TGF-β1 stimulates human at₁ receptor expression in lung fibroblasts by cross talk between the Smad, p38 MAPK, JNK, and PI3K signaling pathways

Both angiotensin II (ANG II) and transforming growth factor-β1 (TGF-β1) are thought to be involved in mediating pulmonary fibrosis. Interactions between the reninangiotensin system (RAS) and TGF-β1 have been well documented, with most studies describing the effect of ANG II on TGF-β1 expression. However, recent gene expression profiling experiments demonstrated that the angiotensin II type 1 receptor (AT₁R) gene was a novel TGF-β1 target in human adult lung fibroblasts. In this report, we show that TGF-β1 augments human AT₁R (hAT₁R) steady-state mRNA and protein levels in a dose- and time-dependent manner in primary human fetal pulmonary fibroblasts (hPFBs). Nuclear run-on experiments demonstrate that TGF-β1 transcriptionally activates the hAT₁R gene and does not influence hAT₁R mRNA stability. Pharmacological inhibitors and specific siRNA knockdown experiments demonstrate that the TGF-β1 type 1 receptor (TβRI/ALK5), Smad2/3, and Smad4 are essential for TGF-β1-stimulated hAT₁R expression. Additional pharmacological inhibitor and small interference RNA experiments also demonstrated that p38 MAPK, JNK, and phosphatidylinositol 3-kinase (PI3K) signaling pathways are also involved in the TGF-β1-stimulated increase in hAT₁R density. Together, our results suggest an important role for cross talk among Smad, p38 MAPK, JNK, and PI3K pathways in mediating the augmented expression of hAT₁R following TGF-β1 treatment in hPFB. This study supports the hypothesis that a self-potentiating loop exists between the RAS and the TGF-β1 signaling pathways and suggests that ANG II and TGF-β1 may cooperate in the pathogenesis of pulmonary fibrosis. The synergy between these systems may require that both pathways be simultaneously inhibited to treat fibrotic lung disease.