Both Smad and non-Smad signaling pathways are involved in TGFβ-mediated LOX induction, suggesting complex regulation of these important extracellular matrix cross-linking enzymes. Increased LOX activity may be at least partially responsible for TGFβ-mediated IOP elevation and increased aqueous humor outflow resistance.

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.

Gremlin employs canonical TGFβ2/Smad signaling to induce ECM genes and proteins in cultured human TM cells. Gremlin also induces both TGFβ2 and CTGF, which can act downstream to mediate some of these ECM changes in TM cells.

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 TGFβ signaling pathway activator TGFβ2 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 TGFβ/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFβ/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 5μg/ml TGFβ2 and/or 100ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFβ2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFβ2-induced TGFβ/Smad pathway activation (n=6, p<0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or β-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFβ2 and/or Wnt3a on the other pathway (n=6). Our results suggest the existence of a cross-inhibition between the TGFβ/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and β-catenin.

The TGFβ/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 TGFβ2/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 TGFβ/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.