Christopher Broxson
University of Florida
Objective: Cannabis use by patients with inflammatory bowel disease (IBD) has become increasingly popular. Cannabis improves symptoms but may increase the need for surgery in patients with Crohn’s disease (CD). Previously, our group has shown tetrahydrocannabinol (THC), a primary constituent of cannabis, inhibits pro-fibrotic gene expression in human intestinal myofibroblasts (hIMF). Here we studied the mechanism of the major fibrotic signaling pathway involving the TGF-1/Smad family of proteins.
Methods: hIMFs were stimulated with 5ng/ml transforming growth factor beta 1 (TGF) and pretreated with vehicle or 0.5-2µM THC for 24h. Cellular protein and mRNA were isolated, quantified, and analyzed by Western blot and qPCR and expressed as fold vehicle control. For analysis of phosphorylated proteins, the intensity of the phosphorylated protein was normalized by the intensity of the corresponding total protein and expressed as a ratio.
Results: As expected, stimulation with TGF increased gene expression of SMAD7 (fold increase; 4.69±0.15; p<0.0001). THC did not change SMAD7 mRNA levels suggesting that THC was not acting through the canonical SMAD2/3 pathway. This was confirmed at the protein level where TGF increased the ratio of phosphorylated SMAD2/3 protein (P-SMAD2/3) to total SMAD2/3 protein (3.06±0.09-fold; p=0.02). Pretreatment with 1µM THC did not prevent the increased P-SMAD2/3 levels, and THC treatment alone had insignificant effect on P-SMAD2/3 to total SMAD2/3 ratio (0.8±0.02-fold). To address a second pathway, TGF stimulation increased phosphorylated RAC-alpha serine/threonine-protein kinase (AKT) protein. Phosphorylated (P-AKT) to total AKT protein was increased 2.01±0.45-fold (p=0.15), however 24hr pretreatment with 1µM THC reduced P-AKT/AKT levels 0.78-±0.31-fold (p=0.15). THC alone also reduced the P-AKT/AKT to 0.87±0.27-fold.
Conclusion: THC did not alter SMAD7 mRNA or P-SMAD2/3 protein levels suggesting the canonical pathway is not responsible for its anti-fibrotic action. THC may mediate this effect through a SMAD-independent AKT pathway. In hIMF, an important cell type in CD, THC has potentially beneficial effects on fibrosis. It should be noted however, that aberrant AKT signaling is the underlying defect found in several pathologies (cancer, diabetes, cardiovascular disease) and AKT phosphorylates over 100 different substrates, therefore the potential impact of THC in patients could be substantial and not immediately apparent.
Co-authors: Ellen Zimmermann1
1University of Florida