Endogenous TGF-β Research Articles

P44/WDR77 restricts the sensitivity of proliferating cells to TGFβ signaling

We previously reported that a novel WD-40 domain-containing protein, p44/WDR77, drives quiescent epithelial cells to re-enter the cell cycle and plays an essential role for growth of lung and prostate cancer cells. Transforming growth factor beta (TGFβ) signaling is important in the maintenance of non-transformed cells in the quiescent or slowly cycling stage. However, both non-transformed proliferating cells and human cancer cells are non-responsive to endogenous TGFβ signaling. The mechanism by which proliferating cells become refractory to TGFβ inhibition is not well established. Here, we found that silencing p44/WDR77 increased cellular sensitivity to TGFβ signaling and that this was inversely correlated with decreased cell proliferation. Smad2 or 3 phosphorylation, TGFβ-mediated transcription, and TGFβ2 and TGFβ receptor type II (TβRII) expression were dramatically induced by silencing of p44/WDR77. These data support the hypothesis that p44/WDR77 down-regulates the expression of the TGFβ ligand and its receptor, thereby leading to a cellular non-response to TGFβ signaling. Finally, we found that p44/WDR77 expression was correlated with cell proliferation and decreased TGFβ signaling during lung tumorigenesis. Together, these results suggest that p44/WDR77 expression causes the non-sensitivity of proliferating cells to TGFβ signaling, thereby contributing to cellular proliferation during lung tumorigenesis.

TGF-β1 of no avail as prognostic marker in lyme disease.

Background. Within the present in vivo study using the wild type mouse strains C3H/HeN and FVB/N it was intended to (1) measure TGF-β1 expression in the course of lyme disease, (2) examine the potential correlation of TGF-β1 expression with the clinical outcome of a Borrelia infection (with a focus on lyme arthritis), (3) develop a diagnostic tool based on the endogenous factor TGF-β1 to predict the progressivity of lyme disease.Findings. In the course of lyme disease there was an increase in the serum content of active TGF-β1, which became significant 56 days post infection (p < 0.001). The serum concentration of total TGF-β1 in the course of infection initially decreased then rebounded and subsequently dropped again. Despite considerable individual variations in active TGF-β1 serum concentrations there were no identifiable dissimilarities in the clinical appearance of the mice. Likewise, no correlation could be seen between the serum content of active TGF-β1 and the severity of lyme arthritis of tibiotarsal joints of infected mice.Conclusions. The present study clearly shows that TGF-β1 is of no avail as prognostic marker in lyme disease. Hence, the search for an endogenous predictive factor, which can be determined in an easy and reliable manner, remains open.

Microenvironment Is Involved in Cellular Response to Hydrostatic Pressures During Chondrogenesis of Mesenchymal Stem Cells

Chondrocytes integrate numerous microenvironmental cues to mount physiologically relevant differentiation responses, and the regulation of mechanical signaling in chondrogenic differentiation is now coming into intensive focus. To facilitate tissue-engineered chondrogenesis by mechanical strategy, a thorough understanding about the interactional roles of chemical factors under mechanical stimuli in regulating chondrogenesis is in great need. Therefore, this study attempts to investigate the interaction of rat MSCs with their microenvironment by imposing dynamic and static hydrostatic pressure through modulating gaseous tension above the culture medium. Under dynamic pressure, chemical parameters (pH, pO2, and pCO2) were kept in homeostasis. In contrast, pH was remarkably reduced due to increased pCO2 under static pressure. MSCs under the dynamically pressured microenvironment exhibited a strong accumulation of GAG within and outside the alginate beads, while cells under the statically pressured environment lost newly synthesized GAG into the medium with a speed higher than its production. In addition, the synergic influence on expression of chondrogenic genes was more persistent under dynamic pressure than that under static pressure. This temporal contrast was similar to that of activation of endogenous TGF-β1. Taken altogether, it indicates that a loading strategy which can keep a homeostatic chemical microenvironment is preferred, since it might sustain the stimulatory effects of mechanical stimuli on chondrogenesis via activation of endogenous TGF-β1.

Fibroblasts induce epithelial to mesenchymal transition in breast tumor cells which is prevented by fibroblasts treatment with histamine in high concentration

Epithelial to mesenchymal transition (EMT) of cancer cells is an essential process in cancer progression. Cancer cells that undergone EMT loose cell–cell contacts, acquire mesenchymal properties and develop migratory and invasive abilities.In previous studies we have demonstrated that histamine may modify the invasive phenotype of pancreatic and mammary tumor cells. In this work we proposed to investigate whether histamine may also influence the interaction between tumor cells and normal fibroblasts. The potential activation of normal CCD-1059Sk fibroblasts by histamine and EMT phenotypic changes induced in MCF-7 and MDA-MB-231 breast tumor cells by the conditioned media (CM) derived from fibroblasts were evaluated. Initially, we determined the presence of H1, H2 and H4 histamine receptors and matrix metalloproteinase 2 (MMP2) mRNA in CCD-1059Sk fibroblasts. MMP2 gelatinolytic activity, cell migration and alpha-smooth muscle actin expression were increased in fibroblasts by low doses (<1μM) and decreased by high doses (20μM) of histamine. MCF-7 cells cultured with CM from fibroblasts exhibited spindle-shaped morphology, cell spreading and cytoplasmic expression of β-catenin but there was no change in MMP2 activity and cell migration. MDA-MB-231 cells cultured with CM from fibroblasts showed a more elongated phenotype, cell spreading, cytoplasmic β-catenin, increased MMP2 activity and endogenous TGF-β1 expression, and enhanced cell migration and invasion. Notably, all these features were reversed when mammary tumor cells were cultured with CM from fibroblasts treated with 20μM histamine. In conclusion, high doses of histamine may prevent the activation of fibroblasts and also avert the EMT related changes induced in epithelial tumor cells by fibroblasts CM.

The inhibitory effect of salvianolic acid B on TGF-β1-induced proliferation and differentiation in lung fibroblasts

ABSTRACTSalvianolic acid B (Sal B), one of the major water-soluble compounds of Danshen (a popular Chinese herb), possesses many of the biological activities, such as antifibrogenic effect in liver and renal diseases. Transforming growth factor-β1 (TGF-β1) plays a central role in the development of pulmonary fibrosis by stimulating extracellular matrix (ECM) accumulation and activating fibroblasts. Here, we investigated the effects of Sal B on cell proliferation, collagen synthesis, endogenous TGF-β1 production, and α-smooth muscle actin (α-SMA, a marker of myofibroblasts) expression in human lung fibroblasts stimulated by TGF-β1 in vitro. The cell proliferation rates were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. The expression of TGF-β1 and type I collagen at both the mRNA and protein levels was detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay, respectively. The α-SMA expression was detected by Western blot. TGF-β1 treatment of lung fibroblasts increased cell proliferation rates, and enhanced the expression level of type I collagen, endogenous TGF-β1 production, and α-SMA expression (P < .05). The treatment with only Sal B did not affect the proliferation and differentiation of lung fibroblasts. Interestingly, Sal B was found to inhibit TGF-β1-induced cell proliferation, expression of type I collagen, endogenous TGF-β1 production, and α-SMA expression in lung fibroblasts. Moreover, the inhibitory effect of Sal B on TGF-β1-induced proliferation and differentiation in lung fibroblasts was more significant when treated with high-dose Sal B (1 μmol/L versus 10 μmol/L, P < .05). These data demonstrate that Sal B inhibits TGF-β1-induced cell proliferation and differentiation in vitro experiment.

Fibronectin and transforming growth factor beta contribute to erythropoietin resistance and maladaptive cardiac hypertrophy

The use of recombinant human erythropoietin (rhEPO) to promote repair and minimize cardiac hypertrophy after myocardial infarction has had disappointing outcomes in clinical trials. We hypothesized that the beneficial non-hematopoietic effects of rhEPO against cardiac hypertrophy could be offset by the molecular changes initiated by rhEPO itself, leading to rhEPO resistance or maladaptive hypertrophy. This hypothesis was investigated using an isoproterenol-induced model of myocardial infarct and cardiac remodelling with emphasis on hypertrophy. In h9c2 cardiomyocytes, rhEPO decreased isoproterenol-induced hypertrophy, and the expression of the pro-fibrotic factors fibronectin, alpha smooth muscle actin and transforming growth factor beta-1 (TGF-β1). In contrast, by itself, rhEPO increased the expression of fibronectin and TGF-β1. Exogenous TGF-β1 induced a significant increase in hypertrophy, which was further potentiated by rhEPO. Exogenous fibronectin not only induced hypertrophy of cardiomyocytes, but also conferred resistance to rhEPO treatment. Based on these findings we propose that the outcome of rhEPO treatment for myocardial infarction is determined by the baseline concentrations of fibronectin and TGF-β1. If endogenous fibronectin or TGF-β levels are above a certain threshold, they could cause resistance to rhEPO therapy and enhancement of cardiac hypertrophy, respectively, leading to maladaptive hypertrophy.

Proteolytic and Non-proteolytic Activation of Keratinocyte-Derived Latent TGF-β1 Induces Fibroblast Differentiation in a Wound-Healing Model Using Rat Skin

Transforming growth factor-β1 (TGF-β1) reportedly causes the differentiation of fibroblasts to myofibroblasts during wound healing. We investigated the mechanism underlying the activation of latent TGF-β1 released by keratinocytes in efforts to identify promising pharmacological approaches for the prevention of hypertrophic scar formation. A three-dimensional collagen gel matrix culture was prepared using rat keratinocytes and dermal fibroblasts. Stratified keratinocytes promoted the TGF receptor–dependent increase in α-smooth muscle actin (α-SMA) immunostaining and mRNA levels in fibroblasts. Latent TGF-β1 was found to be localized suprabasally and secreted. α-SMA expression was inhibited by an anti-αv-integrin antibody and a matrix metalloproteinase (MMP) inhibitor, GM6001. In a two-dimensional fibroblast culture, α-SMA expression depended on the production of endogenous TGF-β1 and required αv-integrin or MMP for the response to recombinant latent TGF-β1. In keratinocyte-conditioned medium, MMP-dependent latent TGF-β1 secretion was detected. Applying this medium to the fibroblast culture enhanced α-SMA production. This effect was decreased by GM6001, the anti-αv-integrin antibody, or the preabsorption of latent TGF-β1. These results indicate that keratinocytes secrete latent TGF-β1, which is liberated to fibroblasts over distance and is activated to produce α-SMA with the aid of a positive-feedback loop. MMP inhibition was effective for targeting both keratinocytes and fibroblasts in this model. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.13209FP]

TGF-β1 Attenuates Spinal Neuroinflammation and the Excitatory Amino Acid System in Rats With Neuropathic Pain

Previous studies have reported that the intrathecal (i.t.) administration of transforming growth factor β1 (TGF-β1) prevents and reverses neuropathic pain. However, only limited information is available regarding the possible role and effects of spinal TGF-β1 in neuropathic pain. We aimed to investigate the antinociceptive effects of exogenous TGF-β1 on chronic constriction injury (CCI)-induced neuropathic pain in rats. We demonstrated that sciatic nerve injury caused a downregulation of endogenous TGF-β1 levels on the ipsilateral side of the lumbar spinal dorsal gray matter, and that the i.t. administration of TGF-β1 (.01–10 ng) significantly attenuated CCI-induced thermal hyperalgesia in neuropathic rats. TGF-β1 significantly inhibited CCI-induced spinal neuroinflammation, microglial and astrocytic activation, and upregulation of tumor necrosis factor-α. Moreover, i.t. TGF-β1 significantly attenuated the CCI-induced downregulation of glutamate transporter 1, the glutamate aspartate transporter, and the excitatory amino acid carrier 1 on the ipsilateral side. Furthermore, i.t. TGF-β1 significantly decreased the concentrations of 2 excitatory amino acids, aspartate and glutamate, in the spinal dialysates in CCI rats. In summary, we conclude that the mechanisms of the antinociceptive effects of i.t. TGF-β1 in neuropathy may include attenuation of spinal neuroinflammation, attenuation, or upregulation of glutamate transporter downregulation, and a decrease of spinal extracellular excitatory amino acids. PerspectiveClinically, medical treatment is usually initiated after the onset of intractable pain. Therefore, in the present study, i.t. TGF-β1 was designed to be administered 2 weeks after the establishment of CCI pain. Compared to the continuous TGF-β1 infusion mode, single-dose administration seems more convenient and practical to use.

Conditional Expression of TGF-β1 in Skeletal Muscles Causes Endomysial Fibrosis and Myofibers Atrophy

To study the effects of transforming growth factor beta 1 (TGF-β1) on fibrosis and failure of regeneration of skeletal muscles, we generated a tet-repressible muscle-specific TGF-β1 transgenic mouse in which expression of TGF-β1 is controlled by oral doxycycline. The mice developed muscle weakness and atrophy after TGF-β1 over-expression. We defined the group of mice that showed phenotype within 2 weeks as early onset (EO) and the rest as late onset (LO), which allowed us to further examine phenotypic differences between the groups. While only mice in the EO group showed significant muscle weakness, pathological changes including endomysial fibrosis and smaller myofibers were observed in both groups at two weeks after the TGF-β1 was over-expressed. In addition, the size of the myofibers and collagen accumulation were significantly different between the two groups. The amount of latent and active TGF-β1 in the muscle and circulation were significantly higher in the EO group compared to the LO or control groups. The up-regulation of the latent TGF-β1 indicated that endogenous TGF-β1 was induced by the expression of the TGF-β1 transgene. Our studies showed that the primary effects of TGF-β1 over-expression in skeletal muscles are muscle wasting and endomysial fibrosis. In addition, the severity of the pathology is associated with the total amount of TGF-β1 and the expression of endogenous TGF-β1. The findings suggest that an auto-feedback loop of TGF-β1 may contribute to the severity of phenotypes.

A novel peptide‐modified and gene‐activated biomimetic bone matrix accelerating bone regeneration

The osteogenic differentiation of bone marrow stromal cells (BMSCs) can be regulated by systemic or local growth factor, especially by transforming growth factor beta 1 (TGF-β1). However, how to maintain the bioactivity of exogenous TGF-β1 is a great challenge due to its short half-life time. The most promising solution is to transfer TGF-β1 gene into seed cells through transgenic technology and then transgenic cells to continuously secret endogenous TGF-β1 protein via gene expression. In this study, a novel non-viral vector (K)16GRGDSPC was chemically linked to bioactive bone matrices PLGA-[ASP-PEG]n using cross-linker to construct a novel non-viral gene transfer system. TGF-β1 gene was incubated with this system and subsequently rabbit-derived BMSCs were co-cultured with this gene-activated PLGA-[ASP-PEG]n, while co-cultured with PLGA-[ASP-PEG]n modified with (K)16GRGDSPC only and original PLGA-[ASP-PEG]n as control. Thus we fabricated three kinds of composites: Group A (BMSCs-TGF-β1DNA-(K)16GRGDSPC-PLGA-[ASP-PEG]n composite); Group B (BMSCs-(K)16GRGDSPC-PLGA-[ASP-PEG]n composite); and Group C (BMSCs-PLGA-[ASP-PEG]n composite). TGF-β1 and other osteogenic phenotype markers of alkaline phosphatase, osteocalcin, osteopontin and type I collagen in Group A were all significantly higher than the other two groups ex vivo. In vivo, 15-mm long segmental rabbit bone defects were created and randomly implanted the aforementioned composites separately, and then fixed with plate-screws. The results demonstrated that the implants in Group A significantly accelerated bone regeneration compared with the other implants based on X-rays, histological and biomechanical examinations. Therefore, we conclude this novel peptide-modified and gene-activated biomimetic bone matrix of TGF-β1DNA-(K)16GRGDSPC-PLGA-[ASP-PEG]n is a very promising scaffold biomaterial for accelerating bone regeneration.

Endogenous versus exogenous growth factor regulation of articular chondrocytes

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-β1 stimulated these reparative functions, while endogenous TGF-β1 had little effect. Endogenous TGF-β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form.

Scube regulates synovial angiogenesis-related signaling

Angiogenesis is particularly driven in the synovial microenvironment of Rheumatoid arthritis (RA), and considered as the fundamental cause for the persistent injury and chronic damage. Therefore, exploring the pathomechanism of synovial angiogenesis may provide promising prospects for vascular-targeting treatment of RA. The noval family of Scube proteins is confirmed to overlap significantly in structure characterized by epidermal growth factor (EGF)-like domains and CUB (complement subcomponents C1r/C1s, Uegf, bone morphogenetic protein-1) domain. As secreted glycoprotein and peripheral membrane protein, Scube increases its serum level in response to stimuli of inflammation and hypoxia. In rheumatoid angiogenesis-related signaling system defined by hedgehog (Hh), transforming growth factor (TGF)β and bone morphogenetic protein 2 (BMP2), Scube1 and 2 antagonize BMP2 signaling, suppressing BMP2-induced phospho-Smad1/5/8 level in vivo. Scube3 functions as an endogenous TGFβ receptor ligand, increasing Smad2/3 phosphorylation, and thus upregulates target genes involved in angiogenesis. Via obligate assistance of Scube1 and 3, Scube2 plays a center role to recruit dually lipid-modified Hh transferred from Dispatched A (DispA), increasing Hh secretion by promoting its solubility. These findings support the hypothesis that Scube may regulate synovial angiogenesis may be the ideal vascular targets for anti-rheumatic treatment of RA.

Inhibition of TGF-β1 signaling promotes central memory T cell differentiation.

This study affirmed that isolated CD8(+) T cells express mRNA and produce TGF-β following cognate peptide recognition. Blockage of endogenous TGF-β with either a TGF-β-blocking Ab or a small molecule inhibitor of TGF-βRI enhances the generation of CD62L(high)/CD44(high) central memory CD8(+) T cells accompanied with a robust recall response. Interestingly, the augmentation within the central memory T cell pool occurs in lieu of cellular proliferation or activation, but with the expected increase in the ratio of the Eomesoderm/T-bet transcriptional factors. Yet, the signal transduction pathway(s) seems to be noncanonical, independent of SMAD or mammalian target of rapamycin signaling. Enhancement of central memory generation by TGF-β blockade is also confirmed in human PBMCs. The findings underscore the role(s) that autocrine TGF-β plays in T cell homeostasis and, in particular, the balance of effector/memory and central/memory T cells. These results may provide a rationale to targeting TGF-β signaling to enhance Ag-specific CD8(+) T cell memory against a lethal infection or cancer.

Resveratrol Sensitizes Tamoxifen in Antiestrogen-Resistant Breast Cancer Cells with Epithelial-Mesenchymal Transition Features

Tamoxifen resistance remains to be a huge obstacle in the treatment of hormone-dependent breast cancer, and this therefore highlights the dire need to explore the underlying mechanisms. The epithelial-mesenchymal transition (EMT) is a molecular process through which an epithelial cell transfers into a mesenchymal phenotype. Roles of EMT in embryo development, cancer invasion and metastasis have been extensively reported. Herein, we established tamoxifen-resistant MCF-7/TR breast cancer cells and showed that MCF-7/TR cells underwent EMT driven by enhanced endogenous TGF-β/Smad signaling. Ectopic supplement of TGF-β promoted in MCF-7 cells a mesenchymal and resistant phenotype. In parallel, we demonstrated that resveratrol was capable of synergizing with tamoxifen and triggering apoptosis in MCF-7/TR cells. Further Western blot analysis indicated that the chemosensitizing effects of resveratrol were conferred with its modulation on endogenous TGF-β production and Smad phosphorylation. In particular, 50 μM resveratrol had minor effects on MCF-7/TR cell proliferation, but could significantly attenuate endogenous TGF-β production and the Smad pathway, ultimately leading to reversion of EMT. Collectively, our study highlighted distinct roles of EMT in tamoxifen resistance and resveratrol as a potential agent to overcome acquired tamoxifen resistance. The molecular mechanism of resveratrol chemosensitizing effects is, at least in part, TGF-β/Smad-dependent.

In vivo, in vitro, and in silico studies suggest a conserved immune module that regulates malaria parasite transmission from mammals to mosquitoes

Human malaria can be caused by the parasite Plasmodium falciparum that is transmitted by female Anopheles mosquitoes. “Immunological crosstalk” between the mammalian and anopheline hosts for Plasmodium functions to control parasite numbers. Key to this process is the mammalian cytokine transforming growth factor-β1 (TGF-β1). In mammals, TGF-β1 regulates inducible nitric oxide (NO) synthase (iNOS) both positively and negatively. In some settings, high levels of NO activate latent TGF-β1, which in turn suppresses iNOS expression. In the mosquito, ingested TGF-β1 induces A. stephensi NOS (AsNOS), which limits parasite development and which in turn is suppressed by activation of the mosquito homolog of the mitogen-activated protein kinases MEK and ERK. Computational models linking TGF-β1, AsNOS, and MEK/ERK were developed to provide insights into this complex biology. An initial Boolean model suggested that, as occurs in mammalian cells, MEK/ERK and AsNOS would oscillate upon ingestion of TGF-β1. An ordinary differential equation (ODE) model further supported the hypothesis of TGF-β1-induced multiphasic behavior of MEK/ERK and AsNOS. To achieve this multiphasic behavior, the ODE model was predicated on the presence of constant levels of TGF-β1 in the mosquito midgut. Ingested TGF-β1, however, did not exhibit this behavior. Accordingly, we hypothesized and experimentally verified that ingested TGF-β1 induces the expression of the endogenous mosquito TGF-β superfamily ligand As60A. Computational simulation of these complex, cross-species interactions suggested that TGF-β1 and NO-mediated induction of As60A expression together may act to maintain multiphasic AsNOS expression via MEK/ERK-dependent signaling.We hypothesize that multiphasic behavior as represented in this model allows the mosquito to balance the conflicting demands of parasite killing and metabolic homeostasis in the face of damaging inflammation.

Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition

In the mammary gland, the stromal extracellular matrix (ECM) undergoes dramatic changes during development and in tumorigenesis. For example, normal adult breast tissue is largely devoid of the ECM protein fibronectin (FN) whereas high FN levels have been detected in the stroma of breast tumors. FN is an established marker for epithelial-mesenchymal transition (EMT), which occurs during development and has been linked to cancer. During EMT, epithelial cell adhesion switches from cell-cell contacts to mainly cell-ECM interactions raising the possibility that FN may have a role in promoting this transition. Using MCF-10A mammary epithelial cells, we show that exposure to exogenous FN induces an EMT response including up-regulation of the EMT markers FN, Snail, N-cadherin, vimentin, the matrix metalloprotease MMP2, α-smooth muscle actin, and phospho-Smad2 as well as acquisition of cell migratory behavior. FN-induced EMT depends on Src kinase and ERK/MAP kinase signaling but not on the immediate early gene EGR-1. FN initiates EMT under serum-free conditions; this response is partially reversed by a TGFβ neutralizing antibody suggesting that FN enhances the effect of endogenous TGFβ. EMT marker expression is up-regulated in cells on a fragment of FN containing the integrin-binding domain but not other domains. Differences in gene expression between FN and MG are maintained with addition of a sub-threshold level of TGFβ1. Together, these results show that cells interacting with FN are primed to respond to TGFβ. The ability of FN to induce EMT shows an active role for the stromal ECM in this process and supports the notion that the increased levels of FN observed in breast tumors facilitate tumorigenesis.

Notch4-dependent antagonism of canonical TGF-β1 signaling defines unique temporal fluctuations of SMAD3 activity in sheared proximal tubular epithelial cells

Transforming growth factor-β1 (TGF-β1) is thought to drive fibrogenesis in numerous organ systems. However, we recently established that ectopic expression of TGF-β1 abrogates collagen accumulation via canonical SMAD signaling mechanisms in a shear-induced model of kidney fibrosis. We herein delineate the temporal control of endogenous TGF-β1 signaling that generates sustained synchronous fluctuations in TGF-β1 cascade activation in shear-stimulated proximal tubule epithelial cells (PTECs). During 8-h exposure to physiological shear stress (0.3 dyn/cm²), PTECs experience in situ oscillatory concentrations of active endogenous TGF-β1 that are ~10-fold greater than those detected under higher stress regimes (2-4 dyn/cm²). The elevated levels of intrinsic TGF-β1 maturation observed under physiological conditions are accompanied by persistent downstream SMAD3 activation. Pathological shear stresses (2 dyn/cm²) first elicit temporal variations in phosphorylated SMAD3 with an apparent period of ~6 h, whereas even higher stresses (4 dyn/cm²) abolish SMAD3 activation. These divergent patterns of SMAD3 activation are attributed to varying levels of Notch4-dependent phospho-SMAD3 degradation. Depletion of Notch4 in shear-stimulated PTECs eventually increases the levels of active TGF-β1 protein by approximately fivefold, recovers stable SMAD phosphorylation and ubiquitinated SMAD species, and attenuates collagen accumulation. Collectively, these data establish Notch4 as a critical mediator of shear-induced fibrosis and further reinforce the renoprotective effects of canonical TGF-β1 signaling.

IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-β1-induced fibrotic responses

TGF-β1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-β1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localisation of IHG-1 is pivotal in the amplification of TGF-β1 signalling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-β1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (Δmts-IHG-1) repressed TGF-β1 fibrotic signalling in renal epithelial cells. In cells expressing Δmts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-β1. Δmts-IHG-1 modulation of TGF-β1 signalling was associated with increased Smad7 protein expression. Δmts-IHG-1 modulated TGF-β1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-β1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-β1 signal transduction and that IHG-1 is a key player in this modulation.

TGF‐β regulated miRNAs: therapeutic targets for cardiac hypertrophy

Heart failure (HF) is one of the most frequent causes of death worldwide. The underlying causes of HF are diverse but often relate to cardiac hypertrophy. The role of TGF‐β signaling in cardiac hypertrophy has been extremely contradictory. We have previously showed that targeted deletion of Smad4 unexpectedly leads to cardiac hypertrophy, demonstrating that the endogenous cardiomyocyte Smad4‐dependent TGF‐β pathway protects heart from cardiac hypertrophy and fibrosis.Recently, we have revealed that the function of endogenous TGF‐β/Smad signaling in maintaining cardiac homeostasis involves the downregulation of miRNAs inducing cardiac hypertrophy. We show that TGF‐β1 inhibits the expression of miR‐23a/miR‐27a/miR‐24–2 and miR‐23b/miR‐27b/miR‐24–1 clusters at the transcriptional level. Transgenic mice with cardiomyocyte‐specific overexpression of miR‐27b exhibit cardiac hypertrophy and dysfunction by directly targeting PPAR‐¦Ã. Most importantly, in vivo silencing of miR‐27b in a pressure‐overload‐induced mouse model of HF attenuates cardiac hypertrophy and dysfunction. The function and mechanisms of other miRNAs regulated by TGF‐β/Smad signaling in cardiac hypertrophy have also been investigated. All these results provide critical genetic evidence showing that TGF‐β‐regulated miRNAs might serve as efficient therapeutic targets for cardiac diseases.

Endogenous TGF‐β activity limits TSLP expression in the intervertebral disc tissue by suppressing NF‐κB activation

Thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, is highly expressed in herniated disc (HD) tissue and may act as a key molecule for the initiation of macrophage recruitment into the tissue and natural resorption of HD. However, it remains unclear how TSLP expression is regulated in the intervertebral discs. This study showed that expression of TSLP and phosphorylated NF-κB in HD tissue samples was inversely correlated with expression of phosphorylated Smad2/3 (an indicator of active TGF-β signaling) and vice versa in posterior lumbar spinal fusion samples. The pharmacological blockades of endogenous TGF-β activity induced TSLP expression in mouse intervertebral disc tissue culture, which was inhibited by NF-κB inhibitors. Additionally, phosphorylation of Smad2/3 was constitutively detected in mouse intervertebral disc tissue in the steady states. Collectively, these results suggest that endogenous TGF-β activity limits TSLP expression in intervertebral disc tissue in the steady states by suppressing NF-κB activation. The findings reveal a regulatory mechanism how TSLP expression is induced in the intervertebral disc tissue and suggest a novel role of TGF-β in maintaining the homeostasis of intervertebral disc tissue.