Inhibition of Autoregulated TGFβ Signaling Simultaneously Enhances Proliferation and Differentiation of Kidney Epithelium and Promotes Repair Following Renal Ischemia

We studied autocrine transforming growth factor (TGF)β signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFβ receptor and Smad7 levels, but not with active TGFβ, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFβ, suggesting that TGFβ signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFβ type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFβ signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFβ signaling is redundant and maladaptive during tubule repair by epithelial regeneration.Regeneration of an adult epithelium such as those lining the kidney tubules involves not only proliferation but also de-differentiation, followed by growth arrest and re-differentiation.^1,2,3 The signaling cues that coordinate these processes are largely unknown. Endocrine and paracrine factors affect epithelial repair following injury in vivo. Nevertheless, epithelial homeostasis is also regulated by density-dependent contact-inhibition and attendant differentiation. The mechanisms that mediate these processes are poorly understood, but likely involve transforming growth factor (TGF)β, in addition to signals generated by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. These considerations prompted us to investigate how endogenously generated signals might control epithelial regeneration in terms of cell proliferation and differentiation.Increased TGFβ signaling can lead to apoptosis, growth inhibition, or epithelial-mesenchymal transitions (EMT) of epithelial cells, including kidney epithelial cells.^4,5,6,7,8,9 Prolonged exposure to high concentrations of active TGFβ is frequently used to model these alterations. While these effects of sustained high intensity TGFβ signaling are well studied, much less is known about physiologically regulated TGFβ signals and how they become increased by epithelial injury and subsequent regeneration. Autocrine TGFβ signals are antiproliferative for epithelial cells and cultures from TGFβ₁ null kidney tubules display enhanced proliferative rates.¹⁰ Nevertheless, TGFβ signaling was found to be increased rather than decreased during the proliferation of surviving kidney epithelium following cell loss by ischemia, and this was accompanied by increased expression of TGFβ and its receptors in regenerating cells.¹¹ Similarly, proliferating keratinocytes in skin wounds show enhanced TGFβ signaling¹². It has been puzzling why antiproliferative TGFβ signaling becomes enhanced in rapidly growing cells under pathological conditions.In this study, we have investigated the functional relevance of cell-autonomous, ie, endogenously generated, TGFβ signals for regenerating kidney epithelial cells in culture and in vivo. Fully differentiated proximal tubule (PT) cells retain the ability to undergo mitotic division,^13,14,15 and, following cell loss by injury, survivors dedifferentiate, proliferate, and then redifferentiate to reconstitute the lost cell mass.^1,2,3,13,16 We found that cell-autonomous TGFβ signals are tightly autoregulated during repeated cycles of proliferation and contact-inhibition in PT cultures. Signaling was high during log phase growth and became progressively suppressed as cultures became contact-inhibited and differentiated. It was decreased in growing subconfluent cultures by neutralizing TGFβ antibodies, indicating a requirement for extracellular ligand. However, in the absence of TGFβ antibodies, increases and decreases of signaling were determined solely by cell density, and occurred independently of the concentrations of barely measurable active TGFβ in growth medium. Instead, the signaling fluctuations were associated with increased and decreased expression of TGFβ receptor and reciprocal alterations of inhibitory Smad7. Moreover, saturating concentrations of exogenous TGFβ were found to elicit blunted signaling responses from contact-inhibited differentiated cells relative to growing undifferentiated cells. These observations suggested that: (1) extracellular TGFβ ligand played a permissive role but did not, by itself, determine the intensity of signaling fluctuations during the epithelial growth cycle; and (2) signaling homeostasis during growth and quiescence was related to the modulation of TGFβ receptors and Smad7. Functionally, we found that inhibition of cell-autonomous TGFβ signals resulted in remarkably accelerated differentiation and concurrent stimulation of proliferation in growing PT cultures. Importantly, we extended our observations to demonstrate that treatment with small molecule Alk5 inhibitors not only promoted differentiation in regenerating PT epithelium during wound healing in vitro, but also improved the repair of kidney damage with greater restoration of epithelial differentiation and tubule integrity following ischemia in vivo. These unprecedented findings have direct relevance to the development of treatments that might promote repair and recovery following loss of epithelium by acute kidney injury (AKI).     

Macrophages Regulate Renal Fibrosis Through Modulating TGFβ Superfamily Signaling

Renal fibrosis is the fundamental pathway leading to end-stage renal disease, while its exact molecular basis remains incompletely elucidated. Previous studies have demonstrated that transforming growth factor beta 1 (TGFβ1) is an inducer of the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, while bone morphogenic protein 7 (BMP7) counteracts TGFβ1-induced EMT and reverses chronic renal injury. Although macrophage recruitment is believed to play an important role during the whole pathogenesis, the mechanism underlying their activate involvement in the formation of renal fibrosis besides phagocytosizing extracellular matrix and apoptotic cells is largely unknown. Here, in a mouse unilateral ureteral obstruction (UUO) model, we show that the recruited macrophages are mainly M1 macrophages at early stage. However, these F4/80-positive and CD301-negative M1 macrophages were shortly polarized into F4/80-positive and CD301-positive M2 macrophages, respectively, which released high levels TGFβ1, to contradict the local expression of BMP7 to facilitate EMT-induced renal fibrosis. M2 macrophages depletion specifically inhibited EMT, and subsequently the renal fibrosis. Adoptive transplantation of M2 macrophages increased the features of renal fibrosis. Our study thus identified double-edged effects of macrophages in the formation of renal fibrosis, which suggest that modulation of macrophage polarization may substantially improve the treatment of renal fibrosis.     

The Transcription Factor IRF8 Activates Integrin-Mediated TGF-β Signaling and Promotes Neuroinflammation

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The Effects of TGFβ on Haemopoietic Cells

Abstract

Transforming growth factor β (TGFβ) suppresses the growth of differentiation inducible, murine IL-3-dependent multipotential cell lines but has no growth inhibitory effect upon an IL-3-independent (leukaemic) cell line arising from one of them, nor on IL-3-dependent cell lines that are unable to undergo differentiation. TGFβ inhibits in vitro colony formation by normal multipotential haemopoietic progenitor cells. Bipotential progenitors recruited by GM-CSF are, however, more resistant to the inhibitory effects of TGFβ, whereas progenitors recruited by the lineage restricted factor, M-CSF, are sensitive to the inhibitory effects. These data indicate that responsiveness to TGFβ is differentiation linked and studies with the cell lines suggest that response (or lack of response) is not determined solely by levels of expression of TGFβ receptors. Furthermore, the effects of TGFβ2 on haemopoietic progenitors are very similar to those induced by TGFβ.     

Inhibition of GSK-3β Decreases the Ischemia-Induced Death of Renal Cells

Pharmacological preconditioning with insulin and lithium ions prevented the death of renal cells under conditions of ischemia/reperfusion. Preincubation of cells with insulin or lithium ions decreased production of reactive oxygen species after ischemia/reoxygenation. These agents also prevented the development of mitochondrial dysfunction in renal cells induced by ischemia/reoxygenation. It was hypothesized that the protective effects of these agents are related to inhibition of glycogen synthase kinase-3(. This enzyme is inactivated upon phosphorylation of serine residue in position 9. We found that in vivo administration of lithium ions to animals before renal ischemia prevents the development of kidney failure.     

Inhibition of Lupus by Genetic Alteration of the Interferon-α/β Receptor

Type I interferons (IFN-αβ) are immunoregulatory cytokines that promote both innate and adaptive immune responses. Although they have been implicated in human SLE, recent studies in mice have helped solidify this connection. By using lupus-prone mice with knockout of the IFN-αβ receptor, we and others have documented that lack of IFN-αβ leads to a marked reduction in disease manifestations, including autoantibody production, target organ damage and mortality. Furthermore, IFN-αβ was found to potentially contribute to several levels of disease pathogenesis. These included the differentiation and activation of dendritic cells, the activation and proliferation of T cells, T cell survival and the activation and survival of autoantibody-producing B cells. These findings strongly support the targeting of IFN-αβ in SLE and suggest that definition of the specific pathways critical for disease induction will be important for optimal intervention.     

Inhibition of NF-κB Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are diseases characterized by IgA deposits in the kidney and/or skin. Both may arise after upper respiratory tract infections, but the pathogenic mechanisms governing these diseases remain unclear. Patients with IgAN (n = 16) and HSP (n = 17) were included in this study aimed at examining whether IgA-binding M proteins of group A streptococci could be involved. As M proteins vary in sequence, the study focused on the IgA-binding-region (IgA-BR) of three different M proteins: M4, M22, and M60. Renal tissue from IgAN and HSP patients and skin from HSP patients were examined for deposits of streptococcal IgA-BR by immunohistochemistry and electron microscopy using specific antibodies, and a skin sample from a HSP patient was examined by mass spectrometry. IgA-BR deposits were detected in 10/16 IgAN kidneys and 7/13 HSP kidneys. Electron microscopy demonstrated deposits of IgA-BRs in the mesangial matrix and glomerular basement membrane, which colocalized with IgA. Skin samples exhibited IgA-BR deposits in 4/5 biopsies, a result confirmed by mass spectrometry in one patient. IgA-BR deposits were not detected in normal kidney and skin samples. Taken together, these results demonstrate IgA-BR from streptococcal M proteins in patient tissues. IgA-BR, would on gaining access to the circulation, encounter circulatory IgA and form a complex with IgA-Fc that could deposit in tissues and contribute to the pathogenesis of IgAN and HSP.Tissue deposits containing IgA characterize IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP), two conditions affecting kidney function. IgAN is the most common primary glomerulonephritis worldwide. Its predominant clinical feature is episodic macroscopic hematuria usually coinciding with upper respiratory tract infections. Symptoms may, however, vary from microscopic hematuria to a severe nephritic-nephrotic syndrome. End-stage kidney disease occurs in 30% to 40% of patients within 20 years. Histopathologically IgAN is characterized by mesangial cell proliferation and in progressive cases crescent formation as well as glomerular sclerosis, interstitial fibrosis, and tubular atrophy. Ultramorphologic findings show mesangial deposits of immune complexes containing predominantly IgA.^1,2HSP is the most common form of vasculitis in childhood. It may affect many organs, but usually presents as skin lesions, varying from purpura to bullous intradermal bleedings, arthritis, gastrointestinal involvement with pain and/or bleeding. Renal involvement occurs in up to 50% of cases³ and is known as Henoch-Schönlein nephropathy (HSN). HSN may manifest as microscopic or macroscopic hematuria as well as glomerulonephritis or nephrotic syndrome. Approximately 20% of HSN cases will develop renal failure.⁴ The histopathological lesion termed leukocytoclastic vasculitis is characterized by inflammation of small vessels with perivascular polymorphonuclear leukocyte and mononuclear cell infiltrates. Immune deposits in affected organs contain IgA, and renal pathology resembles that seen in IgAN.^1,3The IgA mesangial deposits in kidneys of patients with IgAN and HSP are primarily composed of galactose-deficient IgA1.^5,6,7 The mechanism by which under-glycosylated IgA1 deposits in the mesangium, possibly in complex with IgG,^8,9 has not been determined. Environmental antigens have been proposed to contribute to the disease but have not been consistently associated with mesangial deposits.⁹ Although the etiology of IgAN and HSP is unclear, these diseases are often preceded by infections, primarily of the upper respiratory tract, and an infectious agent has therefore been suspected. There is circumstantial evidence for involvement of group A streptococcus (GAS, Streptococcus pyogenes),^{10,11,12,13,14,15} but infections with other bacteria^16,17 as well as viruses¹⁸ have been implicated as well.In this study we hypothesized that GAS infection could trigger IgAN and/or HSN, because GAS is a very common cause of upper respiratory tract infection, and because many GAS strains bind IgA-Fc.^19,20,21 The ability of a GAS strain to bind human IgA results from the presence of an IgA-binding region (IgA-BR) in the surface-localized M protein.^22,23 The fibrillar M protein, which is a major virulence factor of GAS, varies in sequence between strains²⁴ allowing classification of GAS isolates into more than 120 M serotypes.²⁵ The exact function of the IgA-BR in an M protein is not known, but there is evidence that it contributes to bacterial phagocytosis resistance.²⁶ The IgA-BR of an M protein represents a distinct domain that can be studied in isolated form, as a peptide that binds IgA.^27,28 Such IgA-binding peptides, designated Sap (streptococcal IgA-binding peptide), were used in the experiments described herein.To analyze whether IgA-binding streptococcal M proteins are present in affected tissues of patients with IgAN and/or HSP, and colocalize with IgA, we used antibodies to the IgA-BR of three different M proteins M4, M22, and M60. Of note, M4 and M22 are among the most common serotypes of clinical GAS isolates.²⁹ As the IgA-BRs of different M proteins vary extensively in sequence,^22,23 the use of antibodies to three different serotypes enhanced our chances to detect tissue deposition of an IgA-BR.     

Attenuation of the Progression of Articular Cartilage Degeneration by Inhibition of TGF-β1 Signaling in a Mouse Model of Osteoarthritis

Transforming growth factor beta 1 (TGF-β1) is implicated in osteoarthritis. We therefore studied the role of TGF-β1 signaling in the development of osteoarthritis in a developmental stage-dependent manner. Three different mouse models were investigated. First, the Tgf-β receptor II (Tgfbr2) was specifically removed from the mature cartilage of joints. Tgfbr2-deficient mice were grown to 12 months of age and were then euthanized for collection of knee and temporomandibular joints. Second, Tgfbr2-deficient mice were subjected to destabilization of the medial meniscus (DMM) surgery. Knee joints were then collected from the mice at 8 and 16 weeks after the surgery. Third, wild-type mice were subjected to DMM at the age of 8 weeks. Immediately after the surgery, these mice were treated with the Tgfbr2 inhibitor losartan for 8 weeks and then euthanized for collection of knee joints. All joints were characterized for evidences of articular cartilage degeneration. Initiation or acceleration of articular cartilage degeneration was not observed by the genetic inactivation of Tgfbr2 in the joints at the age of 12 months. In fact, the removal of Tgfbr2 and treatment with losartan both delayed the progression of articular cartilage degeneration induced by DMM compared with control littermates. Therefore, we conclude that inhibition of Tgf-β1 signaling protects adult knee joints in mice against the development of osteoarthritis.Transforming growth factor beta 1 (TGF-β1) is considered an anabolic factor to articular chondrocytes, based largely on results from in vitro and ex vivo experiments in which TGF-β1 can stimulate chondrocytes to synthesize and release extracellular matrix molecules, including proteoglycans and type II collagen.^{1, 2} In addition, results from other studies indicate that the genetic inactivation of Smad3 or disruption of the interaction of Tgf-β1 with its receptor, Tgf-β receptor type II (Tgfbr2), during early development results in osteoarthritis (OA)-like knee joints in mice.^{3, 4, 5} Moreover, a human genetic study reports that a two-nucleotide deletion, 741-742del AT (nonsense mutation), in SMAD3 causes early-onset OA in a human family.⁶ These investigations indicate that TGF-β1 is, at least, required for the normal development of a joint.The lack of TGF-β1 signaling during early development can cause a normal joint to develop into an osteoarthritic joint. However, observations from studies of adult mice as opposed to developing mice suggest that the increase in the activity of TGF-β1 signaling may initiate and accelerate articular cartilage degeneration in adult joints. First, studies in animal models by Itayem et al^{7, 8} suggest that intra-articular injection of TGF-β1 into adult rat knee joints causes early onset of OA. Second, a study by Bakker et al⁹ reports that the constitutive overexpression of active TGF-β1 in adult mouse knee joints results in OA associated with increase in the production of proteoglycans in articular cartilage and hyperplasia of synovium and chondro-osteophyte formation. Note that the enhanced production of extracellular matrix molecules is not necessarily beneficial or physiologic to adult articular cartilage. For instance, one of the earliest pathologic signs in articular cartilage degeneration is the overproduction of proteoglycans in mouse models of OA.^{10, 11} Thus, the overproduction of the proteoglycans could disrupt the homeostasis of adult articular cartilage. Third, the above-mentioned human genetic study reports that a nucleotide change, 859C>T or 782C>T in SMAD3, increases the level of TGF-β1 and activity of the TGF-β1 signaling pathway in two human families associated with early-onset OA.⁶ In addition, data from a human genetic association study suggest that an increase in the expression of SMAD3 is a risk factor for the development of OA.¹² This is in agreement with the observation from studies showing that the level of TGF-β1 is significantly higher in human osteoarthritic tissues than in healthy articular cartilage.^{13, 14} Fourth, we found that the protein level of Tgf-β1 was significantly increased in the articular chondrocyte of adult knee joints in two mouse models of OA, collagen type XI gene-deficient mice and destabilization of the medial meniscus (DMM).¹⁵ On the basis of results from all of the aforementioned studies, a question remains: what is the exact role of TGF-β1 in the development of OA? We hypothesized that TGF-β1 signaling in the development of OA acts in a developmental stage-dependent manner. In this scenario, TGF-β1 is required for the development of articular cartilage; however, once a joint is formed, TGF-β1 is no longer needed. Therefore, induction of TGF-β1 in an adult joint causes articular cartilage degeneration, which eventually leads to OA.To support our hypothesis, we evaluated the articular cartilage of knee joints for evidence of changes in structural characteristics and protein expression of genes in three different conditions of adult mice. First, Tgfbr2 was specifically removed from the articular cartilage of knee and temporomandibular (TM) joints of mice at the age of 8 weeks. The mice were grown to the age of 12 months, at which point knee and TM joints were collected. Second, adult mice (8 weeks old) without Tgfbr2 in the articular cartilage of their knee joints were subjected to DMM surgery (known to induce OA) and were euthanized at 8 and 16 weeks after DMM for the collection of knee joints. Third, adult wild-type C57BL/6 mice were subjected to DMM and then treated with a Tgfbr2 inhibitor, losartan. The mice were euthanized at 8 weeks after DMM for collection of knee joints. The articular cartilage of joints from the mice and their corresponding controls were analyzed.     

Inhibition of Mycobacterium tuberculosis-induced signalling by transforming growth factor-β in human mononuclear phagocytes

Tuberculosis (TB) is associated with excessive production and bioactivation of transforming growth factor bets (TGF-β) in situ. Here, modification of expression of components of plasminogen/plasmin pathway in human monocytes (MN) by inhibitors of TGF-β signalling was examined. Smad3 siRNA effectively inhibited TGF-β-induced urokinase plasminogen activator receptor (uPAR). Agents known to interfere with TGF-β signalling, including the Smad inhibitors SIS3 and erythromycin derivatives, and ALK5 receptor inhibitor (SB 431542) in inhibition of uPAR expression in response to Mycobacterium tuberculosis (MTB) were examined. Inhibition by SIS3 only inhibited uPAR mRNA significantly. SIS3 may prove to be an effective adjunct to TB therapy.