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gap junctional intercellular communication (GJIC) exhibited by cells is highly regulated (Giaume and McCarthy, 1996). One mechanism for regulation is connexin phosphorylation, which has been extensively studied in cells coupled by Cx43 (Bruzzone et al., 1996; Goodenough et al., 1996). Cx43 phosphorylation is correlated with alterations in gap junction uncoupling (Warn-Cramer et al., 1998) and its dephosphorylation is correlated with either decreased GJIC (Oelze et al., 1995) or increased channel conductance (Moreno et al., 1994).
Immunohistochemistry of Cx43 in the monkey normal and glaucomatous optic nerve shows that Cx43 is associated with astrocyte membranes in the lamina cribrosa in normal ONH (Fig. 2A). In contrast Cx43 is intracellular in astrocytes in the glaucomatous ONH (Fig. 2B). Real time RT-PCR comparing primary cultures of astrocytes from normal and glaucomatous human eyes indicate that gene expression of GJA1 is higher in glaucomatous astrocytes compared to normal age matched controls (Fig. 2C). Next we examined the effects of elevated hydrostatic pressure on gap junction intracellular communication GJIC in human ONH astrocytes in vitro providing evidence that astrocytes decrease intercellular communication upon exposure to elevated HP, a mechanical stress (Fig. 3) (Malone et al., 2007). The data further demonstrates that activation of epidermal growth factor receptor (EGFR) mediates long-term phosphorylation of Cx43 on tyrosine in response to pressure (Fig. 4). Activation of EGFR by pressure induces phosphorylation of Cx43 on tyrosine residues and subsequent closure of gap junction communication between neighboring astrocytes. In the normal optic nerve, astrocyte GJIC is maintained so that astrocyte– astrocyte cellular coupling in the synctitium is continuous, allowing propagation of extracellular cues to obtain coordinated responses. Under exposure to elevated pressure in vitro and perhaps in vivo, gap junctions close via activation of the EGFR pathway. Closure of gap junctions will interrupt the continuity of astrocyte intercellular communication causing loss of cell–cell contact and loss of homeostatic regulation. Under these conditions, astrocytes may adopt the reactive
astrocyte phenotype characteristic of response to injury in the CNS. In summary, if astrocyte– astrocyte gap junction communication is interrupted in vivo by chronic elevated IOP, as in glaucoma, the homeostasis of the RGC nonmyelinated axons may be altered and the blood nerve barrier may be impaired leading to axonal loss and optic disc remodeling characteristic of glaucomatous neuropathy.
Signal transduction in glaucomatous astrocytes
Protein tyrosine kinases (PTKs)
PTKs are the primary mediators of the signaling network that transmit extracellular signals into the cell. PTK signaling activates several small G proteins, including Ras, Rap-1, and the cdc42- Rac-Rho family, as well as pathways regulated by mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. Receptor-type PTKs are activated by ligand binding and directly transduce the extracellular information into intracellular tyrosine phosphorylation events, whereas nonreceptor tyrosine kinases (nrPTK) function as signal transducers in concert with receptor-like molecules that lack tyrosine kinase activity. Members of the receptor PTK family include the EGFR (Wieduwilt and Moasser, 2008), the FGF receptor (FGFR) (Rusnati and Presta, 2007), the PDGF receptor (PDGFR) (Fruttiger et al., 1996), neurotrophin receptors (Lykissas et al., 2007), and ephrin receptors (Himanen et al., 2007). NrPTKs include Src, Fyn, TEC, TXK, JAK1-3, and FAK. In mouse brain astrocytes, members of Src family tyrosine regulate cell adhesion via a FAK-depen- dent mechanism (Beggs et al., 2003). Among many stimuli, PTKs are activated by mechanical stress (Sawada et al., 2006). Inhibitors of PTKs, such as genistein, block the proliferation through an autocrine response of release of soluble factors in response to transmural compression in astrocytoma cells. Recently, Liu and Neufeld (2007) demonstrated that phosphorylation of EGFR is a necessary step for induction of nitric oxide synthase (iNOS) in astrocytes exposed to HP.
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Fig. 2. Expression of connexin-43 in the monkey optic nerve head. (A) Immunofluorescent staining for GFAP shows astrocytes (green) forming lamellae in the cribriform plates of the lamina cribrosa (CP) stained with Cx43 (red). Fine lines of colocalization of Cx43 on astrocytes membranes (yellow, arrows), V: blood vessel, NB: nerve bundles. Inset: Cx43 immunoreactivity following the fine lamellar processes of astrocytes in the normal lamina cribrosa. (B) In the contralateral eye with experimental glaucoma, astrocytes (green) are disorganized and appear rounded with intracellular Cx43 immunoreactivity (yellow, arrows) in the lamina cribrosa. A and B: Magnification bar ¼ 35 mm. For methods see (Agapova et al., 2006a). (C) Relative amount of Cx43 (GJA1) mRNA in human normal and glaucomatous ONH astrocytes measured by quantitative RT-PCR. Bar graphs represent relative expression of Cx43 mRNA normalized to 18S in normal (n ¼ 8) and glaucomatous (n ¼ 8) ONH astrocyte cultures. Two-tailed t test was used. Indicates po0.0002. (See Colour Plate 25.2 in the colour plate section.)
Serine/threonine protein mitogen-activated kinases (MAPKs)
MAPKs phosphorylate specific serines and threonines of target proteins and regulate gene
expression, mitosis, movement, metabolism, and programmed cell death. MAPK-catalyzed phosphorylation of proteins functions as a switch to turn on or off activity. Substrates include other protein kinases, phospholipases, transcription
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Fig. 3. Effects of hydrostatic pressure (HP) on GJIC measured by the SLDT technique. Fluorescent micrographs of cultured ONH astrocytes exposed to (A) CP or (B) 30 min, (C) 90 min, (D) 3 h, (E) 6 h, and (F) 24 h HP conditions. (G) GJIC in control astrocytes exposed to CP. (H) Carbenoxolone (CBX) inhibited GJIC in ONH astrocytes exposed to the same conditions (Magnification bar ¼ 30 mm). (I) Exposure to HP induces a decrease in astrocyte GJIC determined by the SLDT technique. Quantitation of the LY fluorescent area was determined using OPTIMAS software and expressed as a percentage of the control (100%) exposed to CP (solid bar) and to HP (gray bar) for 30 min, 90 min, 3 h, 6 h, and 24 h (mean7standard deviation). HP induced a decrease in GJIC by 3677, 5678, 5774, 7177, and 78%79, respectively, compared to controls. Indicates statistical significance at po0.05 from three independent experiments.
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Fig. 4. EGFR mediates GJIC and Cx43 tyrosine phosphorylation in response to HP. (A) GJIC analysis of ONH astrocytes under HP conditions with AG1478 (3 nM), an EGFR inhibitor, results in complete opening of gap junctions from 0–6 h compared to astrocytes exposed to HP without the inhibitor ( po0.05 from three different experiments). (B) Micrographs of a scratch assay in astrocytes exposed to HP for 90 min with (a) and without (b) AG1478 demonstrate reversal of inhibition of GJIC due to HP in the presence of EGFR inhibitor. (C) Immunoblot analysis of Cx43 immunoprecipitates from ONH astrocytes exposed to HP with and without AG1478. Astrocytes were preincubated with AG1478 for 30 min and protein lysates were collected at 30 min, 90 min, 3 h, and 6 h. Lysates from untreated controls were collected at time 0. Immunoblots on Cx43 immunoprecipitates using phosphotyrosine antibody show a unique band of Cx43, which disappears in the presence of AG1478, indicating tyrosine phosphorylation of Cx43 under HP. (D) Immunoblot with monoclonal anti-Cx43 (BD) on immunoprecipitates of polyclonal anti-Cx43 (Sigma) showed an increase in Cx43 in astrocytes exposed to HP. Ag1478 treatment did not inhibit the increase in Cx43 under pressure. (E) Western blot of phosphorylated EGFR (pEGFR) from astrocytes lysates exposed to HP with and without AG1478. Note that a single band at 175 kDa corresponding to pEGFR appears in lysates from ONH astrocytes exposed to HP, which disappears in the presence of AG1478. Total EGFR levels were not affected. (F) Upper blots: detection of EGFR in Cx43 immunoprecipitates using a polyclonal antibody against human EGFR (Cell Signaling #2232). Note a band at 175 kDa corresponding to EGFR associated with Cx43 that appears in samples exposed to HP. Lower blots: detection of Cx43 in EGFR immunoprecipitates using a polyclonal antibody against human Cx43 (Sigma). Note a darker band at 43 kDa corresponding to Cx43 associated with EGFR in samples exposed to HP for 90 min (from Malone et al., 2007).
factors, and cytoskeletal proteins. There are three well-characterized subfamilies of MAPKs: extracellular signal-regulated kinases, ERK1 and ERK2; c-Jun NH2-terminal kinases, JNK-1, JNK-2, and JNK-3; and the four p38 enzymes — p38a, p38b, p38g, and p38d. Studies of tissue samples from
patients with various CNS diseases have demonstrated activation of the ERK/MAPK pathway, suggesting a role for the triggering and/or persistence of reactive astrogliosis (Tichauer et al., 2007). Mechanical trauma induces rapid activation of ERK/MAPK pathway in astrocytes in vitro