Cell type and receptor identity regulate cholera toxin subunit B (CTB) internalization.

Cholera toxin (CT) is a secreted bacterial toxin that binds to glycoconjugate receptors on the surface of mammalian cells, enters mammalian cells through endocytic mechanisms and intoxicates mammalian cells by activating cytosolic adenylate cyclase. CT recognizes cell surface receptors through its B subunit (CTB). While the ganglioside GM1 has been historically described as the sole receptor, CTB is also capable of binding to fucosylated glycoconjugates, and fucosylated molecules have been shown to play a functional role in host cell intoxication by CT. Here, we use colonic epithelial and respiratory epithelial cell lines to examine how two types of CT receptors-gangliosides and fucosylated glycoconjugates-contribute to CTB internalization. We show that fucosylated glycoconjugates contribute to CTB binding to and internalization into host cells, even when the ganglioside GM1 is present. The contributions of the two classes of receptors to CTB internalization depend on cell type. Additionally, in a cell line that harbours both classes of receptors, gangliosides dictate the efficiency of CTB internalization. Together, the results lend support to the idea that fucosylated glycoconjugates play a functional role in CTB internalization, and suggest that CT internalization depends on both receptor identity and cell type.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

Crosstalk between TGFß and Wnt signaling pathways in the human trabecular meshwork.

Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFß signaling pathway activator TGFß2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFß/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFß/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 µg/ml TGFß2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFß2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFß2-induced TGFß/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or ß-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFß2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFß/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and ß-catenin.

A Novel Inhibitor of Topoisomerase I Is Selectively Toxic for a Subset of Non-Small Cell Lung Cancer Cell Lines.

SW044248, identified through a screen for chemicals that are selectively toxic for non-small cell lung cancer (NSCLC) cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive, cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was nontoxic to immortalized human bronchial cell lines. The acute transcriptional response to SW044248 in sensitive HCC4017 cells correlated significantly with inhibitors of topoisomerases and SW044248 inhibited topoisomerase 1 (Top1) but not topoisomerase 2. SW044248 inhibited Top1 differently from camptothecin and camptothecin did not show the same selective toxicity as SW044248. Elimination of Top1 by siRNA partially protected cells from SW044248, although removing Top1 was itself eventually toxic. Cells resistant to SW044248 responded to the compound by upregulating CDKN1A and siRNA to CDKN1A sensitized those cells to SW044248. Thus, at least part of the differential sensitivity of NSCLC cells to SW044248 is the ability to upregulate CDKN1A.

Gremlin utilizes canonical and non-canonical TGFß signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells.

The TGFß/BMP signaling pathways are involved in glaucomatous damage to the trabecular meshwork (TM) leading to elevated intraocular pressure (IOP), which is a major risk factor for the development and progression of glaucoma. The BMP antagonist gremlin is elevated in glaucomatous TM cells and tissues and can directly elevate IOP. Gremlin utilizes the TGFß2/SMAD pathway to induce TM extracellular matrix (ECM) proteins. The purpose of this study is to determine whether expression of the ECM cross-linking lysyl oxidase (LOX) genes is regulated by gremlin in cultured human TM cells. Human TM cells were treated with recombinant gremlin, and expression of the LOX genes was examined by quantitative RT-PCR and western immunoblotting. TM cells were pretreated with TGFBR inhibitors (LY364947 or SB431542), an inhibitor of the SMAD signaling pathway (SIS3), or with JNK (SP600125) and p38 MAPK (SB203580) inhibitors to identify the signaling pathway(s) involved in gremlin induction of LOX protein expression. All five LOX genes (LOX and LOXL1-4) were induced by gremlin. Gremlin induction of LOX genes and protein expression was blocked by TGFBR inhibitors as well as by inhibitors of the SMAD3, JNK and p38 MAPK signaling pathways. We conclude that gremlin employs both canonical TGFß/SMAD and the non-canonical JNK and p38 MAPK signaling pathways to induce LOX genes and proteins in cultured human TM cells. Increased LOX levels may be at least partially responsible for gremlin-mediated IOP elevation and increased aqueous humor outflow resistance leading to glaucoma.

Transforming growth factor-ß2 increases extracellular matrix proteins in optic nerve head cells via activation of the Smad signaling pathway.

PURPOSE: Transforming growth factor-ß2 (TGF-ß2) is associated with glaucomatous neuropathy, primarily via the increased synthesis and secretion of extracellular matrix (ECM) proteins and remodeling of the optic nerve head (ONH). Here, we investigated the signaling pathways used by TGF-ß2 to stimulate ECM expression by ONH astrocytes and lamina cribrosa (LC) cells. METHODS: TGF-ß2 localization and secretion was examined in human donor tissues and ONH astrocytes and LC cells. To examine TGF-ß2 signaling, ONH astrocytes and LC cells were treated with recombinant TGF-ß2, and phosphorylation of Smad and non-Smad signaling proteins were examined by western blot analyses and immunostaining. RESULTS: TGF-ß2 is significantly increased in the LC region of the ONH in glaucomatous eyes compared to age-matched normal eyes (n=4, p<0.0013). ONH astrocytes and LC cells secrete TGF-ß2, indicating that these cells may be an in vivo source of TGF-ß2 in the human ONH. In addition, treatment of ONH astrocytes and LC cells with exogenous TGF-ß2 increased ECM protein synthesis and secretion. With respect to TGF-ß2 signaling, recombinant TGF-ß2 induced phosphorylation of canonical signaling proteins Smad2/3 but did not alter phosphorylation of non-canonical signaling proteins extracellular signal-regulated kinases (ERK)1/2, p38, and c-Jun N-terminal kinases (JNK)1/2 proteins in ONH astrocytes and LC cells. Exogenous TGF-ß2 increased co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells further indicating activation of the canonical Smad signaling pathway. Furthermore, inhibition of TGF-ß I receptor activity by SB431542 or inhibition of Smad3 phosphorylation by SIS3 blocked TGF-ß2 stimulated ECM expression as well as activation of downstream canonical pathway signaling molecules. Knockdown of either Smad2 or Smad3 via small interfering RNA (siRNA) reduced TGF-ß2 stimulated ECM proteins in ONH astrocytes and LC cells. CONCLUSIONS: These studies indicate that TGF-ß2 utilizes the canonical Smad signaling pathway to stimulate ECM synthesis in human ONH cells. Our studies also indicate that pSmad2/3 is required for TGF-ß2 stimulation of ECM remodeling.

Role of TGFbeta/Smad signaling in gremlin induction of human trabecular meshwork extracellular matrix proteins.

PURPOSE: The bone morphogenic protein (BMP) antagonist gremlin is elevated in glaucomatous trabecular meshwork (TM) cells and tissues and elevates intraocular pressure (IOP). Gremlin also blocks BMP4 inhibition of transforming growth factor (TGF)-ß2 induction of TM extracellular matrix (ECM) proteins. The purpose of this study was to determine whether Gremlin regulates ECM proteins in cultured human TM cells. METHODS: Human TM cells were treated with recombinant gremlin to determine the effects on ECM gene and protein expression. Expression of the ECM genes FN, COL1, PAI1, and ELN was examined in cultured human TM cells by quantitative RT-PCR and Western immunoblot analysis. TM cells were pretreated with TGFBR inhibitors (LY364947, SB431542 or TGFBR1/TGFB2 siRNAs), inhibitors of the Smad signaling pathway (SIS3 or Smad2/3/4 siRNAs), or CTGF siRNA to identify the signaling pathway(s) involved in gremlin induction of ECM gene and protein expression. RESULTS: All ECM genes analyzed (FN, COL1, PAI1, and ELN) were induced by gremlin. This gremlin induction of ECM genes and protein expression was blocked by inhibitors of TGFBR and the canonical Smad2/3/4 and CTGF signaling pathways. CONCLUSIONS: Gremlin employs canonical TGFß2/Smad signaling to induce ECM genes and proteins in cultured human TM cells. Gremlin also induces both TGFß2 and CTGF, which can act downstream to mediate some of these ECM changes in TM cells.

Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells.

PURPOSE: The profibrotic cytokine TGFß is associated with glaucoma and plays an important role in the regulation of extracellular matrix metabolism in the trabecular meshwork (TM). The purpose of this study was to determine whether expression of ECM cross-linking LOX genes is regulated by TGFß in TM cells. METHODS: Expression of the five LOX genes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) was examined in cultured human TM cells by using RT-PCR, quantitative RT-PCR, and Western immunoblot analysis. TM cells were treated with recombinant TGFß1, -2, and -3, to determine the effects on LOX and LOXL1 to -4 expression. The TM cells were pretreated with TGFBR inhibitors (LY364947, SB431542), canonical Smad signaling pathway (SIS3 or Smad2, -3, and -4 siRNAs) inhibitors, or inhibitors of the non-Smad signaling pathways (SP600125, SR11302), to identify the signaling pathway(s) involved in TGFß induction of LOX and LOXL gene and protein expression. A novel LOX activity assay was used to determine the effects of the LOX inhibitor BAPN on tropoelastin cross-linking. RESULTS: All five LOX genes (LOX, LOXL1 to -4) were expressed in cultured human TM cells and were induced by all three isoforms of TGFß. This TGFß induction of LOX and LOXL expression was blocked by TGFß inhibitors as well as by inhibitors of the canonical Smad2, -3, and -4 signaling and non-Smad JNK/AP-1 signaling pathways (P < 0.05). CONCLUSIONS: Both Smad and non-Smad signaling pathways are involved in TGFß-mediated LOX induction, suggesting complex regulation of these important extracellular matrix cross-linking enzymes. Increased LOX activity may be at least partially responsible for TGFß-mediated IOP elevation and increased aqueous humor outflow resistance.