TGF-β Signaling via TAK1 Pathway: Role in Kidney Fibrosis

In progressive kidney diseases, fibrosis represents the common pathway to end-stage kidney failure. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that has been established as a central mediator of kidney fibrosis. Emerging evidence shows a complex scheme of signaling networks that enable multifunctionality of TGF-β1 actions. Specific targeting of the TGF-β signaling pathway is seemingly critical and an attractive molecular therapeutic strategy. TGF-β1 signals through the interaction of type I and type II receptors to activate distinct intracellular pathways involving the Smad and the non-Smad. The Smad signaling axis is known as the canonical pathway induced by TGF-β1. Importantly, recent investigations have shown that TGF-β1 also induces various non-Smad signaling pathways. In this review, we focus on current insights into the mechanism and function of the Smad-independent signaling pathway via TGF-β-activated kinase 1 and its role in mediating the profibrotic effects of TGF-β1.     

Crosstalk of TGF-β and Estrogen Receptor Signaling in Breast Cancer

Estrogen receptor-α (ERα) and transforming growth factor (TGF)-β signaling pathways are major regulators during mammary gland development, function and tumorigenesis. Predominantly, they have opposing roles in proliferation and apoptosis. While ERα signaling supports growth and differentiation and is antiapoptotic, mammary gland epithelia cells are very sensitive to TGF-β—induced cell cycle arrest and apoptosis. Their regulatory pathways intersect, and ERα blocks TGF-β pathway by multiple means, including direct interactions of its signaling components, Smads. However, relatively little is known of the dysfunction of their interactions in cancer. A better understanding would help to develop new strategies for breast cancer treatment.     

TGF-β Signaling in Breast Cancer Cell Invasion and Bone Metastasis

The contribution of transforming growth factor β (TGF-β) signaling to breast cancer has been studied for more than two decades. In an early phase TGF-β may act as a tumour suppressor, while later, when cells have become resistant to its anti-mitogenic effects, the role of TGF-β switches towards malignant conversion and progression. TGF-β stimulates cell invasion and modifies the microenvironment to the advantage of cancer cells. Studies have shown that TGF-β promotes bone and lung metastasis via different mechanisms. The therapeutic strategies to target the TGF-β pathway in breast cancer are becoming increasingly clear. This review will focus on the role TGF-β in breast cancer invasion and metastasis.     

BAMBI Elimination Enhances Alternative TGF-β Signaling and Glomerular Dysfunction in Diabetic Mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI^−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI^−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI^+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI^−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI^−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI^+/+ or BAMBI^−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI^−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.     

Noncanonical TGF-β Signaling During Mammary Tumorigenesis

Breast cancer is a heterogeneous disease comprised of at least five major tumor subtypes that coalesce as the second leading cause of cancer death in women in the United States. Although metastasis clearly represents the most lethal characteristic of breast cancer, our understanding of the molecular mechanisms that govern this event remains inadequate. Clinically, ~30% of breast cancer patients diagnosed with early-stage disease undergo metastatic progression, an event that (a) severely limits treatment options, (b) typically results in chemoresistance and low response rates, and (c) greatly contributes to aggressive relapses and dismal survival rates. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that regulates all phases of postnatal mammary gland development, including branching morphogenesis, lactation, and involution. TGF-β also plays a prominent role in suppressing mammary tumorigenesis by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Genetic and epigenetic events that transpire during mammary tumorigenesis conspire to circumvent the tumor suppressing activities of TGF-β, thereby permitting late-stage breast cancer cells to acquire invasive and metastatic phenotypes in response to TGF-β. Metastatic progression stimulated by TGF-β also relies on its ability to induce epithelial-mesenchymal transition (EMT) and the expansion of chemoresistant breast cancer stem cells. Precisely how this metamorphosis in TGF-β function comes about remains incompletely understood; however, recent findings indicate that the initiation of oncogenic TGF-β activity is contingent upon imbalances between its canonical and noncanonical signaling systems. Here we review the molecular and cellular contributions of noncanonical TGF-β effectors to mammary tumorigenesis and metastatic progression.     

How Does TGF-β Mediate Tubulointerstitial Fibrosis?

Tubulointerstitial fibrosis mediates the development of end-stage renal disease from renal injuries of all etiologies and is considered an important predictor of renal survival. Transforming growth factor-β (TGF-β) is one of the most important growth factors that promotes tubulointerstitial fibrosis, but the mechanisms whereby this occurs are not well defined. This is because TGF-β has pleiotropic effects that depend on the target cell type. This review discusses how TGF-β signaling in each of the relevant cell types (eg, tubular epithelium, fibroblasts) may contribute to tubulointerstitial fibrosis progression and suggests ways in which future research can improve our understanding of TGF-β-mediated tubulointerstitial fibrosis.     

IFT80 Is Required for Fracture Healing Through Controlling the Regulation of TGF-β Signaling in Chondrocyte Differentiation and Function

Primary cilia are essential cellular organelles that are anchored at the cell surface membrane to sense and transduce signaling. Intraflagellar transport (IFT) proteins are indispensable for cilia formation and function. Although major advances in understanding the roles of these proteins in bone development have been made, the mechanisms by which IFT proteins regulate bone repair have not been identified. We investigated the role of the IFT80 protein in chondrocytes during fracture healing by creating femoral fractures in mice with conditional deletion of IFT80 in chondrocytes utilizing tamoxifen inducible Col2α1-CreER mice. Col2α1^creIFT80^f/f mice had smaller fracture calluses than IFT80^f/f (control) mice. The max-width and max-callus area were 31% and 48% smaller than those of the control mice, respectively. Col2α1^creIFT80^f/f mice formed low-density/porous woven bony tissue with significantly lower ratio of bone volume, Trabecular (Tb) number and Tb thickness, and greater Tb spacing compared to control mice. IFT80 deletion significantly downregulated the expression of angiogenesis markers-VEGF, PDGF and angiopoietin and inhibited fracture callus vascularization. Mechanistically, loss of IFT80 in chondrocytes resulted in a decrease in cilia formation and chondrocyte proliferation rate in fracture callus compared to the control mice. Meanwhile, IFT80 deletion downregulated the TGF-β signaling pathway by inhibiting the expression of TGF-βI, TGF-βR, and phosphorylation of Smad2/3 in the fracture callus. In primary chondrocyte cultures in vitro, IFT80 deletion dramatically reduced chondrocyte proliferation, cilia assembly, and chondrogenic gene expression and differentiation. Collectively, our findings demonstrate that IFT80 and primary cilia play an essential role in fracture healing, likely through controlling chondrocyte proliferation and differentiation, and the TGF-β signaling pathway. © 2019 American Society for Bone and Mineral Research.     

Autophagy Regulates TGF-β Expression and Suppresses Kidney Fibrosis Induced by Unilateral Ureteral Obstruction

Autophagy is an evolutionarily conserved process that cells use to degrade and recycle cellular proteins and remove damaged organelles. During the past decade, there has been a growing interest in defining the basic cellular mechanism of autophagy and its roles in health and disease. However, the functional role of autophagy in kidney fibrosis remains poorly understood. Here, using GFP-LC3 transgenic mice, we show that autophagy is induced in renal tubular epithelial cells (RTECs) of obstructed kidneys after unilateral ureteral obstruction (UUO). Deletion of LC3B (LC3^−/− mice) resulted in increased collagen deposition and increased mature profibrotic factor TGF-β levels in obstructed kidneys. Beclin 1 heterozygous (beclin 1^+/−) mice also displayed increased collagen deposition in the obstructed kidneys after UUO. We also show that TGF-β1 induces autophagy in primary mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. LC3 deficiency resulted in increased levels of mature TGF-β in primary RTECs. Under conditions of TGF-β1 stimulation and autoinduction, inhibition of autolysosomal protein degradation by bafilomycin A1 increased mature TGF-β protein levels without alterations in TGF-β1 mRNA. These data suggest a novel intracellular mechanism by which mature TGF-β1 protein levels may be regulated in RTECs through autophagic degradation, which suppresses kidney fibrosis induced by UUO. The dual functions of TGF-β1, as an inducer of TGF-β1 autoinduction and an inducer of autophagy and TGF-β degradation, underscore the multifunctionality of TGF-β1.In the kidney, fibrosis is responsible for chronic progressive kidney failure, and the prevalence of CKD is increasing worldwide.^1,2 Extracellular matrix (ECM) protein production and progressive accumulation are hallmarks of renal tubulointerstitial fibrosis in progressive kidney disease. Collagens are the main components of the ECM in the kidney, and type I collagen (Col-I) is the major type associated with disease states.^3,4 The cellular mechanisms that facilitate tubulointerstitial fibrosis after injury remain incompletely defined. Recent lineage tracing or genetic fate mapping studies have strongly challenged the theory that renal tubular epithelial cells (RTECs) traverse the tubular basement membrane to become myofibroblasts in a process of epithelial-to-mesenchymal transition (EMT), but rather, that interstitial pericytes/perivascular fibroblasts are the myofibroblast progenitor cells.^5–7 It also has been proposed that profibrotic factors, such as TGF-β1, are upregulated in the tubular interstitial area on injury, leading to kidney fibrosis.⁸ TGF-β1 induces production of ECM proteins, including fibronectin and collagens, and inhibits degradation of ECM proteins mainly by matrix metalloproteinases.^9–11 Given the recent evidence that casts doubts about the role of EMT in vivo, how RTECs contribute to the development of renal tubulointerstitial fibrosis is not entirely clear.TGF-β is synthesized as a single polypeptide precursor that includes a preregion signal peptide, which is removed by proteolytic cleavage, and pro–TGF-β, containing a proregion called the latency-associated peptide and a mature TGF-β, and it converts to homodimeric pro–TGF-β through disulfide bonds.¹² After cleavage by proprotein convertases, such as furin, latency-associated peptide remains noncovalently associated with the dimeric form of mature TGF-β as the small latent complex (SLC).¹³ SLC formation occurs in the Golgi apparatus, and mature TGF-β is secreted as part of SLC and associated with latent TGF-β–binding protein to form TGF-β large latent complex, which interacts with ECM. On stimulus, the dimeric form of mature TGF-β is dissociated from large latent complex and becomes the bioactive mature TGF-β ligand, which can then bind TGF-β receptors to trigger downstream Smad-dependent or -independent signaling pathways.^12,13 Thus, the availability of mature TGF-β is the limiting factor of TGF-β activity and not TGF-β synthesis per se, because the body generates more pro–TGF-β than necessary. Whereas TGF-β/TGF-β receptor downstream signaling pathways have been extensively investigated, the regulation of TGF-β maturation and bioavailability has not been well studied but may serve as an important target for fibrotic diseases that alter TGF-β signaling.Macroautophagy, hereafter referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. The process of autophagy involves the formation of double membrane–bound vesicles called autophagosomes that envelop and sequester cytoplasmic components, including macromolecular aggregates and cellular organelles, for bulk degradation by a lysosomal degradative pathway.¹⁴ Autophagy can be induced in response to either intracellular or extracellular factors, such as amino acid or growth factor deprivation, hypoxia, low cellular energy state, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection.^15–22 To date, over 30 genes involved in autophagy have been identified in yeast, and they have been termed autophagy-related genes (Atgs). The mammalian ortholog of Atg8 is comprised of a family of proteins known as microtubule-associated protein 1 light chain 3 (LC3) that functions as a structural component in the formation of autophagosomes.²³ LC3B (herein referred to as LC3) is the best characterized form and the most widely used as an autophagic marker. The conversion of the cytosolic form of LC3 (LC3-I) to lipidated form (LC3-II) indicates autophagosome formation. In contrast to LC3, Beclin 1, encoded by the beclin 1 gene, is the mammalian ortholog of yeast Atg6 that is required for the initiation of autophagy through its interaction with Vps34. Homozygous deletion of beclin 1 (beclin 1^−/−) exhibits early embryonic lethality, whereas heterozygous deletion (beclin 1^+/−) results in increased incidence of spontaneous tumorigenesis, abnormal proliferation of mammary epithelial cells and germinal center B lymphocytes, and increased susceptibility to neurodegeneration.^24–27We previously reported that autophagy promotes intracellular degradation of Col-I induced by TGF-β1 in glomerular mesangial cells.²⁸ In the present study, we explored the functional role of autophagy in an in vivo model of progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) in autophagy-deficient LC3 null (LC3^−/−) and heterozygous (beclin 1^+/−) mice and green fluorescent protein (GFP)-LC3 transgenic mice. We also performed functional studies in primary cultured mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. We hypothesized that induction of autophagy in RTECs promotes TGF-β degradation and thereby reduces TGF-β secretion and suppresses development of kidney fibrosis.     

Sugar, Sex, and TGF-β in Diabetic Nephropathy

TGF-β is well known to play a critical role in diabetic kidney disease, and ongoing clinical studies are testing the potential therapeutic promise of inhibiting TGF-β production and action. An aspect of TGF-β action that has not received much attention is its potential role in explaining sex-related proclivity for kidney disease. In this review, we discuss recent studies linking TGF-β signaling to sex-related effects in diabetic kidney disease and suggest targets for future studies.     

Is there added value to adding ARB to ACE inhibitors in the management of CKD?

Antagonism of the rennin-angiotensin-aldosterone-system (RAAS) decreases BP and reduces proteinuria in chronic kidney disease. BP is decreased approximately 5 mmHg when angiotensin II blockers are added to angiotensin-converting enzyme (ACE) inhibitors and is less than typically seen when other agents are added to existing ACE inhibitor regimens. Dual RAAS blockade results in additional reduction in proteinuria. Clinically insignificant increases in hyperkalemia and modest decreases in GFR occur. Data regarding long-term preservation of renal function are lacking. We suggest dual RAAS blockade be used in patients with chronic kidney disease with residual proteinuria on maximal ACE inhibitor or angiotensin II blocker therapy, anticipating additional data with ongoing trials.