Downstream TGFβ Research Articles

Bruceine A inhibits TGF-β1/Smad pathway in pulmonary fibrosis by blocking gal3/TGF-β1 interaction

BackgroundBruceine A(BA) has many pharmacological activities and significantly inhibits fibrosis in keloid fibroblasts. However, the underlying mechanisms have not yet been fully elucidated. ObjectiveThis study aimed to investigate the effects of BA on pulmonary fibrosis(PF) and explore its underlying mechanisms. MethodsPF models were constructed by BLM-induced C57BL/6 J mice, TGF-β1- induced MRC-5 and HFL-1 cells. Cell proliferation, MMP, apoptosis, and ROS levels were analyzed in vitro. In vivo, experiments were performed to evaluate the therapeutic effect of BA on PF by detecting respiratory function, histopathology, and collagen level. Fibro-associated, ECM, and EMT key proteins were used to assess the degree of PF. To predict the target of BA by molecular docking technology, and verified by DARTS, CETSA, MST,and SPR. Then overexpression gal3-lentivirus, GB1107 gal3 inhibitor, and BA addition were used to verify the TGF-β1/Smad pathway key protein by western blot. ResultsWe found that BA inhibited PF both in vitro and in vivo. The predicted and validated results showed that gal3 was the target of BA, and the binding site was Arg144, His158, and Trp181. Mechanistically, BA disrupts the interaction between gal3 and TGF-β1. BA reduced Smad2/3 and p-Smad2/3 protein content and inhibited TGF-β1/Smad pathway in the overexpressing gal3 HFL-1 cells. After adding GB1107, the inhibitory effect of BA on TGF-β1/Smad pathway disappeared. ConclusionThis study is the first to demonstrate that BA can target gal3, interfere with the interaction between gal3 and TGF-β1 protein, inhibit the downstream TGF-β1/Smad pathway, and act as a “brake” to reverse the PF process. These findings provide a solid scientific basis for the clinical application of BA in the prevention and treatment of PF.

Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

Oral submucous fibrosis (OSF) is a chronic and inflammatory mucosal disease caused by betel quid chewing, which belongs to oral potentially malignant disorders. Abnormal fibroblast differentiation leading to disordered collagen metabolism is the core process underlying OSF development. The epithelium, which is the first line of defense against the external environment, can convert external signals into pathological signals and participate in the remodeling of the fibrotic microenvironment. However, the specific mechanisms by which the epithelium drives fibroblast differentiation remain unclear. In this study, we found that Arecoline-exposed epithelium communicated with the fibrotic microenvironment by secreting exosomes. MiR-17-5p was encapsulated in epithelial cell-derived exosomes and absorbed by fibroblasts, where it promoted cell secretion, contraction, migration and fibrogenic marker (α-SMA and collagen type I) expression. The underlying molecular mechanism involved miR-17-5p targeting Smad7 and suppressing the degradation of TGF-β receptor 1 (TGFBR1) through the E3 ubiquitination ligase WWP1, thus facilitating downstream TGF-β pathway signaling. Treatment of fibroblasts with an inhibitor of miR-17-5p reversed the contraction and migration phenotypes induced by epithelial-derived exosomes. Exosomal miR-17-5p was confirmed to function as a key regulator of the phenotypic transformation of fibroblasts. In conclusion, we demonstrated that Arecoline triggers aberrant epithelium-fibroblast crosstalk and identified that epithelial cell-derived miR-17-5p mediates fibroblast differentiation through the classical TGF-β fibrotic pathway, which provided a new perspective and strategy for the diagnosis and treatment of OSF.

The Role of TGF-β1/SMAD in Diabetic Nephropathy: Mechanisms and Research Development

Diabetic nephropathy (DN) is a common complication of diabetes and a leading cause of end-stage renal disease. Transforming growth factor-β1 (TGF-β1)/SMAD signaling activation plays an important role in the onset and progression of DN. Reported findings suggest that the activation of TGF-β1 (including the latent form, the active form, and the receptors) and its downstream signaling proteins (SMAD3, SMAD7, etc.) plays a critical role in DN. In addition, TGF-β1/SMAD signaling may mediate the pathogenesis and progression of DN via various microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Emerging evidence shows that TGF-β1, SMAD3, and SMAD7 are the main signaling proteins that contribute to the development of DN, and that they can be potential targets for the treatment of DN. However, recent clinical trials have shown that the anti-TGF-β1 monoclonal antibody treatment fails to effectively alleviate DN, which suggests that upstream inhibition of TGF-β1/SMAD signaling does not alleviate clinical symptoms and that this may be related to the fact that TGF-β1/SMAD has multiple biological effects. Targeted inhibition of the downstream TGF-β1 signaling (e.g., SMAD3 and SMAD7) may be an effective approach to attenuate DN. This article discussed the current understanding of the molecular mechanisms and potential targets for the treatment and prevention of DN by focusing on TGF-β1/SMAD signaling.

Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-β-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-β (TGF-β)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-β receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-β signaling. Pharmacological inhibition of the downstream TGF-β effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-β1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-β receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-β-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-β-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-β signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.

Jiangtang Tongmai Prescription Reduced Diabetic Lung Injury Through SnoN and TGF-β1/Smads Signaling Pathway.

By establishing a rat diabetes model in rats with intervening treatment by Jiangtang Tongmai Prescription (JTTMP), this study explored the restorative pairing effect of JTTMP on diabetic lung injury. The model of type II diabetes model was used to establish the rat diabetes model, using a high-fat diet and streptozotocin (STZ) induction. Different doses of JTTMP and metformin were administered as a therapeutic to intervene, and blood was collected to assess the blood glucose level of each group of rats. HE (Hematoxylin and eosin (H&E) staining was performed to detect the morphological changes in rat lung tissue and enzyme-linked immunoassay ELISA was used to detect and quantify the expression of interleukin (IL)-6, TNF tumor necrosis factor-ɑa, and IL-1β in serum and the lung tissue of each group of rats. The level expression of TGF-β1 [transforming growth factor (TGF)-β1), SnoN (transcriptional co-repressor Ski-N terminal (SnoN)], Smad2, Smad3, Smad7, and other signaling pathway proteins were assessed by Western blot. In comparison with the normal control (NC) group, rats in the diabetes model (DM) group lost weight and showed significantly increased blood sugar levels. The levels of TGF-β1 and Smad2/3 were increased in the DM group but Smad7 decreased. After 8 weeks of JTTMP intervention, the level of TGF-β1 and Smad2/3 decreased but Smad7 increased, blood sugar decreased significantly and the expression of inflammatory factors in lung tissue decreased. Therefore, JTTMP may activate SnoN and the downstream TGF-β1/Smads signaling pathway to repair diabetic lung injury, which suggests its application has potential for future clinical treatment of diabetes with lung injury.

METTL3-mediated macrophage exosomal NEAT1 contributes to hepatic fibrosis progression through Sp1/TGF-β1/Smad signaling pathway

Hepatic fibrosis (HF) is caused by chronic hepatic injury and is characterized by hepatic stellate cells (HSCs) activation. Studies focusing on the function of exosomes derived from macrophages in HF progression are limited. This study aims to identify the roles of exosomal NEAT1 derived from macrophages on HF and the underlying mechanisms. Our studies showed that METTL3 targeted and enhanced NEAT1 expression in macrophages. Exosomal NEAT1 originating from LPS-treated macrophages promoted HSCs proliferation and migration, and induced the expression of fibrotic proteins including collagen I, α-SMA, and fibronectin. Macrophage exosomal NEAT1 contributed to HSCs activation by sponging miR-342. MiR-342 directly targeted Sp1 and suppressed its downstream TGF-β1/Smad signaling pathway, which eventually led to the inhibition of HSCs activation. Depletion of NEAT1 in the macrophage exosomes inhibited HF progression both in vitro and in vivo. Altogether, our study proved that silence of NEAT1 in the macrophage exosomes exerted protective roles against HF through the miR-342/Sp1/TGF-β1/Smad signaling pathway, suggesting a potential therapeutic target in HF treatment.

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.

Dimethyl fumarate ameliorates stress urinary incontinence by reversing ECM remodeling via the Nrf2-TGF-β1/Smad3 pathway in mice.

Mechanical trauma and oxidative injury are involved in the pathogenesis of stress urinary incontinence (SUI), and oxidative stress (OS) is considered a potential therapeutic target. The antioxidant properties of dimethyl fumarate (DMF), a potent activator of Nrf2, have been highlighted recently. We therefore predicted that DMF might have therapeutic effects on mechanical trauma-induced SUI. The SUI mice model was established by vaginal distension (VD). Leak point pressure (LPP), serum OS biomarkers, cell proliferation and apoptosis, collagen, elastin, matrix metalloproteinases (MMP), Nrf2, the TGF-β1/Smad3 signaling pathway, and the associated tissue growth factors in the anterior vaginal wall were measured in either wild-type or Nrf2-knockout (Nrf2-/-) female C57BL/6 mice. The results showed that DMF improved the VD-induced LPP reduction, alleviated oxidative injury, stimulated cell proliferation and inhibited apoptosis in the anterior vaginal wall tissue of mice. Moreover, DMF treatment reduced the hydrolysis of ECM proteins by MMP2 and MMP9. The above effects may be mediated by a series of tissue growth factors, including α-SMA, PAI-1, and TIMP-2, with the TGF-β1/Smad3 signaling pathway as the core regulatory mechanism. In further study, Nrf2-/- mice were used to replicate the SUI model. And the difference is that DMF failed to reactivate the TGF-β1/Smad3 pathway, nor did it improve LPP. Dimethyl fumarate can ameliorate urethra closure dysfunction in the VD-induced SUI mice model, and the therapeutic effect of DMF is mediated by the Nrf2-dominated antioxidant system and its downstream TGF-β1/Smad3 signaling pathway.

Pilot Study of Combination Gemogenovatucel-T (Vigil) and Durvalumab in Women With Relapsed BRCA-wt Triple-Negative Breast or Ovarian Cancer

Background:Gemogenovatucel-T (Vigil) is a triple-function autologous tumor cell immunotherapy which expresses granulocyte-macrophage colony-stimulating factor and decreases expression of furin and downstream TGF-β1 and TGF-β2. Vigil has suggested survival benefit in frontline maintenance ovarian cancer patients who are BRCA-wt. In addition, Vigil demonstrates relapse-free and overall survival advantage in homologous recombination-proficient patients with OC. Further evidence of clinical benefit and safety has been demonstrated in combination with atezolizumab.Methods:In this pilot study (NCT02725489), the concurrent combination of the programmed death-ligand 1 (PD-L1) inhibitor durvalumab and Vigil was explored in advanced BRCA-wt relapsed triple-negative breast cancer (TNBC) patients and stage III-IV recurrent/refractory OC patients. Patients received the combination regimen of Vigil (1 × 10e6-10e7 cells/dose intradermally, up to 12 doses) and durvalumab (1500 mg/dose intravenous infusion, up to 12 months) once every 4 weeks. The primary objective was to evaluate safety of this combination. The study included 13 BRCA-wt patients (TNBC, n = 8; OC, n = 5).Results:The most common treatment-emergent adverse events (⩾20%) in all patients included injection-site reaction (92.3%), myalgia (38.5%), bruise at injection site (23.1%), and pruritus (23.1%). Three grade 3 treatment-related adverse events were observed and related to durvalumab. There were no grade 4/5 treatment-related adverse events. Median progression-free survival was 7.1 months and the median overall survival was not reached. Prolonged progression-free survival was improved in patients with PD-L1+ tumors (n = 8, hazard ratio = 0.304, 95% confidence interval, 0.0593-1.56, 1-sided P = .04715) compared with those with PD-L1− tumors.Conclusions:Vigil plus durvalumab was well tolerated and showed promising clinical activity in advanced BRCA-wt TNBC and stage III-IV recurrent/refractory OC patients.

Revisiting the Role of TGFβ Receptor Internalization for Smad Signaling: It is Not Required in Optogenetic TGFβ Signaling Systems.

Endocytosis is an important process by which many signaling receptors reach their intracellular effectors. Accumulating evidence suggests that internalized receptors play critical roles in triggering cellular signaling, including transforming growth factor β (TGFβ) signaling. Despite intensive studies on the TGFβ pathway over the last decades, the necessity of TGFβ receptor endocytosis for downstream TGFβ signaling responses is a subject of debate. In this study, mathematical modeling and synthetic biology approaches are combined to re-evaluate whether TGFβ receptor internalization is indispensable for inducing Smad signaling. It is found that optogenetic systems with plasma membrane-tethered TGFβ receptors can induce fast and sustained Smad2 activation upon light stimulations. Modeling analysis suggests that endocytosis is precluded for the membrane-anchored optogenetic TGFβ receptors. Therefore, this study provides new evidence to support that TGFβ receptor internalization is not required for Smad2 activation.

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Translational pharmacology of an inhaled small molecule \u03b1v\u03b26 integrin inhibitor for idiopathic pulmonary fibrosis

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Galectin-3 Induces Atrial Fibrosis by Activating the TGF-β1/Smad Pathway in Patients with Atrial Fibrillation

Background: Atrial fibrosis plays a critical role in the occurrence and maintenance of atrial fibrillation. The role of TGF-β1 in mediating atrial fibrosis is well documented. The β-galactoside-binding lectin galectin-3 (Gal-3) is mainly produced by macrophages in biological events such as inflammation and angiogenesis. Previous studies have shown that Gal-3 is associated with atrial fibrosis, but the relationship between TGF-β1 and Gal-3 in atrial fibrosis remains unclear. Objective: To determine whether Gal-3 induces atrial fibrosis and atrial fibrillation by activating the TGF-β1/Smad pathway and whether the expression of Gal-3 is mediated by TGF-β1, which can enable assessing the relationship between Gal-3 and TGF-β1 in atrial fibrosis. Methods: In this study, 30 patients’ right atrial appendages were collected and divided into 3 groups: congenital heart disease sinus rhythm group (n = 10, as a control group), rheumatic heart disease sinus rhythm group (n = 10), and rheumatic heart disease atrial fibrillation group (n = 10). Rat atrial fibroblasts were cultured in vitro, and recombinant Gal-3 and recombinant TGF-β1 proteins were added to the cell culture. The expression of Gal-3, TGF-β1, Smad2, and collagen I was detected by Western blotting and quantitative real-time PCR. Atrial tissues were stained with Masson’s trichrome stain to evaluate the extent of atrial fibrosis. The expression of Gal-3 and TGF-β1 was detected by immunohistochemical staining and immunofluorescence staining. Gal-3 and TGF-β1 interaction was demonstrated by immunoprecipitation. Results: The expression levels of Gal-3, TGF-β1, Smad2, and collagen I were elevated in the rheumatic heart disease atrial fibrillation group compared with the congenital heart disease sinus rhythm group and the rheumatic heart disease sinus rhythm group. In cultured atrial fibroblasts, there is a synergistic interaction between Gal-3 and TGF-β1. Gal-3 stimulated the TGF-β1/Smad pathway, and overexpression of TGF-β1 induced Gal-3 expression. Conclusions: Gal-3 and TGF-β1 interact with each other and stimulate the downstream TGF-β1/Smad pathway. This finding suggests that Gal-3 could be an important factor in TGF-β1-induced fibrosis in atrial fibrillation.

Abstract 39: Novel Lineage Tracing System To Identify Site-specific Ectopic Skeletal Stem Cells

Purpose: Traumatic heterotopic ossification (HO) is the formation of bone outside of the healthy skeletal system after traumatic injury causing decreased range of motion and discomfort in affected patients. Previous research has identified potential progenitor cells of HO, however, this is still contended. Better understanding of the cells contributing to HO will allow for targeted treatment to reduce HO in high-risk patients. It was recently discovered that cells expressing Hoxa11, an embryonic patterning transcription factor expressed exclusively in the zeugopod, is expressed by mesenchymal stem cells (MSCs) into adulthood and all skeletal lineages are marked downstream of these progenitors using a CreERT2 lineage tracing system. Currently, most lineage tracing systems label MSCs throughout the body, making it difficult to determine if genetic deletion is due to systemic or local effects. We hypothesize, using a mouse model of HO that Hoxa11+ MSCs will differentiate into site-specific HO-progenitor cells, and eventual form HO. Methods:Hoxa11-CreERT2;ROSA-TdTomato mice were treated with tamoxifen at 6 weeks of age, chased for one week, then injured with an established and reproducible HO-forming Achilles’ tenotomy with concurrent 30% total body surface area back burn. Injured and uninjured hind limb samples were harvested one week, three weeks, and nine weeks post-injury, sectioned for immunofluorescent histology, and imaged by confocal microscopy. Results: Examining the uninjured hindlimb of the Hoxa11-CreERT2;ROSA-TdTomato mouse, tdTomato-marked Hoxa11 lineage-positive cells were found throughout the zeugopod within tendon, enthesis, and bone. These cells were PDGFRα+ (not shown). Three weeks after injury, Hoxa11 lineage-positive cells are found at regions of heterotopic ossification in the burn/tenotomy model, specifically at the cut end of the tendon and calcaneus. After injury, Hoxa11 lineage+/PDGFRα+ cells express chondrocyte marker, SOX9, and pSMAD3, a downstream TGFβ1 signaling pathway transcription factor central to HO formation. Three weeks after injury, condensing chondrocytes within the HO anlagen are Hoxa11 lineage-positive and are colocalized with early bone marker, RUNX2. At nine weeks post-injury, SP7+ osteoblast and RUNX2+ pre-osteoblasts are marked with tdTomato. Hoxa11 cells were also able to differentiate into adipocytes and showed colocalization with perilipin and adipocyte morphology. Conclusions: Our results suggest that Hoxa11+ stem cells differentiate through endochondral ossification into heterotopic bone after injury, highlighting that local cells within the hind limb form the majority of HO bone, rather than circulating progenitor cells. Additionally, this lineage tracing system can be a useful tool to study skeletal stem cells and tendon pathology within the zeugopod.

Senescence of Alveolar Stem Cells Drives Progressive Pulmonary Fibrosis

SummaryTissue fibrosis is a common pathological outcome of chronic disease that markedly impairs organ function leading to morbidity and mortality. In the lung, idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Here, we show that alveolar type 2 (AT2) stem cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence with associated loss of SIN3A, a critical determinant of endodermal progenitor cell function in the developing lung. Conditional loss of Sin3a in adult mouse AT2 cells initiated a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We establish that senescence rather than loss of epithelial stem cells serves as a proximal driver of Tgfβ activation and progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation, senescence, or downstream Tgfβ activation, block fibrogenesis.

Neotuberostemonine inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1α signaling.

Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 μmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-β, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-β, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

Activation of TGF-β1/α-SMA/Col I Profibrotic Pathway in Fibroblasts by Galectin-3 Contributes to Atrial Fibrosis in Experimental Models and Patients

Background/Aims: This study aimed to evaluate whether galectin-3 (Gal-3) contributes actively to atrial fibrosis both in patients and experimental atrial fibrillation (AF) models. Methods: Mouse HL-1 cardiomyocytes were subjected to rapid electrical stimulation (RES) to explore Gal-3 expression and secretion levels by western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Neonatal rat cardiac fibroblasts were treated with conditioned culture medium and recombinant human Gal-3 to evaluate the activation of the transforming growth factor (TGF)-β1/α-smooth muscle actin (SMA)/collagen I (Col I) profibrotic pathway (WB) and fibroblast proliferation with a Cell Counting Kit-8 (CCK-8). Furthermore, in the rapid atrial pacing (RAP) rabbit AF model, atrial Gal-3 expression and its effects on the profibrotic pathway were evaluated (WB and Masson’s trichrome staining). Moreover, 44 consecutive patients who underwent single mitral valve repair/replacement were included, consisting of 28 patients with persistent AF (PeAF) and 16 with sinus rhythm (SR). Coronary sinus blood was also sampled to test circulating Gal-3 levels (ELISA), and atrial myocardium Gal-3 and its downstream TGF-β1/α-SMA pathway were also measured by WB and immunohistochemical staining. Results: Gal-3 expression in HL-1 cells and its secretion level in culture medium were greatly increased after 24 h RES. Treatment of neonatal rat cardiac fibroblasts with conditioned media collected from the RES group or recombinant human Gal-3 protein (10 and 30 µg/mL) for 72 h induced the activation of the TGF-β1/α-SMA/Col I profibrotic pathway. RAP increased Gal-3 levels and activated the TGF-β1/α-SMA/Col I pathway in rabbit left atria, while the Gal-3 inhibitor N-acetyllactosamine, injected at 4.5 mg/kg every 3 days, mitigated these adverse changes. Furthermore, Gal-3 levels in coronary sinus blood samples and myocardial Gal-3 expression levels were higher in the PeAF patients than in the SR patients, and higher level profibrotic pathway activation was also confirmed. Conclusions: Activation of Gal-3 expression in the atria can subsequently activate the TGF-β1/α-SMA/Col I pathway in cardiac fibroblasts, which may enhance atrial fibrosis.

R-Spondin1/LGR5 Activates TGFβ Signaling and Suppresses Colon Cancer Metastasis.

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, is known to exhibit tumor suppressor activity in colon cancer, the mechanism of which is not understood. Here we show that R-spondin 1 (RSPO1)/LGR5 directly activates TGFβ signaling cooperatively with TGFβ type II receptor in colon cancer cells, enhancing TGFβ-mediated growth inhibition and stress-induced apoptosis. Knockdown of LGR5 attenuated downstream TGFβ signaling and increased cell proliferation, survival, and metastasis in an orthotopic model of colon cancer in vivo Upon RSPO1 stimulation, LGR5 formed complexes with TGFβ receptors. Studies of patient specimens indicate that LGR5 expression was reduced in advanced stages and positively correlated with markers of TGFβ activation in colon cancer. Our study uncovers a novel cross-talk between LGR5 and TGFβ signaling in colon cancer and identifies LGR5 as a new modulator of TGFβ signaling able to suppress colon cancer metastasis. Cancer Res; 77(23); 6589-602. ©2017 AACR.

MicroRNA-20b promotes cell proliferation via targeting of TGF-β receptor II and upregulates MYC expression in Ewing's sarcoma cells.

Transforming growth factor-β receptor II (TGFBR2) is implicated in various types of cancer. Most molecules involved in the TGF-β pathway can be degraded by one or more microRNAs (miRNAs). In the present study, we show that miRNA plays an important role in downregulating TGFBR2 expression in Ewing's sarcoma (ES) cells. Microarray-based analyses revealed that the expression of miR-20b was significantly increased, whereas TGFBR2 and MYC were significantly downregulated and upregulated, respectively, in all ES cells compared to their expression in human mesenchymal stem cells (hMSCs). In ES cell lines, anti-miR-20b increased TGFBR2 expression and significantly decreased MYC expression, showing an inverse relationship with TGFBR2. The induction by anti-miR-20b further prohibited ES cell growth and cell cycle progression. Moreover, decreased miR-20b in ES cells significantly inhibited tumor growth invivo. Taken together, these results suggest that miR-20b behaves as an oncogene in ES when its overexpression is unregulated by targeting TGFBR2. Because downstream TGFBR2 and TGF-β signaling regulate cell cycle, apoptosis, and tumor proliferation via MYC, our findings may contribute to new targeted therapies for ES.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells

BackgroundChondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. ResultsCo-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2–4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA12-luciferase activity. ConclusionsTGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation.