Attenuated TGF-β Signaling Research Articles

The Neurodevelopmental Protein POGZ Suppresses Metastasis in Triple-Negative Breast Cancer by Attenuating TGFβ Signaling.

The pogo transposable element-derived zinc finger protein, POGZ, is notably associated with neurodevelopmental disorders through its role in gene transcription. Many proteins involved in neurological development are often dysregulated in cancer, suggesting a potential role for POGZ in tumor biology. Here, we provided experimental evidence that POGZ influences the growth and metastatic spread of triple-negative breast cancers (TNBC). In well-characterized models of TNBC, POGZ exerted a dual role, both as a tumor promoter and metastasis suppressor. Mechanistically, loss of POGZ potentiated TGFβ pathway activation to exert cytostatic effects while simultaneously increasing the mesenchymal and migratory properties of breast tumors. Although POGZ levels are elevated in human breast cancers, the most aggressive forms of TNBC tumors, including those with increased mesenchymal and metastatic properties, exhibit dampened POGZ levels, and low POGZ expression was associated with inferior clinical outcomes in these tumor types. Taken together, these data suggest that POGZ is a critical suppressor of the early stages of the metastatic cascade. Significance: The POGZ neurodevelopmental protein plays dual functions in triple-negative breast cancers as a tumor promoter and metastasis suppressor, inhibiting TGFβ-regulated EMT to limit breast cancer metastatic progression.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-β (transforming growth factor-β) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-β family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-βR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-β-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-β signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-β signaling by downregulating TGF-β expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-β signaling in PASMCs. Such rebalance of BMP/TGF-β pathways is translationally important for PAH alleviation.

Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFβ signaling

TGFβ is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFβ stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFβ signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFβ1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFβ signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFβ signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFβ signaling targets, consistent with its extracellular binding to TGFβ. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFβ1 and attenuates (but importantly does not eliminate) TGFβ signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

Targeting stromal cell Syndecan-2 reduces breast tumour growth, metastasis and limits immune evasion.

Tumour stromal cells support tumourigenesis. We report that Syndecan‐2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan‐2 modulated TGFβ signalling (SMAD7, PAI‐1), migration and immunosuppression of patient‐derived tumour‐associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan‐2 in TASCs significantly enhanced TGFβ signalling (SMAD7, PAI‐1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFβ signalling (SMAD7, PAI‐1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan‐2‐peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFβ‐regulated immunosuppressive genes, such as CXCR4 and PD‐L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan‐2‐peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan‐2 within the TME inhibits tumour growth and metastasis due to decreased TGFβ signalling and increased immune control.

TGF-β Signaling Contributes to the Clonal Dominance of Jak2V617F Hematopoietic Stem/Progenitor Cells

Increased TGF-β expression in the bone marrow microenvironment is present in most cases of myeloproliferative neoplasms (MPNs), however, its contribution to disease pathogenesis is not well understood. Multiple prior studies show that TGF-β suppresses the proliferation of hematopoietic stem/progenitor cells (HSPCs) in vitro. However, its impact on HSCs in vivo is less clear, with conflicting data showing that TGF-β suppresses or induces HSPC proliferation, depending upon the dose of TGF-β and cell context. Here, we explore the hypothesis that HSPCs carrying a Jak2V617F mutation are resistant to the suppressive effects of TGF-β, conferring a fitness advantage that contributes to their expansion in MPNs. To test this hypothesis, we first assessed TGF-β signaling in wildtype (WT) and Jak2V617F HSPCs. Whereas TGF-β1 induced robust Smad2/3 phosphorylation in WT phenotypic hematopoietic stem cells (HSCs), it was barely detectable in Jak2V617F HSCs. Interestingly, this phenotype was lost in lineage-restricted progenitors, where TGF-β1-induced Smad2/3 phosphorylation was comparable in WT and Jak2V617F cells. These data suggest that TGF-β signaling, at least Smad2/3 phosphorylation, is selectively loss in Jak2V617F HSCs but not lineage-restricted progenitors. Competitive repopulation assays (CRAs) were performed using WT and Jak2V617F HSPCs in which Tgfbr2 was deleted to abrogate TGF-β signaling in HSCs. We first established bone marrow chimeras containing equal amounts of WT and UBC-CreERT2; Tgfbr2f/f HSPCs (hereafter referred to as TR2KO cells). Tamoxifen was given 6 weeks after transplantation to activate Cre expression and delete Tgfbr2. Multilineage donor chimerism of the Tgfbr2-deleted cells increased over time, suggesting that TGF-β signaling negatively regulates HSPC repopulating activity. However, when competing Jak2V617F cells with Jak2V617F TR2KO cells, the TR2KO compartment no longer had competitive advantage, suggesting that Jak2 mutated HSPC are insensitive to the suppressive effect of TGF-β. To extend these findings, CRAs were established that included the following 4 cohorts: 1) WT vs. Jak2V617F; 2) TR2KO vs. Jak2V617F; 3) TR2KO vs. Jak2V617FTR2KO; and 4) WT vs. Jak2V617FTR2KO. In each case, a 5:1 ratio of competitor to Jak2-mutated cells was used and tamoxifen was given 6 weeks after transplantation to induce both Cre-mediated deletion of Tgfbr2 and activation of the Jak2V617F allele. In cohort 1, as expected, donor chimerism with Jak2 mutated cells increased over time (Figure 1A, black). Consistent with this finding, Jak2 mutant chimerism in HSPCs was increased at 12-14 weeks (Figure 1B). In cohort 2 (orange), the loss of TGF-β signaling in WT HSPCs completely abrogated the competitive advantage of Jak2V617F HSPCs. In fact, the TR2KO cells outcompeted Jak2V617F HSPCs. In cohort 3 (red), we asked whether loss of TGF-β signaling in both WT and Jak2V617F HSPCs "leveled the playing field". Indeed, no expansion of Jak2 mutant peripheral blood leukocytes or HSPCs was observed in these mice. Finally, in cohort 4 (blue), we observed that loss of TGF-β signaling in Jak2 mutant cells had little impact on their repopulating activity. To assess the impact of TGF-β signaling on WT and Jak2V617F HSPC proliferation, we generated bone marrow chimeras containing either WT vs. Jak2V617F HSPCs or TR2KO vs. Jak2V617FTR2KO HSPCs. Of note, in both types of bone marrow chimeras, the presence of Jak2 mutant cells is expected to increase the local level of endogenous TGF-β. As expected, in the WT vs. Jak2V617F chimeras, the Jak2V617F HSPCs were less quiescent than WT cells. Surprisingly, loss of TGF-β signaling resulted in an increase in HSC quiescence in both TR2KO and Jak2V617F TR2KO HSCs. Of note, the loss of TGF-β signaling again "leveled the playing field" with a similar percentage of quiescent TR2KO vs. Jak2V617FTR2KO HSCs. Collectively, these data suggest that attenuated TGF-β signaling in Jak2V617F HSPCs plays a key role in the pathogenesis of MPN. Specifically, the data support a model in which increased local production of TGF-β by Jak2V617F hematopoietic cells selectively inhibits WT HSPCs, thereby promoting the expansion of Jak2V617F HSPCs. Targeting TGF-β signaling early in the course of MPN or in persons with Jak2V617F clonal hematopoiesis may prevent or delay clonal progression. Disclosures No relevant conflicts of interest to declare.

Syndecan-4 Inhibits the Development of Pulmonary Fibrosis by Attenuating TGF-β Signaling.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and its heparan sulfate glycosaminoglycan side chains bind to several proteins exhibiting various biological roles. The authors have previously demonstrated syndecan-4′s critical roles in pulmonary inflammation. In the current study, however, its role in pulmonary fibrosis was evaluated. Wild-type and syndecan-4-deficient mice were injected with bleomycin, and several parameters of inflammation and fibrosis were analyzed. The mRNA expression of collagen and α-smooth muscle action (α-SMA) in lung tissues, as well as the histopathological lung fibrosis score and collagen content in lung tissues, were significantly higher in the syndecan-4-deficient mice. However, the total cell count and cell differentiation in bronchoalveolar lavage fluid were equivalent between the wild-type and syndecan-4-deficient mice. Although there was no difference in the TGF-β expression in lung tissues between the wild-type and syndecan-4-deficient mice, significantly more activation of Smad3 in lung tissues was observed in the syndecan-4-deficient mice compared to the wild-type mice. Furthermore, in the in vitro experiments using lung fibroblasts, the co-incubation of syndecan-4 significantly inhibited TGF-β-induced Smad3 activation, collagen and α-SMA upregulation. Moreover, syndecan-4 knock-down by siRNA increased TGF-β-induced Smad3 activation and upregulated collagen and α-SMA expression. These findings showed that syndecan-4 inhibits the development of pulmonary fibrosis, at least in part, through attenuating TGF-β signaling.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.

Sparstolonin B (SsnB) attenuates liver fibrosis via a parallel conjugate pathway involving P53-P21 axis, TGF-beta signaling and focal adhesion that is TLR4 dependent

SsnB previously showed a promising role to lessen liver inflammation observed in a mouse model of NAFLD. Since NAFLD can progress to fibrosis, studies were designed to unravel its role in attenuating NAFLD associated fibrosis. Using both in vivo and in vitro approaches, the study probed the possible mechanisms that underlined the role of SsnB in mitigating fibrosis. Mechanistically, SsnB, a TLR4 antagonist, decreased TLR4-PI3k akt signaling by upregulating PTEN protein expression. It also decreased MDM2 protein activation and increased p53 and p21 gene and protein expression. SsnB also downregulated pro-fibrogenic hedgehog signaling pathway, inhibited hepatic stellate cell proliferation and induced apoptosis in hepatic stellate cells, a mechanism that was LPS dependent. Further, SsnB decreased fibrosis by antagonizing TLR4 induced TGFβ signaling pathway. Alternatively, SsnB augmented BAMBI (a TGFβ pseudo-receptor) expression in mice liver by inhibiting TLR4 signaling pathway and thus reduced TGFβ signaling, resulting in decreased hepatic stellate cell activation and extracellular matrix deposition. In vitro experiments on human hepatic stellate cell line showed that SsnB increased gene and protein expression of BAMBI. It also decreased nuclear co-localization of phospho SMAD2/3 and SMAD4 protein and thus attenuated TGFβ signaling in vitro. We also observed a significant decrease in phosphorylation of SMAD2/3 protein, decreased STAT3 activation, alteration of focal adhesion protein and stress fiber disassembly upon SsnB administration in hepatic stellate cells which further confirmed the antagonistic effect of SsnB on TLR4-induced fibrogenesis.

Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 editing inhibits epithelial to mesenchymal transition in ovarian cancer cells.

BIRC5 encodes the protein survivin, a member of the inhibitor of apoptosis family. Survivin is highly expressed in a variety of cancers but has very low expression in the corresponding normal tissues, and its expression is often associated with tumor metastasis and chemoresistance. We report that survivin was highly expressed in ovarian cancer and strongly correlated with patient overall poor survival. For the first time, we provide experimental evidence that survivin is involved in epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 gene editing led to the inhibition of EMT by upregulating epithelial cell marker, cytokeratin 7 and downregulating mesenchymal markers: snail2, β-catenin, and vimentin in both ovarian cancer SKOV3 and OVCAR3 cells. Consistent with this molecular approach, pharmacological treatment of ovarian cancer cells using a small molecule survivin inhibitor, YM155 also inhibited EMT in these ovarian cancer cell lines. Overexpression of BIRC5 promoted EMT in SKOV3 cells. Using molecular or pharmacological approaches, we found that cell proliferation, migration, and invasion were significantly inhibited following BIRC5 disruption in both cell lines. Inhibition of BIRC5 expression also sensitized cell responses to paclitaxel treatment. Moreover, loss of BIRC5 expression attenuated TGFβ signaling in both SKOV3 and OVCAR3 cells. Collectively, our studies demonstrated that disruption of BIRC5 expression inhibited EMT by attenuating the TGFβ pathway in ovarian cancer cells.

Abstract 3421: Pharmacological inhibition of histone methylation attenuates TGFβ induced EMT characteristics in pancreatic cancer

Abstract Epithelial-to-Mesenchymal Transition (EMT) is a tumor cell remodeling process that drives progression of several epithelial solid tumors including pancreatic tumors. Transforming growth factor (TGF) β signaling is a key promoter of EMT pathway and remains an attractive target for curtailing tumor progression. Here we present a strategy for investigating inhibitors of TGFβ signaling in pancreatic cancer via reprograming of miRNAs. We find a carbocyclic adenosine analog and histone methyl transferase inhibitor, 3-deazaneplanocin A (DZNep) to significantly attenuate the TGFβ signaling induced EMT characteristics in pancreatic ductal adenocarcinoma (PDAC). Specifically, we find DZNep treatment to restore expression of selective epigenetically silenced miRNAs (mir-663a, miR-4787-5p, and miR-202) that inhibit endogenous TGFβ1 ligand and receptor expression via RNA interference. Consequently, DZNep significantly attenuated both exogenous and endogenous TGFβ-induced EMT response. Lentiviral overexpression of mir-663a, miR-4787-5p or miR-202 reduced tumor cellular proliferation and EMT phenotypic characteristics similar to DZNep in cultured pancreatic cancer cells, whereas locked nucleic acids inhibition of miRNAs abrogated such effects. In an orthotopic pancreatic cancer mouse model, DZNep and all three miRs reduced overall tumor burden in pancreas and metastatic load in liver, lungs and spleen. Together, these findings suggest epigenetic reprograming of miRNAs by synthetic histone methylation reversal agents as a potentially viable approach for attenuating TGFβ signaling pathway involved in pancreatic cancer progression. Citation Format: Hardik R. Mody, Rajgopal Govindarajan. Pharmacological inhibition of histone methylation attenuates TGFβ induced EMT characteristics in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2017-3421

TGF-β signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Vascular mimicry formation is promoted by paracrine TGF-β and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma

Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM+ human HCC tissues compared to VM− ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-β or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-β1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-β and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5γ2 via TGF-βR1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-β signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-β signaling transduction by targeting TGF-βR1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-β/SDF1-VE-cadherin/MMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy.

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

Purpose: The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro.Methods: The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.Results: Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.Conclusions: Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.

σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila.

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling.

Abstract 9967: Altered TGF-β Signalling and Myofibroblast-like Cell Differentiation Contribute to Myxomatous Mitral Valve Disease

Upregulated Transforming Growth Factor-beta (TGF-β) signaling has been implicated in the pathogenesis of myxomatous mitral valve disease (MMVD) in both humans and canines. Moreover, it has been postulated that the phenotypic transformation of valve interstitial cells (VICs) in the mitral valve into alpha-smooth muscle actin (α-SMA)-positive myofibroblast-like cells contribute to elevated levels of proteolytic enzymes such as matrix metalloproteinases (MMPs), which subsequently induce pathological remodeling of the extracellular matrix (ECM) in the mitral valve. We hypothesized that, in MMVD, upregulation of TGF-β signaling would induce the transformation of VICs into myofibroblast-like cells, which subsequently express increased levels of MMPs. Computational immunohistochemistry was used to quantify protein expression in mitral valve leaflet tissue from canines affected by MMVD (n=4) and from control canines (n=5). TGF-β and α-SMA expression was significantly increased (p<0.001) in MMVD tissue compared to control tissue. Moreover, a significant positive correlation was found between TGF-β expression and α-SMA expression (p<0.05) across the entire cohort. There were also significant increases in MMP-2 and MMP-9 expression (p<0.05) in MMVD tissue compared to control tissue. No change in MMP-3 expression was detected. These findings are consistent with abnormal TGF-β signaling inducing the differentiation of VICs into α-SMA-positive myofibroblast-like cells. Moreover, the increases in expression of MMP-2 and MMP-9 in MMVD indicate the likely role of these myofibroblast-like cells in mediating pathological remodeling of the ECM throughout the disease process. The paradoxical finding of no change in MMP-3 expression may represent a compensatory mechanism that attempts to limit further degenerative damage to the mitral valve. In conclusion, MMVD is characterized by TGF-β-induced differentiation of VICs into myofibroblast-like cells, which subsequently facilitate ECM degradation via the increased expression of MMPs. This supports the rationale for the use of angiotensin II receptor blockers, capable of attenuating TGF-β signaling, in the treatment of MMVD.

Systemic attenuation of the TGF-β pathway by a single drug simultaneously rejuvenates hippocampal neurogenesis and myogenesis in the same old mammal

Stem cell function declines with age largely due to the biochemical imbalances in their tissue niches, and this work demonstrates that aging imposes an elevation in transforming growth factor β (TGF-β) signaling in the neurogenic niche of the hippocampus, analogous to the previously demonstrated changes in the myogenic niche of skeletal muscle with age. Exploring the hypothesis that youthful calibration of key signaling pathways may enhance regeneration of multiple old tissues, we found that systemically attenuating TGF-β signaling with a single drug simultaneously enhanced neurogenesis and muscle regeneration in the same old mice, findings further substantiated via genetic perturbations. At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-β1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of β2 microglobulin (B2M), a component of MHC class I molecules. These findings suggest that at high levels typical of aged tissues, TGF-β1 promotes inflammation instead of its canonical role in attenuating immune responses. In agreement with this conclusion, inhibition of TGF-β1 signaling normalized B2M to young levels in both studied tissues.

Zinc-α2-Glycoprotein Exerts Antifibrotic Effects in Kidney and Heart.

Zinc-α2-glycoprotein (AZGP1) is a secreted protein synthesized by epithelial cells and adipocytes that has roles in lipid metabolism, cell cycling, and cancer progression. Our previous findings in AKI indicated a new role for AZGP1 in the regulation of fibrosis, which is a unifying feature of CKD. Using two models of chronic kidney injury, we now show that mice with genetic AZGP1 deletion develop significantly more kidney fibrosis. This destructive phenotype was rescued by injection of recombinant AZGP1. Exposure of AZGP1-deficient mice to cardiac stress by thoracic aortic constriction revealed that antifibrotic effects were not restricted to the kidney but were cardioprotective. In vitro, recombinant AZGP1 inhibited kidney epithelial dedifferentiation and antagonized fibroblast activation by negatively regulating TGF-β signaling. Patient sera with high levels of AZGP1 similarly attenuated TGF-β signaling in fibroblasts. Taken together, these findings indicate a novel role for AZGP1 as a negative regulator of fibrosis progression, suggesting that recombinant AZGP1 may have translational effect for treating fibrotic disease.

Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis.

IntroductionTransforming growth factor beta (TGFβ) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFβ receptor (TβRII) for signaling. Previously, we have shown that deletion of TβRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFβ signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFβ signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.MethodsAll studies were performed on MMTV-Neu mice with and without dominant-negative TβRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.ResultsAttenuated TGFβ signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.ConclusionOur studies provide insights into a novel mechanism by which epithelial TGFβ signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFβ pathway in vivo during tumor progression remain controversial. The targeting of TGFβ signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFβ signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0425-7) contains supplementary material, which is available to authorized users.

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

Glucosamine hydrochloride exerts a protective effect against unilateral ureteral obstruction-induced renal fibrosis by attenuating TGF-β signaling

Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-β1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-β signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-β1-induced renal fibrosis. In the obstructed kidneys and TGF-β1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-β signaling by inhibiting N-linked glycosylation of the type II TGF-β receptor (TβRII), leading to an inefficient trafficking of TβRII to the membrane surface. Defective N-glycosylation of TβRII further suppressed the TGF-β1-binding to TβRII, thereby decreasing TGF-β signaling. Notably, GS-HCl treatment significantly reduced TGF-β1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-β signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-β1-induced renal fibrosis in kidney diseases.Key message Glucosamine-mediated attenuation of TGF-β signaling ameliorates renal fibrosis in vivoTGF-β1-induced fibrogenic action is reduced by glucosamine in vitro N-glycosylation of the type II TGF-β receptor is suppressed by glucosamineGlucosamine-induced defective N-glycosylation of TβRII decreases TGF-β signaling. Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-013-1086-1) contains supplementary material, which is available to authorized users.