Smad Activation Research Articles

Targeting selective E3 ligase for healing cardiac fibrosis

Protein quality control is often disrupted in heart diseases. In this issue of Cell Chemical Biology, Yang etal.1 describe the selective E3 ligase SPOP as a pro-fibrotic factor for inducing cardiac fibrosis through RACK1 degradation-mediated Smad3 activation.

Catechin hydrate ameliorates cerulein‑induced chronic pancreatitis via the inactivation of TGF‑β/Smad2 signaling.

Chronic pancreatitis (CP) is a pancreatic inflammatory disease associated with histological changes, including fibrosis, acinar cell loss and immune cell infiltration, and leads to damage of the pancreas, which results in pain, weight loss and loss of pancreas function. Catechin or catechin hydrate (CH) has antioxidant, anticancer and immune‑regulatory effects. However, unlike other catechins, the antifibrotic effects of (+)‑CH have not been widely studied in many diseases, including CP. Therefore, the anti‑fibrotic effects of (+)‑CH against CP were evaluated in the present study. To assess the prophylactic effects of CH, (+)‑CH (1, 5 or 10mg/kg) or ethanol was administered 1h before first cerulein (50µg/kg) injection. To assess the therapeutic effects, (+)‑CH (5mg/kg) or ethanol was administered after cerulein injection for one or two weeks. In both methods, cerulein was injected intraperitoneally into mice once every hour, six times a day, four times a week, for a total of three weeks, to induce CP. The data showed that (+)‑CH markedly inhibited glandular destruction and inflammation during CP. Moreover, (+)‑CH prevented pancreatic stellate cell (PSC) activation and the production of extracellular matrix components, such as fibronectin 1 and collagens, which suggested that it may act as a novel therapeutic agent. Furthermore, the mechanism and effectiveness of (+)‑CH on pancreatic fibrosis were investigated in isolated PSCs. (+)‑CH suppressed the activation of Smad2 and fibrosis factors that act through transforming growth factor‑β (TGF‑β) or platelet‑derived growth factor. These findings suggest that (+)‑CH exhibits antifibrotic effects in cerulein‑induced CP by inactivating TGF‑β/Smad2 signaling.

SUMOylation of Smad2 mediates TGF-β-regulated endothelial–mesenchymal transition

Endothelial-mesenchymal transition (EndoMT) is a complex biological process in which endothelial cells are transformed into mesenchymal cells, and dysregulated EndoMT causes a variety of pathological processes. Transforming growth factor beta (TGF-β) signaling effectively induces the EndoMT process in endothelial cells, and Smad2 is the critical protein of the TGF-β signaling pathway. However, whether small ubiquitin-like modifier modification (SUMOylation) is involved in EndoMT remains unclear. Here, we show that Smad2 is predominantly modified by SUMO1 at two major SUMOylation sites with PIAS2α as the primary E3 ligase, whereas SENP1 (sentrin/SUMO-specific protease 1) mediates the deSUMOylation of Smad2. In addition, we identified that SUMOylation significantly enhances the transcriptional activity and protein stability of Smad2, regulating the expression of downstream target genes. SUMOylation increases the phosphorylation of Smad2 and the formation of the Smad2-Smad4 complex, thus promoting the nuclear translocation of Smad2. Ultimately, the wildtype, but not SUMOylation site mutant Smad2 facilitated the EndoMT process. More importantly, TGF-β enhances the nuclear translocation of Smad2 by enhancing its SUMOylation and promoting the EndoMT process. These results demonstrate that SUMOylation of Smad2 plays a critical role in the TGF-β-mediated EndoMT process, providing a new theoretical basis for the treatment and potential drug targets of EndoMT-related clinical diseases.

TGF-β1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

The TGF-β/UCHL5/Smad2 Axis Contributes to the Pathogenesis of Placenta Accreta.

Placenta accreta is a high-risk condition causing obstetric crisis and hemorrhage; however, its pathogenesis remains unknown. We aimed to identify the factors contributing to trophoblast invasiveness and angiogenic potential, which in turn drive the pathogenesis of placenta accreta. We focused on the transforming growth factor (TGF)-β1-Smad pathway and investigated the intrinsic relationship between the time- and dose-dependent inhibition of the ubiquitinating enzyme UCHL5 using bAP15, a deubiquitinase inhibitor, after TGF-β1 stimulation and the invasive and angiogenic potential of two cell lines, gestational choriocarcinoma cell line JEG-3 and trophoblast cell line HTR-8/SVneo. UCHL5 inhibition negatively regulated TGF-β1-induced Smad2 activation, decreasing extravillous trophoblast invasiveness. Smad1/5/9 and extracellular signal-regulated kinase (ERK) were simultaneously activated, and vascular endothelial growth factor was secreted into the trophoblast medium. However, extravillous trophoblast culture supernatant severely impaired the vasculogenic potential of human umbilical vein endothelial cells. These results suggest that the downstream ERK pathway and Smad1/5/9 potentially regulate the TGF-β1-Smad pathway in extravillous trophoblasts, whereas Smad2 contributes to their invasiveness. The abnormal invasive and angiogenic capacities of extravillous cells, likely driven by the interaction between TGF-β1-Smad and ERK pathways, underlie the pathogenesis of placenta accreta.

TGF-β uncouples glycolysis and inflammation in macrophages and controls survival during sepsis.

Changes in metabolism of macrophages are required to sustain macrophage activation in response to different stimuli. We showed that the cytokine TGF-β (transforming growth factor-β) regulates glycolysis in macrophages independently of inflammatory cytokine production and affects survival in mouse models of sepsis. During macrophage activation, TGF-β increased the expression and activity of the glycolytic enzyme PFKL (phosphofructokinase-1 liver type) and promoted glycolysis but suppressed the production of proinflammatory cytokines. The increase in glycolysis was mediated by an mTOR-c-MYC-dependent pathway, whereas the inhibition of cytokine production was due to activation of the transcriptional coactivator SMAD3 and suppression of the activity of the proinflammatory transcription factors AP-1, NF-κB, and STAT1. In mice with LPS-induced endotoxemia and experimentally induced sepsis, the TGF-β-induced enhancement in macrophage glycolysis led to decreased survival, which was associated with increased blood coagulation. Analysis of septic patient cohorts revealed that the expression of PFKL, TGFBRI (which encodes a TGF-β receptor), and F13A1 (which encodes a coagulation factor) in myeloid cells positively correlated with COVID-19 disease. Thus, these results suggest that TGF-β is a critical regulator of macrophage metabolism and could be a therapeutic target in patients with sepsis.

Wnt7b expressed by hypertrophic chondrocytes is a stimulatory factor for endochondral ossification that is regulated by Smad4 activity.

Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor β family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which β-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-β-catenin axis during endochondral ossification.

Human Induced Hepatocytes-Derived Extracellular Vesicles Ameliorated Liver Fibrosis in Mice via Suppression of TGF-β1/Smad Signaling and Activation of Nrf2/HO-1 Signaling.

Liver fibrosis is a wound-healing response caused by persistent liver injury and often occurs in chronic liver diseases. Effective treatments for liver fibrosis are still pending. Recent studies have revealed that extracellular vesicles (EVs) derived from primary hepatocytes (Hep-EVs) have therapeutic potential for multiple liver diseases. However, Hep-EVs are difficult to manufacture in bulk because of the limited sources of primary hepatocytes. Human-induced hepatocytes (hiHep) are hepatocyte-like cells that can expand in vitro, and their cell culture supernatant is thus an almost unlimited resource for EVs. This study aimed to investigate the potential therapeutic effects of EVs derived from hiHeps. hiHep-EVs inhibited the expression of inflammatory genes and the secretion of inflammation-related cytokines, and suppressed the activation of hepatic stellate cells by inhibiting the TGF-β1/Smad signaling pathway. The anti-inflammatory and antifibrotic effects of hiHep-EVs were similar to those of mesenchymal stem cell (MSC)-EVs. Furthermore, the administration of hiHep-EVs ameliorated oxidative stress, inflammation, and fibrosis in a CCl4-induced liver fibrosis mouse model. The expression of α smooth muscle actin (α-SMA), collagen I, and collagen III was reduced, which may be attributed to the regulation of metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 by hiHep-EVs, and the protein expression of Nrf2, HO-1, and NQO1 was increased. Taken together, our results suggested that hiHep-EVs alleviated liver fibrosis by activating the Nrf2/HO-1 signaling pathway and inhibiting the TGF-β1/Smad signaling pathway. This study revealed the hepatoprotective effect of hiHep-EVs, and provided a new approach to treating liver fibrosis.

The CaMKII inhibitory peptide AIP alleviates renal fibrosis through the TGF-β/Smad and RAF/ERK pathways.

Renal fibrosis is characterized by the excessive deposition of extracellular matrix that destroys and replaces the functional renal parenchyma, ultimately leading to organ failure. It is a common pathway by which chronic kidney disease (CKD) can develop into end-stage renal disease (ESRD), which has high global morbidity and mortality, and there are currently no good therapeutic agents available. CaMKII has been indicated to be closely related to the occurrence of renal fibrosis, and its specific inhibitory peptide AIP, was shown to directly bind the active site of CaMKII. In this study, we examined the effect of AIP on the progression of renal fibrosis and its possible mechanism. The results showed that AIP could inhibit the expression of the fibrosis markers Fibronectin, Collagen I, MMP2 and α-SMA in vivo and in vitro. Further analysis revealed that AIP could inhibit the expression of various epithelial-to-mesenchymal transformation (EMT)-related markers, such as Vimentin and Snail 1, in vivo and in vitro. Mechanistically, AIP could significantly inhibit the activation of CaMKII, Smad 2, Raf and ERK in vitro and in vivo and reduce the expression of TGF-β in vivo. These results suggested that AIP could alleviate renal fibrosis by inhibiting CaMKII and blocking activation of the TGF-β/Smad2 and RAF/ERK pathways. Our study provides a possible drug candidate and demonstrates that CaMKII is a potential pharmacological target for the treatment of renal fibrosis. Significance Statement We have demonstrated that AIP significantly attenuated TGF-β1-induced fibrogenesis and ameliorated UUO-induced renal fibrosis through the CaMKII/TGF-β/Smad and CaMKII/RAF/ERK signaling pathways in vitro and in vivo. Our study provides a possible drug candidate and demonstrates that CaMKII can be a potential pharmacological target for the treatment of renal fibrosis.

Elevated FBXL18 promotes RPS15A ubiquitination and SMAD3 activation to drive HCC.

F-box and leucine-rich repeat protein 18 (FBXL18) is an E3 ubiquitin ligase that is reported to be involved in the tumorigenesis of various types of cancer. However, it remains unknown whether FBXL18 is correlated with hepatocarcinogenesis. In the current study, we found that FBXL18 was highly expressed in HCC tissues and positively associated with poor overall survival of HCC patients. FBXL18 was an independent risk factor for HCC patients. We observed that FBXL18 drove HCC in FBXL18 transgenic mice. Mechanistically, FBXL18 promoted the K63-linked ubiquitination of small-subunit ribosomal protein S15A (RPS15A) and enhanced its stability, increasing SMAD family member 3 (SMAD3) levels and translocation to the nucleus and promoting HCC cell proliferation. Moreover, the knockdown of RPS15A or SMAD3 significantly suppressed FBXL18-mediated HCC proliferation. In clinical samples, elevated FBXL18 expression was positively associated with RPS15A expression. FBXL18 promotes RPS15A ubiquitination and upregulates SMAD3 expression, leading to hepatocellular carcinogenesis, and this study provides a novel therapeutic strategy for HCC treatment by targeting the FBXL18/RPS15A/SMAD3 pathway.

FSTL1 promotes alveolar epithelial cell aging and worsens pulmonary fibrosis by affecting SENP1-mediated DeSUMOylation.

Alveolar epithelial cell (AEC) senescence-induced changes of lung mesenchymal cells are key to starting the progress of pulmonary fibrosis. Follistatin-like 1 (FSTL1) plays a central regulatory role in the complex process of senescence and pulmonary fibrosis by enhancing transforming growth factor-β1 (TGF-β1) signal pathway activity. Activation of Smad4 and Ras relies on SUMO-specific peptidase 1 (SENP1)-mediated deSUMOylation during TGF-β signaling pathway activation. We hypothesized that SENP1-mediated deSUMOylation may be a potential therapeutic target by modulating FSTL1-regulated cellular senescence in pulmonary fibrosis. In verifying this hypothesis, we found that FSTL1 expression was upregulated in the lung tissues of patients with idiopathic pulmonary fibrosis and that SENP1 was overexpressed in senescent AECs. TGF-β1-induced FSTL1 not only promoted AEC senescence but also upregulated SENP1 expression. Interfering with SENP1 expression inhibited FSTL1-dependent promotion of AEC senescence and improved pulmonary fibrosis in mouse lungs. FSTL1 enhancement of TGF-β1 signaling pathway activation was dependent on SENP1 in senescent AEC. Our work identifies a novel mechanism by which FSTL1 is involved in AEC senescence. Inhibition of SENP1 in epithelial cells alleviated pulmonary fibrosis by blocking FSTL1-enhanced TGF signaling.

Transforming growth factor-β1 and SMAD signalling pathway in the small airways of smokers and patients with COPD: potential role in driving fibrotic type-2 epithelial mesenchymal transition.

COPD is a common disease characterized by respiratory airflow obstruction. TGF-β1 and SMAD pathway is believed to play a role in COPD pathogenesis by driving epithelial mesenchymal transition (EMT). We investigated TGF-β1 signalling and pSmad2/3 and Smad7 activity in resected small airway tissue from patients with; normal lung function and a smoking history (NLFS), current smokers and ex-smokers with COPD GOLD stage 1 and 2 (COPD-CS and COPD-ES) and compared these with normal non-smoking controls (NC). Using immunohistochemistry, we measured activity for these markers in the epithelium, basal epithelium, and reticular basement membrane (RBM). Tissue was also stained for EMT markers E-cadherin, S100A4 and vimentin. The Staining of pSMAD2/3 was significantly increased in the epithelium, and RBM of all COPD groups compared to NC (p <0.0005). There was a less significant increase in COPD-ES basal cell numbers compared to NC (p= 0.02). SMAD7 staining showed a similar pattern (p <0.0001). All COPD group levels of TGF-β1 in the epithelium, basal cells, and RBM cells were significantly lower than NC (p <0.0001). Ratio analysis showed a disproportionate increase in SMAD7 levels compared to pSMAD2/3 in NLFS, COPD-CS and COPD-ES. pSMAD negatively correlated with small airway calibre (FEF25-75%; p= 0.03 r= -0.36). EMT markers were active in the small airway epithelium of all the pathological groups compared to patients with COPD. Activation of the SMAD pathway via pSMAD2/3 is triggered by smoking and active in patients with mild to moderate COPD. These changes correlated to decline in lung function. Activation of the SMADs in the small airways is independent of TGF-β1, suggesting factors other than TGF-β1 are driving these pathways. These factors may have implications for small airway pathology in smokers and COPD through the process of EMT, however more mechanistic work is needed to prove these correlations.

Macrophage Dectin-1 mediates Ang II renal injury through neutrophil migration and TGF-β1 secretion

Macrophage activation has been shown to play an essential role in renal fibrosis and dysfunction in hypertensive chronic kidney disease. Dectin-1 is a pattern recognition receptor that is also involved in chronic noninfectious diseases through immune activation. However, the role of Dectin-1 in Ang II-induced renal failure is still unknown. In this study, we found that Dectin-1 expression on CD68 + macrophages was significantly elevated in the kidney after Ang II infusion. We assessed the effect of Dectin-1 on hypertensive renal injury using Dectin-1-deficient mice infused by Angiotensin II (Ang II) at 1000 ng/kg/min for 4 weeks. Ang II-induced renal dysfunction, interstitial fibrosis, and immune activation were significantly attenuated in Dectin-1-deficient mice. A Dectin-1 neutralizing antibody and Syk inhibitor (R406) were used to examine the effect and mechanism of Dectin-1/Syk signaling axle on cytokine secretion and renal fibrosis in culturing cells. Blocking Dectin-1 or inhibiting Syk significantly reduced the expression and secretion of chemokines in RAW264.7 macrophages. The in vitro data showed that the increase in TGF-β1 in macrophages enhanced the binding of P65 and its target promotor via the Ang II-induced Dectin-1/Syk pathway. Secreted TGF-β1 caused renal fibrosis in kidney cells through Smad3 activation. Thus, macrophage Dectin-1 may be involved in the activation of neutrophil migration and TGF-β1 secretion, thereby promoting kidney fibrosis and dysfunction.Graphical

SHP-1 alleviates atrial fibrosis in atrial fibrillation by modulating STAT3 activation.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) has a well-established role in myocardial infarction, yet its involvement in atrial fibrosis and atrial fibrillation (AF) has not been elucidated. As cardiac arrhythmias caused by AF are a major global health concern, we investigated whether SHP-1 modulates AF development. The degree of atrial fibrosis was examined using Masson's trichrome staining, and SHP-1 expression in the human atrium was assessed using quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and western blotting (WB). We also examined SHP-1 expression in cardiac tissue from an AF mouse model, as well as in angiotensin II (Ang II)-treated mouse atrial myocytes and fibroblasts. We found that SHP-1 expression was reduced with the aggravation of atrial fibrosis in clinical samples of patients with AF. SHP-1 was also downregulated in the heart tissue of AF mice and Ang II-treated myocytes and fibroblasts, compared with that in the control groups. Next, we demonstrated that SHP-1 overexpression alleviated AF severity in mice by injecting a lentiviral vector into the pericardial space. In Ang II-treated myocytes and fibroblasts, we observed excessive extracellular matrix (ECM) deposition, reactive oxygen species (ROS) generation, and transforming growth factor beta 1 (TGF-β1)/mothers against decapentaplegic homolog 2 (SMAD2) pathway activation, all of which were counteracted by the overexpression of SHP-1. Our WB data showed that STAT3 activation was inversely correlated with SHP-1 expression in samples from patients with AF, AF mice, and Ang II-treated cells. Furthermore, administration of colivelin, a STAT3 agonist, in SHP-1-overexpressing, Ang II-treated myocytes and fibroblasts resulted in higher levels of ECM deposition, ROS generation, and TGF-β1/SMAD2 activation. These findings indicate that SHP-1 regulates AF fibrosis progression by modulating STAT3 activation and is thus a potential treatment target for atrial fibrosis and AF.

FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Estrogen receptor β attenuates renal fibrosis by suppressing the transcriptional activity of Smad3

Renal fibrosis (RF) is a common pathway leading to chronic kidney disease (CKD), which lacks effective treatment. While estrogen receptor beta (ERβ) is known to be present in the kidney, its role in RF remains unclear. The present study aimed to investigate the role and underlying mechanism of ERβ during RF progression in patients and animal models with CKD. We found that ERβ was highly expressed in the proximal tubular epithelial cells (PTECs) in healthy kidneys but its expression was largely lost in patients with immunoglobin A nephropathy (IgAN) and in mice with unilateral ureter obstruction (UUO) and subtotal nephrectomy (5/6Nx). ERβ deficiency markedly exacerbated, whereas ERβ activation by WAY200070 and DPN attenuated RF in both UUO and 5/6Nx mouse models, suggesting a protective role of ERβ in RF. In addition, ERβ activation inhibited TGF-β1/Smad3 signaling, while loss of renal ERβ was associated with overactivation of the TGF-β1/Smad3 pathway. Furthermore, deletion or pharmacological inhibition of Smad3 prevented the loss of ERβ and RF. Mechanistically, activation of ERβ competitively inhibited the association of Smad3 with the Smad-binding element, thereby downregulating the transcription of the fibrosis-related genes without altering Smad3 phosphorylation in vivo and in vitro. In conclusion, ERβ exerts a renoprotective role in CKD by blocking the Smad3 signaling pathway. Thus, ERβ may represent as a promising therapeutic agent for RF.

Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

The cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-β receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.Graphical abstract

Laminaria japonica polysaccharide attenuates podocyte epithelial-mesenchymal transformation via TGF-β1-mediated Smad3 and p38MAPK pathways

In the present work, we explored the interventional effect and potential mechanism of a purified Laminaria japonica polysaccharide (LJP61A) on podocyte epithelial-mesenchymal transition (EMT) in TGF-β1-induced podocytes and adriamycin-treated mice. Results showed that compared to the model groups, LJP61A significantly up-regulated the levels of epithelial markers (Nephrin, WT-1, podocin) and down-regulated the levels of mesenchymal markers (α-SMA, FN1) in vitro and in vivo, thus preventing EMT-like morphological changes of podocytes, proteinuria and kidney injury. Smad3 and p38MAPK are two central pathways mediating podocyte EMT activated by TGF-β1. We found that LJP61A suppressed TGF-β1-induced activation of Smad3, Smad4 and p38MAPK in vitro and in vivo. Moreover, the inhibitory actions of LJP61A on podocyte EMT were synergistically strengthened by Smad3 inhibitor SIS3 and p38MAPK inhibitor SB203580. Taken together, these findings revealed that LJP61A could prevent podocyte EMT, which might be related to the inhibition of TGF-β1-mediated Smad3 and p38MAPK pathways.

Transforming Growth Factor β Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor β (TGF-β) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-β signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-β could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-β-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-β-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-β treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-β upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-β blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-β was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-β promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-β-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-β will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-β blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Abstract 6343: 7E1, a novel blocking antibody targeting human anti-Müllerian-hormone-receptor II (AMHRII), elicits potent anti-tumor activity in vivo

Abstract Anti-Müllerian hormone (AMH), is a TGF-β family autocrine hormone and cognate ligand for the type II AMH receptor (AMHR2). AMH signaling through AMHR2 plays an important role in fetal sexual development by inducing Müllerian duct regression in males. AMHR2 is aberrantly expressed in various human malignancies, including colon, testis, gastric, and ovarian cancers. Functionally, AMH has complex effects on cell cycle regulation; at physiological concentrations it promotes survival and chemoresistance of cell lines, whereas at supraphysiological concentrations it inhibits tumor cell viability. Like other TGF-β family members, binding of its target receptor effects changes in gene expression through activation of intracellular SMAD, NF-κB, and Akt signaling. We developed a fully human anti-AMHR2 monoclonal antibody, 7E1, from RenMab™ mice, which contain the full human immunoglobulin variable domain. 7E1 was found to contain a distinct binding epitope associated with efficient inhibition of AMH binding and signaling. 7E1 also displayed a better ADCC effect than the reference AMHR2 antibody. The in vivo half-life of 7E1 in AMHR2- and FcRn-humanized mice exceeded the reference antibody as well and is in a desirable range from a development perspective. To evaluate the safety and efficacy of 7E1 in vivo, we established syngeneic murine tumor models in AMHR2-humanized mice. We found that 7E1 monotherapy significantly inhibited tumor growth in a dose-dependent manner. Moreover, 7E1 was well tolerated in mice, and no adverse effects were observed even at extremely high doses (e.g. 100 mg/kg). Taken together, these data demonstrate that 7E1 is a novel anti-human AMHR2 blocking antibody with desirable pharmacokinetic and pharmacodynamic properties. 7E1 may potentially find broad application for a range of cancer indications in which AMH signaling is implicated. Citation Format: Yongfei Yang, Huilin Li, Hao Li, Yue Yan, Jianhui Li, Qingcong Lin, Huijun Yin, Xiangshan Zhou, Miao Zhang, Kunbao Wu, Leqiao Sun, Shan Zhong. 7E1, a novel blocking antibody targeting human anti-Müllerian-hormone-receptor II (AMHRII), elicits potent anti-tumor activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6343.