Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis

Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-β1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-β inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-β1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.     

Implication of TGF-β as a survival factor during tumour development

Transforming growth factor (TGF)-β is a pleiotropic secretory protein which inhibits and potentiates tumour progression during early and late stage of tumourigenicity, respectively. However, it still remains veiled how TGF-β signalling reveals its two faces. Hoshino et al. (Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim, J. Biochem. 2011;149:55-65) demonstrated a new aspect of TGF-β as a survival factor in highly metastatic breast cancer cells from which TGF-β1 and TGF-β3 are abundantly expressed. They found that TGF-β suppressed the expression of BH3-only protein Bim which promotes programmed death signalling via release of cytochrome c from mitochondria. Further interestingly, forkhead box C1 (Foxc1) whose expression is suppressed upon TGF-β stimulation is involved in the expression of Bim. Based on their results, autocrine TGF-β signalling in certain breast cancers promotes cell survival via inhibition of apoptotic signalling. Thus, the inhibitors for activin receptor-like kinase (ALK)5 kinase might exert a curative influence on certain types of metastatic breast cancers.     

Murine CD4 T cells produce a new form of TGF-β as measured by a newly developed TGF-β bioassay

Background

It is generally assumed that T cells do not produce active TGF-β since active TGF-β as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-β from T cells may take a special form which is not detectable by ELISA.

Methodology/Principal Findings

We constructed a TGF-β bioassay which can detect both soluble and membrane-bound forms of TGF-β from T cells. For this bioassay, 293T cells were transduced with (caga)₁₂ Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-β. We co-cultured pre-activated murine CD4⁺CD25⁻ T cells or CD4⁺CD25⁺ T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4⁺CD25⁻ T cells induced higher luciferase in the reporter cells than CD4⁺CD25⁺ T cells. This T cell-produced TGF-β is in a soluble form since T cell culture supernatants contained the TGF-β activity. The TGF-β activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-β/pro-TGF-β mAb, but not with anti-active TGF-β Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-β from the T cell culture supernatant, but not the ability to induce TGF-β signaling in the reporter cells. The induction of TGF-β signaling by T cell culture supernatants was cell type-specific.

Conclusions/Significance

A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-β which can induce TGF-β signaling. This new form of TGF-β contains LAP as a component. Our finding of a new form of T cell-produced TGF-β and the newly developed TGF-β bioassay system will provide a new avenue to investigate T cell function of the immune system.     

Regulation of GDF-15, a distant TGF-β superfamily member,in a mouse model of cerebral ischemia

GDF-15 is a novel distant member of the TGF-β superfamily and is widely distributed in the brain and peripheral nervous system. We have previously reported that GDF-15 is a potent neurotrophic factor for lesioned dopaminergic neurons in the substantia nigra, and that GDF-15-deficient mice show progressive postnatal losses of motor and sensory neurons. We have now investigated the regulation of GDF-15 mRNA and immunoreactivity in the murine hippocampal formation and selected cortical areas following an ischemic lesion by occlusion of the middle cerebral artery (MCAO). MCAO prominently upregulates GDF-15 mRNA in the hippocampus and parietal cortex at 3 h and 24 h after lesion. GDF-15 immunoreactivity, which is hardly detectable in the unlesioned brain, is drastically upregulated in neurons identified by double-staining with NeuN. NeuN staining reveals that most, if not all, neurons in the granular layer of the dentate gyrus and pyramidal layers of the cornu ammonis become GDF-15-immunoreactive. Moderate induction of GDF-15 immunoreactivity has been observed in a small number of microglial cells identified by labeling with tomato lectin, whereas astroglial cells remain GDF-15-negative after MCAO. Comparative analysis of the size of the infarcted area after MCAO in GDF-15 wild-type and knockout mice has failed to reveal significant differences. Together, our data substantiate the notion that GDF-15 is prominently upregulated in the lesioned brain and might be involved in orchestrating post-lesional responses other than the trophic support of neurons.     

Role of FoxO3a as a negative regulator of the cardiac myofibroblast conversion induced by TGF-β1

Cardiac fibroblasts (CFs) are necessary to maintain extracellular matrix (ECM) homeostasis in the heart. Normally, CFs are quiescent and secrete small amounts of ECM components, whereas, in pathological conditions, they differentiate into more active cells called cardiac myofibroblasts (CMF). CMF conversion is characteristic of cardiac fibrotic diseases, such as heart failure and diabetic cardiomyopathy. TGF-β1 is a key protein involved in CMF conversion. SMADs are nuclear factor proteins activated by TGF-β1 that need other proteins, such as forkhead box type O (FoxO) family members, to promote CMF conversion. FoxO1, a member of this family protein, is necessary for TGF-β1-induced CMF conversion, whereas the role of FoxO3a, another FoxO family member, is unknown. FoxO3a plays an important role in many fibrotic processes in the kidney and lung. However, the participation of FoxO3a in the conversion of CFs into CMF is not clear. In this paper, we demonstrate that TGF-β1 decreases the activation and expression of FoxO3a in CFs. FoxO3a regulation by TGF-β1 requires activated SMAD3, ERK1/2 and Akt. Furthermore, we show that FoxO1 is crucial in the FoxO3a regulation induced by TGF-β1, as shown by overexpressed FoxO1 enhancing and silenced FoxO1 suppressing the effects of TGF-β1 on FoxO3a. Finally, the regulation of TGF-β1-induced CMF conversion was enhanced by FoxO3a silencing and suppressed by inhibited FoxO3a degradation. Considering these collective findings, we suggest that FoxO3a acts as a negative regulator of the CMF conversion that is induced by TGF-β1.     

Screening and identification of a novel class of TGF-β type 1 receptor kinase inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 μM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 μM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.     

aV integrins and TGF-β-induced EMT: a circle of regulation

Transforming growth factor-β (TGF-β) has roles in embryonic development, the prevention of inappropriate inflammation and tumour suppression. However, TGF-β signalling also regulates pathological epithelial-to-mesenchymal transition (EMT), inducing or progressing a number of diseases ranging from inflammatory disorders, to fibrosis and cancer. However, TGF-β signalling does not proceed linearly but rather induces a complex network of cascades that mutually influence each other and cross-talk with other pathways to successfully induce EMT. Particularly, there is substantial evidence for cross-talk between αV integrins and TGF-β during EMT, and anti-integrin therapeutics are under development as treatments for TGF-β-related disorders. However, TGF-β's complex signalling network makes the development of therapeutics to block TGF-β-mediated pathology challenging. Moreover, despite our current understanding of integrins and TGF-β function during EMT, the precise mechanism of their role during physiological versus pathological EMT is not fully understood. This review focuses on the circle of regulation between αV integrin and TGF-β signalling during TGF-β induced EMT, which pose as a significant driver to many known TGF-β-mediated disorders.     

Cloning and Characterization of a Novel Human TGF-β Activated Kinase-Like Gene

TGF-beta activated kinase (TAK1) plays a critical role in the TGF-beta signaling transduction pathway. By screening a human 18-week fetal brain library, we isolated a novel human TAK1-like (TAKL) gene. The gene encoded a putative protein of 242 amino acids, which shared a homology with human, mouse, and Xenopus TAK1. The TAKL gene was located in chromosome 21q21. Northern blot analysis revealed that the TAKL mRNA was expressed predominantly in peripheral blood leukocytes and ubiquitously in human adult and fetal tissues. TAKL was also expressed strongly in breast carcinoma GI-101, colon adenocarcinoma GI-112, and prostatic adenocarcinoma PC3.     

Integration of a Retrograde Signal during Synapse Formation by Glia-Secreted TGF-β Ligand

1. Download : Download high-res image (219KB)
2. Download : Download full-size image

Highlights? Neuromuscular junction (NMJ) glia release the TGF-β molecule Maverick (Mav) ? Glial Mav is required for activation of P-Mad both pre- and postsynaptically ? The release of glia-derived Mav is essential for normal NMJ growth ? Glial Mav modulates muscle expression of Gbb and thereby retrograde signaling     

Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation.     

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel regulator of TGF-β signaling

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-β signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-β signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-β-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

Regulation of TGF-β receptor activity

Background

Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and offers a unique opportunity to study how T3 controls postembryonic development in vertebrates. Earlier studies have demonstrated that TR mediates the metamorphic effects of T3 in Xenopus laevis. Liganded TR recruits histone modifying coactivator complexes to target genes during metamorphosis. This leads to nucleosomal removal and histone modifications, including methylation of histone H3 lysine (K) 79, in the promoter regions, and the activation of T3-inducible genes.

Results

We show that Dot1L, the only histone methyltransferase capable of methylating H3K79, is directly regulated by TR via binding to a T3 response element in the promoter region during metamorphosis in Xenopus tropicalis, a highly related species of Xenopus laevis. We further show that Dot1L expression in both the intestine and tail correlates with the transformation of the organs.

Conclusions

Our findings suggest that TR activates Dot1L, which in turn participates in metamorphosis through a positive feedback to enhance H3K79 methylation and gene activation by liganded TR.     

Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-β1-mediated gene activation

Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of α-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-β1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against α-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-β1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.     

Aldose reductase is a potent regulator of TGF-β1 induced expression of fibronectin in human mesangial cells

Glomerulosclerosis is considered to be the final pathway leading to the progressive loss of renal function in several kidney diseases, transforming growth factor β1 (TGF-β1) plays a critical role in glomerulosclerosis. However, the mechanisms of TGF-β1 stimulating glomerulosclerosis remain poorly understood. Here we report that TGF-β1-induced expression of fibronectin (FN) depends on the activity of aldose reductase (AR) in human mesangial cells (HMCs).The results show that TGF-β1 increased the expression of FN, which attenuated by pharmacological inhibition of AR or knockdown of the enzyme by small interfering RNA (siRNA). MAPKs (ERK, JNK and p38) signalling pathways were activated in HMCs after stimulated by TGF-β1, inhibition of AR blunted the activation ERK, p38 and JNK signalling pathways. These changes were associated with decreased TGF-β1-induced expression of FN. These results indicate that AR is a potent regulator of TGF-β1 induced expression of FN in human mesangial cells: it suggests that inhibition of this enzyme may be useful to prevented extracellular matrix (ECM) deposition in glomerulosclerosis.