Encoded in mammalian cells by 33 genes, the transforming growth factor-β (TGF-β) family of secreted, homodimeric and heterodimeric proteins controls the differentiation of most, if not all, cell lineages and many aspects of cell and tissue physiology in multicellular eukaryotes. Deregulation of TGF-β family signaling leads to developmental anomalies and disease, whereas enhanced TGF-β signaling contributes to cancer and fibrosis. Here, we review the fundamentals of the signaling mechanisms that are initiated upon TGF-β ligand binding to its cell surface receptors and the dependence of the signaling responses on input from and cooperation with other signaling pathways. We discuss how cells exquisitely control the functional presentation and activation of heteromeric receptor complexes of transmembrane, dual-specificity kinases and, thus, define their context-dependent responsiveness to ligands. We also introduce the mechanisms through which proteins called Smads act as intracellular effectors of ligand-induced gene expression responses and show that the specificity and impressive versatility of Smad signaling depend on cross-talk from other pathways. Last, we discuss how non-Smad signaling mechanisms, initiated by distinct ligand-activated receptor complexes, complement Smad signaling and thus contribute to cellular responses.

Increased activity of transforming growth factor-β (TGF-β), which binds to and stimulates cell surface receptors, contributes to cancer progression and fibrosis by driving epithelial cells toward a migratory mesenchymal phenotype and increasing the abundance of extracellular matrix proteins. The abundance of TGF-β receptors at the cell surface determines cellular responsiveness to TGF-β, which is often produced by the same cells that have the receptors, and thus serves as an autocrine signal. We found that Akt-mediated phosphorylation of AS160, a RabGAP [guanosine triphosphatase (GTPase)-activating protein], promoted the translocation of TGF-β receptors from intracellular stores to the plasma membrane of mouse embryonic fibroblasts and NMuMG epithelial cells. Consequently, insulin, which is commonly used to treat hyperglycemia and activates Akt signaling, increased the amount of TGF-β receptors at the cell surface, thereby enhancing TGF-β responsiveness. This insulin-induced increase in autocrine TGF-β signaling contributed to insulin-induced gene expression responses, attenuated the epithelial phenotype, and promoted the migration of NMuMG cells. Furthermore, the enhanced delivery of TGF-β receptors at the cell surface enabled insulin to increase TGF-β-induced gene responses. The enhancement of TGF-β responsiveness in response to Akt activation may help to explain the biological effects of insulin, the progression of cancers in which Akt is activated, and the increased incidence of fibroses in diabetes.

Insulin signaling governs many processes including glucose homeostasis and metabolism, and is therapeutically used to treat hyperglycemia in diabetes. We demonstrated that insulin-induced Akt activation enhances the sensitivity to TGF-β by directing an increase in cell surface TGF-β receptors from a pool of intracellular TGF-β receptors. Consequently, increased autocrine TGF-β signaling in response to insulin participates in insulin-induced angiogenic responses of endothelial cells. With TGF-β signaling controlling many cell responses, including differentiation and extracellular matrix deposition, and pathologically promoting fibrosis and cancer cell dissemination, we addressed to which extent autocrine TGF-β signaling participates in insulin-induced gene responses of human endothelial cells. Transcriptome analyses of the insulin response, in the absence or presence of a TGF-β receptor kinase inhibitor, revealed substantial positive and negative contributions of autocrine TGF-β signaling in insulin-responsive gene responses. Furthermore, insulin-induced responses of many genes depended on or resulted from autocrine TGF-β signaling. Our analyses also highlight extensive contributions of autocrine TGF-β signaling to basal gene expression in the absence of insulin, and identified many novel TGF-β-responsive genes. This data resource may aid in the appreciation of the roles of autocrine TGF-β signaling in normal physiological responses to insulin, and implications of therapeutic insulin usage.

Angiogenesis, the development of new blood vessels, is a key process in disease. We reported that insulin promotes translocation of transforming growth factor β (TGF-β) receptors to the plasma membrane of epithelial and fibroblast cells, thus enhancing TGF-β responsiveness. Since insulin promotes angiogenesis, we addressed whether increased autocrine TGF-β signaling participates in endothelial cell responses to insulin. We show that insulin enhances TGF-β responsiveness and autocrine TGF-β signaling in primary human endothelial cells, by inducing a rapid increase in cell surface TGF-β receptor levels. Autocrine TGF-β/Smad signaling contributed substantially to insulin-induced gene expression associated with angiogenesis, including TGF-β target genes encoding angiogenic mediators; was essential for endothelial cell migration; and participated in endothelial cell invasion and network formation. Blocking TGF-β signaling impaired insulin-induced microvessel outgrowth from neonatal aortic rings and modified insulin-stimulated blood vessel formation in zebrafish. We conclude that enhanced autocrine TGF-β signaling is integral to endothelial cell and angiogenic responses to insulin.

During epithelial-mesenchymal transition (EMT), reprogramming of gene expression is accompanied by histone modifications. Whether EMT-promoting signaling directs functional changes in histone methylation has not been established. We show here that the histone lysine methyltransferase SETDB1 represses EMT and that, during TGF-β-induced EMT, cells attenuate SETDB1 expression to relieve this inhibition. SETDB1 also controls stem cell generation, cancer cell motility, invasion, metastatic dissemination, as well as sensitivity to certain cancer drugs. These functions may explain the correlation of breast cancer patient survival with SETDB1 expression. At the molecular level, TGF-β induces SETDB1 recruitment by Smad3, to repress Smad3/4-activated transcription of SNAI1, encoding the EMT "master" transcription factor SNAIL1. Suppression of SNAIL1-mediated gene reprogramming by SETDB1 occurs through H3K9 methylation at the SNAI1 gene that represses its H3K9 acetylation imposed by activated Smad3/4 complexes. SETDB1 therefore defines a TGF-β-regulated balance between histone methylation and acetylation that controls EMT.

Rationale

α_v integrins, key regulators of transforming growth factor-β activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (α_vβ₆) and fibroblasts (α_vβ₁) in fibrotic lungs.

Objectives

We evaluated multiple α_v integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis.

Methods

Selective α_vβ₆ and α_vβ₁, dual α_vβ₆/α_vβ₁, and multi-α_v integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-β cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-β signaling. Bleomycin-challenged mice treated with dual α_vβ₆/α_vβ₁ integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation.

Measurements and main results

Inhibition of integrins α_vβ₆ and α_vβ₁ was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional α_v integrins. Antifibrotic efficacy of dual α_vβ₆/α_vβ₁ integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone.

Conclusions

In the fibrotic lung, dual inhibition of integrins α_vβ₆ and α_vβ₁ offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-β.