ggti-286 and farnesyl-pyrophosphate

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.. We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).. Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.. We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Topics: Adult; Airway Remodeling; Alkyl and Aryl Transferases; Asthma; Bronchi; Case-Control Studies; Cells, Cultured; Dose-Response Relationship, Drug; Farnesyltranstransferase; Fibroblasts; Fibronectins; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leucine; Methionine; Mevalonic Acid; Polyisoprenyl Phosphates; Sesquiterpenes; Simvastatin; Time Factors; Transforming Growth Factor beta1; Young Adult

Mevalonate depletion by inhibition of hydroxymethylglutaryl coenzyme A reductase impairs post-translational processing of Ras and Ras-related proteins. We have previously shown that this mevalonate depletion also leads to the upregulation of Ras, Rap1a, RhoA, and RhoB. This upregulation may result from global inhibition of isoprenylation or depletion of key regulatory isoprenoid species. Studies utilizing specific isoprenoid pyrophosphates in mevalonate-depleted cells reveal that farnesyl pyrophosphate (FPP) restores Ras processing and prevents RhoB upregulation while geranylgeranyl pyrophosphate (GGPP) restores Rap1a processing and prevents RhoA and RhoB upregulation. Either FPP or GGPP completely prevents lovastatin-induced upregulation of RhoB mRNA. Inhibition of FPP or squalene synthase allowed for the further identification of the putative regulatory species. Studies involving the specific isoprenyl transferase inhibitors FTI-277 and GGTI-286 demonstrate that selective inhibition of protein isoprenylation does not mimic lovastatin's ability to increase Ras and RhoA synthesis, decrease Ras and RhoA degradation, increase RhoB mRNA, or increase total levels of Ras, Rap1a, RhoA, and RhoB. In aggregate, these findings reveal a novel role and mechanism for isoprenoids to influence levels of Ras and Ras-related proteins.

Topics: Antineoplastic Agents; Blotting, Northern; Blotting, Western; Cycloheximide; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; K562 Cells; Leucine; Lovastatin; Methionine; Mevalonic Acid; Polyisoprenyl Phosphates; Protein Biosynthesis; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; rap1 GTP-Binding Proteins; ras Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA, Messenger; Sesquiterpenes; Time Factors; Transcription, Genetic; Up-Regulation