TGF-ß as a driver of fibrosis: physiological roles and therapeutic opportunities.

Many chronic diseases are marked by fibrosis, which is defined by an abundance of activated fibroblasts and excessive deposition of extracellular matrix, resulting in loss of normal function of the affected organs. The initiation and progression of fibrosis are elaborated by pro-fibrotic cytokines, the most critical of which is transforming growth factor-ß1 (TGF-ß1). This review focuses on the fibrogenic roles of increased TGF-ß activities and underlying signaling mechanisms in the activated fibroblast population and other cell types that contribute to progression of fibrosis. Insight into these roles and mechanisms of TGF-ß as a universal driver of fibrosis has stimulated the development of therapeutic interventions to attenuate fibrosis progression, based on interference with TGF-ß signaling. Their promise in preclinical and clinical settings will be discussed. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß 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-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 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 αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 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 αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

Smad3-mediated recruitment of the methyltransferase SETDB1/ESET controls Snail1 expression and epithelial-mesenchymal transition.

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-ß-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-ß 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-ß-regulated balance between histone methylation and acetylation that controls EMT.

ShcA Protects against Epithelial-Mesenchymal Transition through Compartmentalized Inhibition of TGF-ß-Induced Smad Activation.

Epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-ß (TGF-ß) signaling, and TGF-ß drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-ß also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-ß type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-ß-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-ß receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-ß/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-ß receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-ß receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-ß signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-ß-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation.

The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.

Defects in ErbB-dependent establishment of adult melanocyte stem cells reveal independent origins for embryonic and regeneration melanocytes.

Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre-MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration.

Specificity, versatility, and control of TGF-ß family signaling.

Encoded in mammalian cells by 33 genes, the transforming growth factor-ß (TGF-ß) 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-ß family signaling leads to developmental anomalies and disease, whereas enhanced TGF-ß signaling contributes to cancer and fibrosis. Here, we review the fundamentals of the signaling mechanisms that are initiated upon TGF-ß 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.

Enhanced TGF-ß Signaling Contributes to the Insulin-Induced Angiogenic Responses of Endothelial Cells.

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

Integration of TGF-ß-induced Smad signaling in the insulin-induced transcriptional response in endothelial cells.

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-ß by directing an increase in cell surface TGF-ß receptors from a pool of intracellular TGF-ß receptors. Consequently, increased autocrine TGF-ß signaling in response to insulin participates in insulin-induced angiogenic responses of endothelial cells. With TGF-ß 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-ß 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-ß receptor kinase inhibitor, revealed substantial positive and negative contributions of autocrine TGF-ß signaling in insulin-responsive gene responses. Furthermore, insulin-induced responses of many genes depended on or resulted from autocrine TGF-ß signaling. Our analyses also highlight extensive contributions of autocrine TGF-ß signaling to basal gene expression in the absence of insulin, and identified many novel TGF-ß-responsive genes. This data resource may aid in the appreciation of the roles of autocrine TGF-ß signaling in normal physiological responses to insulin, and implications of therapeutic insulin usage.

Transforming Growth Factor-ß Receptors and Smads: Regulatory Complexity and Functional Versatility.

Transforming growth factor (TGF)-ß family proteins control cell physiology, proliferation, and growth, and direct cell differentiation, thus playing key roles in normal development and disease. The mechanisms of how TGF-ß family ligands interact with heteromeric complexes of cell surface receptors to then activate Smad signaling that directs changes in gene expression are often seen as established. Even though TGF-ß-induced Smad signaling may be seen as a linear signaling pathway with predictable outcomes, this pathway provides cells with a versatile means to induce different cellular responses. Fundamental questions remain as to how, at the molecular level, TGF-ß and TGF-ß family proteins activate the receptor complexes and induce a context-dependent diversity of cell responses. Among the areas of progress, we summarize new insights into how cells control TGF-ß responsiveness by controlling the TGF-ß receptors, and into the key roles and versatility of Smads in directing cell differentiation and cell fate selection.

Regulation of TGF-ß Receptors.

In cells responding to extracellular polypeptide ligands, regulatory mechanisms at the level of cell surface receptors are increasingly seen to define the nature of the ligand-induced signaling responses. Processes that govern the levels of receptors at the plasma membrane, including posttranslational modifications, are crucial to ensure receptor function and specify the downstream signals. Indeed, extracellular posttranslational modifications of the receptors help define stability and ligand binding, while intracellular modifications mediate interactions with signaling mediators and accessory proteins that help define the nature of the signaling response. The use of various molecular biology and biochemistry techniques, based on chemical crosslinking, e.g., biotin or radioactive labeling, immunofluorescence to label membrane receptors and flow cytometry, allows for quantification of changes of cell surface receptor presentation. Here, we discuss recent progress in our understanding of the regulation of TGF-ß receptors, i.e., the type I (TßRI) and type II (TßRII) TGF-ß receptors, and describe basic methods to identify and quantify TGF-ß cell surface receptors.

The insulin response integrates increased TGF-ß signaling through Akt-induced enhancement of cell surface delivery of TGF-ß receptors.

Increased activity of transforming growth factor-ß (TGF-ß), 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-ß receptors at the cell surface determines cellular responsiveness to TGF-ß, 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-ß 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-ß receptors at the cell surface, thereby enhancing TGF-ß responsiveness. This insulin-induced increase in autocrine TGF-ß 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-ß receptors at the cell surface enabled insulin to increase TGF-ß-induced gene responses. The enhancement of TGF-ß 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.

trpm7 regulation of in vivo cation homeostasis and kidney function involves stanniocalcin 1 and fgf23.

The transient receptor potential melastatin 7 (trpm7) channel kinase is a primary regulator of magnesium homeostasis in vitro. Here we show that trpm7 is an important regulator of cation homeostasis as well as kidney function in vivo. Using zebrafish trpm7 mutants, we show that early larvae exhibit reduced levels of both total magnesium and total calcium. Accompanying these deficits, we show that trpm7 mutants express higher levels of stanniocalcin 1 (stc1), a potent regulator of calcium homeostasis. Using transgenic overexpression and morpholino oligonucleotide knockdown, we demonstrate that stc1 modulates both calcium and magnesium levels in trpm7 mutants and in the wild type and that levels of these cations are restored to normal in trpm7 mutants when stc1 activity is blocked. Consistent with defects in both calcium and phosphate homeostasis, we further show that trpm7 mutants develop kidney stones by early larval stages and exhibit increased levels of the anti-hyperphosphatemic factor, fibroblast growth factor 23 (fgf23). Finally, we demonstrate that elevated fgf23 expression contributes to kidney stone formation by morpholino knockdown of fgf23 in trpm7 mutants. Together, these analyses reveal roles for trpm7 in regulating cation homeostasis and kidney function in vivo and implicate both stc1 and fgf23 in these processes.

Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation.

Vertebrate pigment cells are derived from neural crest cells and are a useful system for studying neural crest-derived traits during post-embryonic development. In zebrafish, neural crest-derived melanophores differentiate during embryogenesis to produce stripes in the early larva. Dramatic changes to the pigment pattern occur subsequently during the larva-to-adult transformation, or metamorphosis. At this time, embryonic melanophores are replaced by newly differentiating metamorphic melanophores that form the adult stripes. Mutants with normal embryonic/early larval pigment patterns but defective adult patterns identify factors required uniquely to establish, maintain or recruit the latent precursors to metamorphic melanophores. We show that one such mutant, picasso, lacks most metamorphic melanophores and results from mutations in the ErbB gene erbb3b, which encodes an EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB activities, we treated fish with pharmacological ErbB inhibitors and also knocked down erbb3b by morpholino injection. These analyses reveal an embryonic critical period for ErbB signaling in promoting later pigment pattern metamorphosis, despite the normal patterning of embryonic/early larval melanophores. We further demonstrate a peak requirement during neural crest migration that correlates with early defects in neural crest pathfinding and peripheral ganglion formation. Finally, we show that erbb3b activities are both autonomous and non-autonomous to the metamorphic melanophore lineage. These data identify a very early, embryonic, requirement for erbb3b in the development of much later metamorphic melanophores, and suggest complex modes by which ErbB signals promote adult pigment pattern development.

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes.

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.