Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1β, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1β were detected within 6 h and peaked at 12–24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1−/−Casp4(11)del, Panx1−/− and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas.

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High levels of the Met tyrosine kinase receptor expression are associated with metastatic disease. Met activation by hepatocyte growth factor (HGF) is associated with decreased E-cadherin-dependent cell-cell contacts. The molecular mechanism underlying this process remains unclear. To better understand the relationship between E-cadherin and Met, we assessed Met localization in cells which form mature E-cadherin-dependent adhesion HT-29 and cells which have lost E-cadherin expression BT-549. Met colocalized with E-cadherin at the site of cell-cell adhesion in HT-29 cells, but Met was distributed in an intracellular compartment in BT-549 cells. Forced expression of E-cadherin in BT-549 cells recruited Met to the membrane. Cross-linking studies suggested that Met and E-cadherin interact in the extracellular domain in HT-29 cells. This is the first evidence of a physical interaction between Met and E-cadherin. We suggest that this receptor/cadherin pairing may be a mechanism for cellular presentation of receptors in a manner that localizes them optimally for interaction with ligand.

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Functional state of internalized epidermal growth factor (EGF) receptor in A431 cells has been studied. The use of photoaffinity [12"I]EGF derivative allowed us to establish that inside the cell the EGF retains its connection with the receptor. With the help of polyclonal antibodies to phosphotyrosine, it has been shown that EGF-receptor complexes maintain their phosphorylated state during internalization. The internalized EGF receptor kinase as well as that localized in the plasma membrane appeared to be able to phosphorylate synthetic peptide substrate introduced into the cell.The epidermal growth factor (EGF) receptor is a 170-kilodalton (kDa) integral membrane glycoprotein containing cytosol-oriented tyrosine-specific kinase (for a review, see reference 18). EGF binding stimulates receptor tyrosine kinase, resulting in both receptor and substrate phosphorylation (for a review, see reference 10). There is a lot of data showing that receptor kinase activity plays an important role in the transmission of mitogenic signal (for a review, see references 20 and 25).After EGF binding, the ligand-receptor complexes are rapidly internalized; they are then delivered to various endosomal structures and, finally, degraded within lysosomes (8,9,12,21,22). The role of the internalization process in triggering the biochemical reactions leading to DNA synthesis so far remains obscure. To elucidate this question, the functional state of internalized EGF receptor should be determined.Whether the EGF receptors retain their functional activity during the translocation from plasma membrane into endosomal compartment is yet to be clarified. Thus, the presence of active EGF receptor in the fractions of intracellular components isolated from homogenates of A-431 cells (5) and rat liver cells (13) has been shown. On the other hand, from the results of experiments on intact A-431 cells, Sturani and co-workers concluded that internalized EGF receptors failed to maintain the phosphorylated state of tyrosine residues (24). In this study, we compared the functional state of internalized and membrane-localized EGF-receptor complexes by using two experimental approaches: (i) cell exposure to EGF at low temperature, which is known to completely block internalization (1) and (ii) removal of surface-bound EGF by cell treatment with weak acid buffer (50 mM sodium acetate [pH 4.5 to 5.0] containing 150 mM NaCl). To identify phosphotyrosine-containing proteins, we have used affinity-purified polyclonal antibody to phosphotyrosine (anti-P-Tyr). As has been established previously, the major phosphoprotein interacting with this antibody in the EGF-stimulated A-431 cells is the EGF receptor (17).Effect After that, the cells were solubilized and two equivalent portions of each extract were analyzed. One of them was immunoprecipitated with anti-P-Tyr, and then both the immunoprecipitate and the total extract were processed by electrophoresis. Radioactivity corresponding to 175-kDa bands and total cell-associated radioactivity were measured. The immunop...

Overexpression of the HGF (hepatocyte growth factor) receptor Met is correlated with metastasis and poor outcome in cancer. Upon HGF binding, Met mediates multiple cellular processes, including mitogenesis, motility, and morphogenesis[1]. Met and HGF likely play a key role in regulating many aspects of embryonic development, including epithelial-mesenchymal transition[2], migration of muscle cells[3], and kidney and mammary gland formation[4,5]. Met signaling also facilitates wound healing and tissue regeneration[6]. Met oncogenic activation was shown to play a significant role in promoting tumor cell invasion and metastasis[7]. In vitro, activation of Met by HGF induces disassembly of the cell-cell contacts, followed by cell migration[8]. E-cadherin is the central protein of adhesion complexes in epithelial cells. E-cadherin is a transmembrane protein whose cytoplasmic domains interact with catenins to connect to the cytoskeleton[9]. However, the mechanism involved in down-regulation of E-cadherin function by Met is poorly understood. One important question that has not been resolved is whether E-cadherin and Met have physically interacted at the plasma membrane. There are only a few data suggesting that Met might tightly interact with the E-cadherin-dependent adhesion complex. Met has been shown to be selectively localized at the basolateral area of polarized epithelial cells in vivo[10]. Also, Met and E-cadherin were found colocalized on the membrane of human carcinoma cells in vitro[11,12]. Moreover, in MDCK cells, both proteins were colocalized at the cell-cell adhesion site and underwent coendocytosis upon HGF stimulation[13]. We have now shown that Met is tightly associated with E-cadherin at the site of cell-cell contacts[14]. It appears that the interaction of Met and E-cadherin occurs by direct binding in their extracellular domains as part of the process of maturation of adhesive junctions. The cadherin-mediated cell adhesion divided the plasma membrane into apical and basolateral domains that have different protein compositions[15]. Recently, new data concerning the dynamics of plasma membrane proteins in epithelial cells were published. It was demonstrated that proteins that are normally localized at the plasma membrane in polarized epithelial cells distributed to intacellular compartments when cell polarity is lost or not yet established because of redirection of plasma membrane transport pathways into intracellular vesicles[16,17]. Interestingly, such membrane markers like E-cadherin, β-catenin, occludin, JAM-1, Na/K-ATPase, and syntaxins were accumulated in the unique intracellular storage compartment upon destruction of cell-cell adhesion[18]. Our work demonstrated that E-cadherin-negative BT-549 cells delivered Met to an intracellular location. However, forced expression of E-cadherin restored cell-cell contacts and recruited Met to the membrane.