Cell rounding in cultured human astrocytes and vascular endothelial cells upon inhibition of CK2 is mediated by actomyosin cytoskeleton alterations.

Protein kinase CK2 participates in a wide range of cellular events, including the regulation of cellular morphology and migration, and may be an important mediator of angiogenesis. We previously showed that in the retina, CK2 immunolocalizes mostly to vascular endothelium and astrocytes in association with the cytoskeleton. Additionally, CK2 inhibitors significantly reduced retinal neovascularization and stem cell recruitment in the mouse model of oxygen-induced proliferative retinopathy. We have also shown that CK2 and F-actin co-localized in actin stress fibers in microvascular endothelial cells, and that highly specific CK2 inhibitors caused cell rounding in astrocytes and microvascular endothelial cells, which was alleviated by serum that promotes spreading by Rho/Rho-kinase (RhoK) activation of myosin II. Therefore, we examined a possible role of CK2 in the regulation of actin-myosin II-based contractility. Treatment with CK2 inhibitors correlated with disassembly of actomyosin stress fibers and cell shape changes, including cytoplasmic retraction and process formation that were similar to those occurring during astrocyte stellation. Low doses of specific inhibitors of kinases (RhoK and MLCK) that phosphorylate myosin light chain (MLC) enhanced the effect of suboptimal CK2 inhibition on cell shape. Such striking stellation-like alteration was accompanied by decreased level of phospho-MLC, thus implying a CK2 role in regulation of actomyosin cytoskeleton. Our results suggest an important role of CK2 in the control of cell contractility and motility, which may account for suppressing effect of CK2 inhibition on retinal neovascularization. Together, our data implicate protein kinase CK2 for the first time in stellation-like morphological transformation.

Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton.

Ubiquitous protein kinase CK2 is a key regulator of cell migration, proliferation and tumor growth. CK2 is abundant in retinal astrocytes, and its inhibition suppresses retinal neovascularization in a mouse retinopathy model. In human astrocytes, CK2 co-distributes with GFAP-containing intermediate filaments, which implies its association with cytoskeleton. Contrary to astrocytes, CK2 is co-localized in microvascular endothelial cells (HBMVEC) with microtubules and actin stress fibers, but not with vimentin-containing intermediate filaments. Specific CK2 inhibitors (TBB, TBI, TBCA and DMAT) and nine novel CK2 inhibiting compounds (TID43, TID46, Quinolone-7, Quinolone-39, FNH28, FNH62, FNH64, FNH68 and FNH74) were tested at 10-200 µM for their ability to induce morphological alterations in cultured human astrocytes (HAST-40), and HBMVEC (For explanation of the inhibitor names, see "Methods" section). CK2 inhibitors caused dramatic changes in shape of cultured cells with effective inhibitor concentrations between 50 and 100 µM. Attached cells retracted, acquired shortened processes, and eventually rounded up and detached. CK2 inhibitor-induced morphological alterations were completely reversible and were not blocked by caspase inhibition. However, longer treatment or higher inhibitor concentration did cause apoptosis. The speed and potency of the CK2 inhibitors effects on cell shape and adhesion were inversely correlated with serum concentration. Western analyses showed that TBB and TBCA elicited a significant (about twofold) increase in the activation of p38 and ERK1/2 MAP kinases that may be involved in cytoskeleton regulation. This novel early biological cell response to CK2 inhibition may underlie the anti-angiogenic effect of CK2 suppression in the retina.

Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.

PURPOSE: Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. METHODS: rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. RESULTS: By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. CONCLUSIONS: rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.

Inhibition of protein kinase CK2 suppresses angiogenesis and hematopoietic stem cell recruitment to retinal neovascularization sites.

Ubiquitous protein kinase CK2 participates in a variety of key cellular functions. We have explored CK2 involvement in angiogenesis. As shown previously, CK2 inhibition reduced endothelial cell proliferation, survival and migration, tube formation, and secondary sprouting on Matrigel. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. In this model, CK2 inhibitors had an additive effect with somatostatin analog, octreotide, resulting in marked dose reduction for the drug to achieve the same effect. CK2 inhibitors may thus emerge as potent future drugs aimed at inhibiting pathological angiogenesis. Immunostaining of the retina revealed predominant CK2 expression in astrocytes. In human diabetic retinas, mRNA levels of all CK2 subunits decreased, consistent with increased apoptosis. Importantly, a specific CK2 inhibitor prevented recruitment of bone marrow-derived hematopoietic stem cells to areas of retinal neovascularization. This may provide a novel mechanism of action of CK2 inhibitors on newly forming vessels.

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases.

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.

Mucinoprotein is a universal constituent of stable intercellular bridges in Drosophila melanogaster germ line and somatic cells.

Intercellular bridges formed by incomplete cytokinesis may be important in a variety of processes, including synchronization of mitotic and meiotic divisions in animal cells. Using specific antibodies against a mucin-type glycoprotein (Kramerov et al. [1996] FEBS Lett. 378:213-218) from Drosophila melanogaster cultured embryonic cells, we showed that this glycoprotein is located in all cytoplasmic bridges found in various germline and somatic tissues. In the ovary, immunostaining of ring canals connecting germ cells can be detected in the very early stages at the germarium region 1 where first gonial divisions take place, and the immunostaining appears to persist through late stages when transport of cytoplasm from nurse cells to a growing oocyte occurs. Each ring canal is made up of an outer and an inner rim. Mucin glycoprotein appears to be one of the first proteins localized to the outer rim, which is a derivative of the arrested cleavage furrow. The known ring canal proteins, phosphotyrosine-containing protein(s), F-actin, hts- and kelch proteins, are localized to the inner rim at a later developmental time. Similarly, mucin glycoprotein is recruited early to ring canals connecting mitotic primary spermatocytes in both larval and adult testes. Mucin glycoprotein was found to be present in intercellular bridges (small ring canals) in somatic cells, including follicular epithelium in ovary and imaginal disc cells. Intercellular bridges were observed for the first time in a subset of cells in the larval brain. Thus, mucin glycoprotein is the only protein hitherto found in all known types of stable intercellular bridges and may be an important constituent of a backbone needed for assembly and preservation of this particular type of cell-cell contact.

[The tissue localization and secretion of mucin-D in Drosophila melanogaster]. / Tkanevaia lokalizatsiia i sekretsiia mutsina-D Drosophila melanogaster.

Glycoprotein with biochemical characteristics that allow us to classify it as a glycoprotein of mucin-type was isolated from cultured embryonic cells of Drosophila melanogaster. This is the first finding of mucin-type glycoprotein in insects. Using high-affinity monoclonal antibodies against a carbohydrate epitope, we demonstrated that the accumulation of this glycoprotein in the culture fluid of Drosophila cell line and cultured cells of other insects was inhibited by secretion inhibitors. 20-hydroxyecdysone, a hormone responsible for molting and metamorphosis in insects, inhibits the accumulation of this glycoprotein in the culture fluid, as well as in cells of Drosophila Dm cell line. In another cell line (Schneider-2), where there was practically no secretion of this glycoprotein, the hormone induced its increased accumulation in the cells. Mucin glycoproteins recognized by monoclonal antibodies have been found in embryos, imaginal disc, fat body, testicles, and ovaries, but not in salivary glands or muscles of Drosophila.

Insect mucin-type glycoprotein: immunodetection of the O-glycosylated epitope in Drosophila melanogaster cells and tissues.

A mucin-type glycoprotein (GP) from cultured embryonic cells of Drosophila melanogaster was isolated and used to raise monoclonal antibodies (MAbs). Epitope(s) recognized by MAbs were sensitive to the treatment by O-glycanase, which specifically cleaves off O-linked mucin-type Gal(beta 1,3)GalNAc disaccharide, representing the major part of the carbohydrate moiety of Drosophila GP. Using high-affinity MAbs against carbohydrate epitopes of the Drosophila mucin GP we demonstrated its accumulation in culture medium, as well as in cultured cells, which proved to be regulated by 20-hydroxyecdysone. Mucin GPs carrying Gal(beta 1,3)GalNAc disaccharide recognized by the MAbs were immunochemically localized in several Drosophila tissues of ectodermal, mesodermal and germ line origin, including epidermal and follicle cells capable of their secretion.

Mucin-type glycoprotein from Drosophila melanogaster embryonic cells: characterization of carbohydrate component.

A secreted glycoprotein (GP) with apparent molecular mass of 90 kDa produced by cultured embryonic cells of Drosophila melanogaster was isolated and partially characterized. GP is enriched by Ser + Thr and Pro residues that constitute up to 30% of the total number of amino acids. An abundant carbohydrate moiety (40% of molecular mass) is mainly represented by vertebrate mucin-type O-linked disaccharide units Gal(beta 1-3)-GalNAc, occupying about a half of the total number of Ser+Thr residues and rendering the GP molecule high resistance to protease action. A few of N-glycans are also present in GP. These characteristics allow to consider the Drosophila GP (termed 'mucin-D') as a first representative of invertebrate mucin-type glycoproteins.

[Glycoproteins from Drosophila melanogaster cell culture contains O-bound carbohydrate chains of the Gal(beta1-3)GalNAc type]. / Glikoprotein iz kul'tury kletok Drosophila melanogaster soderzhit O-sviazannye uglevodnye tsepi tipa Gal(beta1-3)GalNAc.

The glycoprotein from cultured cells of D. melanogaster, also detected in various insect tissues as a component of the extracellular matrix, was characterized as a mucin-type glycoprotein not yet described in invertebrates. This glycoprotein with an apparent molecular mass of approximately 90 kDa contains about 40% of carbohydrates, largely represented mainly by GalNAc and Gal; its polypeptide moiety is enriched with Thr, Ser, Pro and Gly. An analysis of oligosaccharides liberated by treatment of the glycoprotein with alkaline NaBH4 or O-glycanase (endo-alpha-N-acetylgalactosaminidase) revealed Gal(beta 1-3)GalNAc as the major type of the sugar chains. About half of the Thr + Ser residues in the glycoprotein were estimated as O-glycosidically linked with the disaccharide units.

[The tissue localization of "chitinoprotein", detectable by using specific antibodies, in the development of Drosophila melanogaster]. / Tkanevaia lokalizatsiia "khitinoproteina", vyiavliaemaia s pomoshch'iu spetsificheskikh antitel, v razvitii Drosophila melanogaster.

The biosynthesis of cognate glycoproteins with chitinase-sensitive carbohydrate moiety ("chitinoproteins") was detected after incubation of cultured cells of different insect species with 3H-glucosamine (Kramerov et al., Insect Biochem. v. 20; 769-775, 1990). It was also demonstrated that production of the specific chitinoprotein takes place during the development of D. melanogaster as revealed by immunoblotting and autoradiographic analysis of crude tissue extracts. An investigation of the developmental pattern of tissue localization of Drosophila chitinoprotein was performed using antibodies raised in rabbit after immunization with a purified preparation of the chitinoprotein (ChiP) from Drosophila embryonic cultured cells. The paraffin-embedded thin (5 microns) sections of organisms fixed in Bouin fixative were stained immunohistochemically with primary antibodies and peroxidase-conjugated secondary antibodies followed by enhancement of the precipitated DAB product with osmium tetroxide. Preimmune serum and antiserum preadsorbed with the purified ChiP preparation were used as negative controls yielding no specific staining of tissue sections. Negative staining with specific anti-ChiP antibodies was demonstrated for salivary glands, gut, muscles, central and peripheral nerve system and some other tissues. A complex pattern of tissue-specific ChiP localization in a variety of tissues of ectodermal, mesodermal and germ line origin was revealed. The mesodermal derivatives--hemocytes and oenocytes capable of producing the components of cuticle as well as epidermal cells of larvae and imago clearly demonstrated staining of cytoplasmic vesicles, which in the latter case were exocytosed and included into the newly formed endocuticle. Another cell type known to produce cuticle--epithelial cells of imaginal discs (primordia of adult organs)--were also stained with antibodies. One can suppose that ChiP is involved in biogenesis of insect cuticle, probably, as a protein precursor of chitin formation. It was quite surprising to observe a rather strong staining of fat body cells and follicle cells of adult ovaries. The follicular epithelium secreted the stained granules into a growing oocyte that accumulated large amounts of this immunopositive material until transformation into a mature egg. In an early embryo ChiP is localized in blastodermal and pole cells, but not in yolk. This is, probably, the result of segregation of ChiP to the periphery of an egg during the final stage of its maturation and subsequent cellularization in the beginning of embryogenesis. Later ChiP can be found in ectodermal cells and hemocyte-like cells. It should be noted that not all amounts of ChiP detected in embryos are maternally inherited, for an active ChiP biosynthesis takes place in dissociated embryonic cells after incubation with labelled sugar precursor.(ABSTRACT TRUNCATED AT 400 WORDS)

[Localization of glycoconjugates in the tissue structures of man and animals]. / Lokalizatsiia glikokon"iugatov v tkanevykh strukturakh cheloveka i zhivotnykh.

By means of concanavalin A (Con A) labelled with fluorescein isotiocyonate in cryostate sections of some human and animal organs (skin, heart, thymus) localization of clycoconjugates containing hexoses and reacting with this lectin has been studied. Glycoconjugates are revealed along the periphery of all cellular elements, in Z-lines and in intercalated disks of the myocardial muscle fibers, in the connective tissue ground substance and also in that of the basal membranes of epithelium, endothelium, muscle fibers (sarcolemma), in cytoplasm of epithelioreticulocytes of the thymus. In fibroblasts of the L-cells culture, glycoconjugates are also localized in cytoplasm. When N-acetylglucosamin is added to the labelled Con A solution, the ability of lectin to react with tissue components is inhibited.