Crosstalk between TGFβ and Wnt signaling pathways in the human trabecular meshwork

Abstract

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.

Impact of Wnt signals on human intervertebral disc cell regeneration

“…Another recent study has provided evidence that several non‐canonical signals in the liver, including WNT5A, can act as antagonists to Wnt3A‐induced β‐catenin/T‐cell factor (TCF) signaling to promote tissue differentiation, suggesting non‐canonical signals can “fine‐tune” the effects of canonical Wnt signaling . Additionally, studies which can uncover further regulatory intricacies and the crosstalk between canonical and non‐canonical Wnt pathways would be beneficial; for example, recent studies have demonstrated that canonical suppression can occur through protein tyrosine kinase 7 (PTK7), as well as crosstalk with the Smad/TGF‐β pathway, known to promote differentiation.…”

Proliferation control of kidney interstitial cells

“…In summary, the histological features that we observed are 1 consistent with CD compression causing increased luminal pressure in the nephron with 2 associated sclerosis of the glomerulus. 3 Loss of Smad4 affects nuclear accumulation of Smad3 but not Smad1/5/8 4 To understand if loss of Smad4 affects nuclear accumulation of R-Smads in regions of medullary 5 interstitial cell expansion, protein localization of Smads was compared in Smad4 con and Smad4 IC 6 mouse kidneys. To localize expression in interstitial cells, we co-stained with aSMA.…”

“…Single cells were washed 2X 1 with ARB to remove residual Digest 2 then incubated with PE-conjugated mouse anti-CD31, 2 anti-CD45, anti-CD326, anti-Ter119 (1:10; Miltenyi) in ARB for 15 min on a nutator in the dark. 3 Cells were washed with ARB then incubated with anti-PE microbeads (1:5; Miltenyi) for 15 min 4 on a nutator in the dark followed by another wash with ARB. Labeled cells were subject to 5 magnetic-activated cell sorting (MACS) and the unlabeled (negative) cellular fraction was 6 cultured in DMEM; 10% FBS; 1% penicillin/strep.…”

“…Interstitial cells are any cells located between the functional cells of a tissue. In the kidney, cells 2 of the nephron and blood vessel are considered the functional components and the interstitial cell 3 population is made up largely of PDGFRb-expressing fibroblasts. These cells derive from the RESULTS 1 2 To understand the role of TGFβ/BMP in the developing renal interstitium, Smad4 was 3 inactivated in interstitial cell progenitors using Foxd1 Cre .…”

“…Sections 21 were incubated in 1.3% triethalamine;0.375% acetic anhydride for 10 minutes, rinsed three times 22 with PBS then incubated in hybridization buffer for 2hr. DIG-labeled riboprobes were diluted to 23 500ng/ml in hybridization buffer (50% formamide, 5X SSC pH 4.5, 1% SDS, 50ug/ml yeast 24 tRNA, 50ug/ml heparin) and sections were hybridized in a humidified chamber at 68°C In vivo proliferation analysis 3 One EdU pulse (20mg/kg) was administered to P0 pups by intraperitoneal injection. After 2hr, 4 kidneys were dissected on ice and fixed in 4% PFA for 30 min.…”