Porcupine is not required for the production of the majority of Wnts from primary human astrocytes and CD8+ T cells

Abstract

Wnts are small secreted glycoproteins that are highly conserved among species. To date, 19 Wnts have been described, which initiate a signal transduction cascade that is either β-catenin dependent or independent, culminating in the regulation of hundreds of target genes. Extracellular release of Wnts is dependent on lipidation of Wnts by porcupine, a membrane-bound-O-acyltransferase protein in the endoplasmic reticulum. Studies demonstrating the requirement of porcupine for Wnts production are based on cell line and non-human primary cells. We evaluated the requirement for porcupine for Wnts production in human primary astrocytes and CD8+ T cells. Using IWP-2, an inhibitor of porcupine, or siRNA targeting porcupine, we demonstrate that porcupine is not required for the release of Wnt 1, 3, 5b, 6,7a, 10b, and 16a. While IWP had no effect on Wnt 2b release, knockdown of porcupine by siRNA reduced Wnt 2b release by 60%. These data indicate that porcupine-mediated production of Wnts is context dependent and is not required for all Wnts production, suggesting that alternative mechanisms exist for Wnts production.

Figure 1. IWP-2 inhibits Wnt mediated β-catenin activity in mouse L-Wnt3a cells.

Mouse L-Wnt3a cells were cultured according to ATCC recommendations. Cells were then transfected with TOPflash or vector control and treated with Vehicle (DMSO), 5 μM IWP-2, or 10 μM IWP-2. Three days later a luciferase assay was performed and data was normalized to vector control. Data is representative of three separate experiments. *p≤0.05 in comparison to vehicle.

Figure 2. IWP-2 does not inhibit production of Wnts from human astrocytes.

PDAs were treated with 5 μM IWP-2 or vehicle (10 μM DMSO). Twenty-four hours later, the media was changed and the cells were treated again with 5 μM IWP-2 for two additional days. On the third day, the supernatant was collected and western blot was performed on 25 μl of supernatant to determine presence of Wnts 1 (A), 2b (B), 3(C), 5b (D), and 10b (E).

Figure 3. IWP-2 does not inhibit Wnt production from primary human CD8+ T cells.

CD8+ T cells were isolated from PBMCs from healthy donors by negative selection and subsequently activated with 1 μg each anti-CD3/anti-CD28 then propagated in presence of 100 units/ml IL-2 for three days. On the third day supernatant was collected and 25 μl was analyzed by western blot for presence of Wnt 1 (A), 3 (B), 6 (C), 7a (D), 10a (E), and 16a (F).

Figure 4. Wnts secreted from HFAs treated with IWP-2 are functional.

HFAs were treated with 5 μM IWP-2 or vehicle (10 μM DMSO). Twenty-four hours later, the media was replaced with cDMEM and HFAs were treated with 5 uM IWP-2 each day for 2 days or vehicle. PBMCs were isolated from a healthy donor and activated overnight with 1 μg each anti-CD3/anti-CD28 and 100 units/ml IL-2. PBMCs were then transfected with either TOPflash or FOPflash, a vector control. PBMCs were then cultured with supernatant from IWP-2 or DMSO treated HFAs (ACM). Three days later, β-catenin activity was determined by luciferase assay. Data is representative of three PBMC donors. *p≤0.05.

Figure 5. PORCN is not essential for Wnt production by PDAs.

PDAs were transfected with smartpool ON TARGET plus siRNA specific for human porcupine or scrambled siRNA. Forty eight hours after transfection cells were lysed and analyzed by western blot to confirm knockdown of porcupine (A). Supernatant was collected from cells at 96 hours post-transfection and analyzed by western blot to determine production of Wnt ligands (B–F). Data is representative of three separate experiments, *p≤0.05.

Figure 6. Wnt/β-catenin activity is not altered by porcupine in U138-MG Cells.

U138 cells were transfected with either a scrambled siRNA and TOPflash or FOP, and either siRNA specific for porcupine and TOP flash or FOP. 72 hours later β-catenin activity was determined by luciferase assay. This was performed three times.