 in cell adhesion: all roads lead to paxillin?

Cell-matrix adhesion is a fundamental biological process that governs survival, migration, and proliferation of living eukaryotic cells. Paxillin is an important central player in a network of adhesome proteins that form focal adhesion complexes. Phosphorylation of tyrosine and serine residues in paxillin is critical for the coordinated sequential recruitment of other adaptor and kinase proteins to adhesion complexes. Recently, the phosphorylation of serine178 in paxillin has been shown to be vital for epithelial cell adhesion and migration. In vivo and in vitro evidence have shown that transglutaminase (TG)-2 positively regulates this phosphorylation. Here, we propose three possible mechanisms that may explain these observations. First, TG-2 itself may be an adhesome member directly interacting with paxillin in a non-covalent way. Second, TG-2 may cross link a mitogen-activated protein kinase kinase kinase (MAP3K), which eventually activates c-Jun N-terminal kinase (JNK), and the latter phosphorylates paxillin. Lastly, TG-2 may have intrinsic kinase activity that phosphorylates paxillin. Future studies investigating these hypotheses on TG-2-paxillin relationships are necessary in order to address this fundamental process in cell matrix adhesion signaling.

Recent Advances: Transglutaminase in Ocular Health and Pathological Processes

Transglutaminase (TG) is a diverse class of crosslinking enzymes involved in the regulation of cytokine production, endocytosis, cell adhesion, migration, apoptosis and autophagy. It has been implicated in inflammatory diseases, neurodegenerative processes and cancer. The eye is a specialized organ which subserves the important function of vision and has distinctive physiological and anatomical properties that differ from other body tissues. Understanding of the roles of various TGs in the eye therefore require studies specific to cells and tissues of ocular origin. We review the advances in TG research in ocular diseases, including pterygium, glaucoma, cataract and proliferative vitreoretinopathy. TG1 is a molecule important for keratinisation in many cicatrizing diseases of the ocular surface, including keratoconjunctivitis sicca. TG2, with multiple functions, has been shown to be important in inflammation and cell adhesion in various ocular diseases. The results of TG research in each region of the eye are critically assessed and the implications of these studies in the treatment of ocular diseases are discussed. By modifying wound healing process and influencing the amount of inflammation in animal models of human ocular diseases, TG-related strategies are now a possibility for selected clinical scenarios. For example, the use of retinoic acid for severe dry eye has undergone clinical trials. However, in many other areas, more research in selection of specific targets, time of intervention, specific method of delivery of interventional molecules, and safety of therapy in humans may be necessary. One promising area in the future is the use of a TG strategy to modify conjunctival wound healing to increase success rates after glaucoma surgery.

A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins

Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.

Comparison of two artificial tear formulations using aberrometry

EDITOR: We commend the authors of a recently published paper, which reported aberrations due to instillation of artificial tears up to 60 seconds. The authors commented that the magnitude of the aberrations appeared to be related to the viscosity of the tear formulation. We measured a variety of commercially available eye-drops of different viscosity, including Systane and TheraTears and found these eye-drops to be of different pH and osmolarity (Table 1). The pH of the formulations was tested using a pH meter (SevenEasy, Mettler Toledo) and the osmolarity using a vapour pressure osmometer 5520 (Wescor Inc). The reproducibility (95% confidence interval of differences) of the osmolarity and pH readings were 9.6 mmol/kg and 0.04, respectively. Differences in pH and osmolarity of instilled eye-drops can influence the comfort or possible stinging sensation in different individuals. These factors could affect reflex tearing, which may in turn affect light transmission and aberrations. As the Systane PF and TheraTears have differences in parameters other than viscosity, the authors should report pH and osmolarity to determine the factors that impact on aberrations. Grant support: SNEC HREF R673/23/ 2009 1. Berger JS, Head KR, Salmon TO. Comparison of two artificial tear formulations using aberrometry. Clin Exp Optom 2009; 92: 206– 211. 2. Fassihi AR, Naidoo NT. Irritation associated with tear-replacement ophthalmic drops. A pharmaceutical and subjective investigation. S Afr Med J 1989; 75: 233–235. 3. Hopkins G, Pearson R. Ophthalmic Drugs Diagnostic and Therapeutic Uses, 5th ed. Edinburgh: Elsevier, 2007. p 247–248.BACKGROUND This study investigates the effect of artificial tears of different viscosities on aberrations of healthy eyes. METHODS We recruited subjects from the student population of the Northeastern State University Oklahoma College of Optometry. Using the Complete Ophthalmic Analysis System (COAS), aberrometry measurements were taken every second for 60 seconds with a blink every 10 seconds. We made measurements of the naked eye, then after instillation of TheraTears non-preserved drops, and after instillation of SystanePF, a more viscous formulation. Zernike coefficients up to the sixth order were recorded and analysed. We evaluated changes in spherical and astigmatic dioptric powers and coma-like, spherical aberration-like and total higher-order aberration (HOA) RMS values as a function of time. RESULTS Both drops yielded significantly higher values than the naked eye for HOA RMS and cylindrical power as well as a hyperopic shift in spherical equivalent (p < 0.001,paired t-test). SystanePF, the more viscous tear, showed significantly higher values than TheraTears in HOA RMS and cylinder in all inter-blink periods (p < 0.001, paired t-test). CONCLUSIONS This study shows artificial tears can have a measurable and significant impact on aberrations of healthy eyes. This effect is sustained for at least one minute and its magnitude appears to be related to the viscosity of the tear formulation.

Mechanistic role of transglutaminase-2 in focal adhesions

Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.

Quantitative Image Analysis of Cellular Morphology Using Amnis® ImageStreamX Mark II Imaging Flow Cytometer: A Comparison againstConventional Methods

Chemotaxis, the directional cell migration guided by chemoattractant gradients, plays essential roles in many physiological processes, such as recruitment of neutrophils to sites of inflammation. Neutrophils detect chemoattractants by G protein-coupled receptors (GPCRs). Chemoattractant stimuli activate multiple signaling pathways to regulate directional migration of neutrophils. Recently, we identified a novel GPCR-mediated PLCβγ/ PKCβ/PKD1 signaling axis that regulates cofilin activity through cofilin phosphatase slingshot 2 (SSH2) and remodels actin cytoskeleton during neutrophil chemotaxis. In the future, it will be important to understand how multiple signaling pathways are spatiotemporally regulated to precisely control the rapid remodeling of actin cytoskeleton in the leading front of chemotaxing neutrophils.

Comment on “The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation”

W e refer to the important article by Kumar et al. (1) This report links the inhibition of ubiquitin ligase and the suppression of NFB in intestinal epithelial cells to fatty acid (FA)/reactive oxygen species (ROS) production, but it is unclear whether this process is sufficient to cause clinical disease. We found that an analogous E3 suppression mechanism, possibly FA/ROS induced, plays a role in human epithelial inflammation in vivo in a similar mucus-producing, goblet cell-containing, stratified squamous epithelium (2). In sun-exposed people, a fibrovascular proliferative condition called pterygium frequently occurs (3). Our studies showed that the ubiquitin ligases UbE3C and UbE3B and the ubiquitin conjugase UbE2K were down-regulated in pterygium epithelia compared with uninvolved conjunctiva epithelia from the same eye (Fig. 1). Expression of other ubiquitin conjugases UbE2A, UbE2B, and UbE2I were unchanged (data not shown). The role of ubiquitin enzymes in pterygium has not been investigated previously, but the function of ubiquitin ligases has been linked to the propagation of inflammation and aberrant Wnt signaling (4). Our findings thus support two key hypotheses in pterygium: T lymphocyte infiltration (5) and epithelial-mesenchymal transition (6). The upstream factors of the ubiquitin ligase-mediated pathway that Kumar et al. (1) reported are also present in human tear film, namely FA (7) and commensal bacteria (8). Their involvement in pterygium is suggested by abnormal FA composition in pterygium (9), the role of FA in ocular surface inflammation (10), and oxidative damage in pterygium due to ROS generated by sunlight (11). In conclusion, the intestinal inflammation phenomenon reported (1) may also be applicable to nonintestinal epithelial inflammation in vivo.