Activation Of TGF-β Signaling Research Articles

Streptomyces spp. Isolated from Rosa davurica Rhizome for Potential Cosmetic Application

Streptomyces species are widely studied and used in different fields, including antibiotics and pesticides, and are spread in several places as soil-derived microorganisms. However, research on anti-aging, including antioxidants obtained from Streptomyces, has not been performed as much. Skin aging due to bacterial infection, especially methicillin-resistant Staphylococcus aureus (MRSA), is challenging to recover, so it is essential to prevent aging by preventing or inhibiting infection. Therefore, this study was conducted to isolate Streptomyces species from Rosa davurica rhizome soil and to determine the effect of the ethyl acetate extract of the isolated strain Streptomyces chattanoogensis THA-663 (THA-663S) on the inhibition of MRSA and UVB-irradiated human skin keratinocytes, to determine whether it could be a treatment for skin aging. The MRSA inhibition and antioxidant activities were evaluated using disc diffusion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and a reactive oxygen species (ROS) assay. The expression of aging-related markers, including mitogen-activated protein kinases/activator protein 1 (MAPK)/AP-1) and transforming growth factor-β/suppressor of mothers against decapentaplegic (TGF-β/Smad) was assessed using Western blotting. The antibacterial effect on four MRSA strains, CCARM 0204, CCARM 0205, CCARM 3855, and CCARM 3089, showed that THA-663S could greatly inhibit MRSA growth. Moreover, the findings showed that THA-663S is efficient in scavenging free radicals and dose-dependently reducing ROS generation. Furthermore, THA-663S notably reduced UVB-induced matrix metalloproteinase-1 (MMP-1) expression by inhibiting the MAPK/AP-1 signaling pathways and blocking extracellular matrix (ECM) degradation in UVB-irradiated HaCaT cells. Additionally, THA-663S improved and enhanced transforming growth factor-beta (TGF-β) signaling activation to promote procollagen type I synthesis, relieving UVB-induced skin cell damage. In conclusion, THA-663S has a high potential to protect skin cells from aging, and, simultaneously, it can prevent or treat aging caused by infection due to pathogen inhibition.

DDDR-33. TARGETING TGFΒ PATHWAY DEPENDENCIES IN GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at < 50%, few recurrent mutations, higher frequency of metastasis, and no targeted therapies. Amplification of MYC (c-myc) and activation of TGFβ signaling are frequent in G3 MB. We hypothesize that the TGFβ pathway and MYC contribute to the intrinsic resistance of G3 MB through deregulation of key genes and pathways. We previously established humanized models for SHH MB by introducing MYCN or PTCH1 deletions into neuroepithelial stem (NES) cells derived from normal human induced pluripotent stem cells (hIPSCs). In this study, we transduced NES cells with TGFb effectors activated in G3 MB (ACVR2A, TGFbR1, TGFb1, TGFb3, and SMAD5) alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors with MYC leading to more aggressive tumors. We thus describe six new humanized isogenic models for both non-MYC and MYC driven G3 MB. We next found that NES cells expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT & RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis of MYC and TGFβ pathway driven NES cells. We discovered a subset of genes upregulated in MYC and TGFb-driven lines that are targets of the histone demethylase KDM2B. We postulate that epigenetic remodeling via MYC and recruitment of other MYC-interacting cofactors culminates in transcriptional changes that lead to aggressive disease. Overall, our studies provide insights on identifying new therapeutic avenues for patients with MYC and TGFβ driven G3 MB.

Delineating the dynamic evolution from preneoplasia to invasive lung adenocarcinoma by integrating single-cell RNA sequencing and spatial transcriptomics

The cell ecology and spatial niche implicated in the dynamic and sequential process of lung adenocarcinoma (LUAD) from adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA) and subsequent invasive adenocarcinoma (IAC) have not yet been elucidated. Here, we performed an integrative analysis of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) to characterize the cell atlas of the invasion trajectory of LUAD. We found that the UBE2C + cancer cell subpopulation constantly increased during the invasive process of LUAD with remarkable elevation in IAC, and its spatial distribution was in the peripheral cancer region of the IAC, representing a more malignant phenotype. Furthermore, analysis of the TME cell type subpopulation showed a constant decrease in mast cells, monocytes, and lymphatic endothelial cells, which were implicated in the whole process of invasive LUAD, accompanied by an increase in NK cells and MALT B cells from AIS to MIA and an increase in Tregs and secretory B cells from MIA to IAC. Notably, for AIS, cancer cells, NK cells, and mast cells were colocalized in the cancer region; however, for IAC, Tregs colocalized with cancer cells. Finally, communication and interaction between cancer cells and TME cell-induced constitutive activation of TGF-β signaling were involved in the invasion of IAC. Therefore, our results reveal the specific cellular information and spatial architecture of cancer cells and TME subpopulations, as well as the cellular interaction between them, which will facilitate the identification and development of precision medicine in the invasive process of LUAD from AIS to IAC.

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transitionof ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.

TrkA+ Neurons Induce Pathologic Regeneration After Soft Tissue Trauma.

Heterotopic ossification (HO) is a dynamic, complex pathologic process that often occurs after severe polytrauma trauma, resulting in an abnormal mesenchymal stem cell differentiation leading to ectopic bone growth in soft-tissues including tendons, ligaments, and muscles. The abnormal bone structure and location induce pain and loss of mobility. Recently, we observed that NGF (Nerve growth factor)-responsive TrkA (Tropomyosin receptor kinase A)-expressing nerves invade sites of soft-tissue trauma, and this is a necessary feature for heterotopic bone formation at sites of injury. Here, we assayed the effects of the partial TrkA agonist Gambogic amide (GA) in peritendinous heterotopic bone after extremity trauma. Mice underwent HO induction using the burn/tenotomy model with or without systemic treatment with GA, followed by an examination of the injury site via radiographic imaging, histology, and immunohistochemistry. Single-cell RNA Sequencing confirmed an increase in neurotrophin signaling activity after HO-inducing extremity trauma. Next, TrkA agonism led to injury site hyper-innervation, more brisk expression of cartilage antigens within the injured tendon, and a shift from FGF to TGFβ signaling activity among injury site cells. Nine weeks after injury, this culminated in higher overall levels of heterotopic bone among GA-treated animals. In summary, these studies further link injury site hyper-innervation with increased vascular ingrowth and ultimately heterotopic bone after trauma. In the future, modulation of TrkA signaling may represent a potent means to prevent the trauma-induced heterotopic bone formation and improve tissue regeneration.

LTBP2 mutation may cause mitral valve prolapse

Abstract Background Mitral valve prolapse (MVP, MIM 157700) is a very common cardiac disorder occurring in 2.4% of the general population. MVP is characterized by myxomatous degeneration resulting in valve thickening, prolapsing and regurgitation. MVP results in mitral regurgitation, arrhythmias, bacterial endocarditis, congestive heart failure and sudden death. Familial studies of MVP suggest an autosomal dominant mode of inheritance with incomplete penetrance. We are wokring on defining the genetic causes of MVP. Methods and results We identified an MVP family with an LTBP-2 Val1506Met rare mutation (minor allele frequency of 0.0001) segregating with the MVP (LOD score of 1.5 out of a maximal potential of 2.6). LTBP2 is an excellent candidate gene for MVP because it regulates TGFβ signaling activity, shown to be disrupted in Marfan's syndrome mitral valvulopathy. High prevalence of MVP was reported in relatives of patients with Weill-Marchesani syndrome, caused by LTBP2 mutations. We then generated two strains of mice using CRISPR technology. A complete knockout (deletion of the first exon) and a knock in mouse. Nineteen mice were dissected so far (figure 3). None of the wt mice (n=8) demonstrated myxomatous degeneration. Of the 11 mice with mutation, 8 homozygote for deletion and 3 homozygote for the human mutation, 8 demostrated myxomatous Degeneration by histology (1 knockin mouse ∼70% pentrance in male mice, p=0.001, Fisher exact test). The pathologist that read the studies was blinded to the genotype. Echocardiography of theses mice demonstrated findings consistant with MVP (deviation of the leaflets above the annluar line in parasternal long axis view) in 50% of affected mice and not in controls Conclusion We demonstrated that an LTBP2 mutation is segretating in a family with MVP. LTBP2 mutation mice model demonstrated myxomatous degeneration of the valve. Thus, LTBP2 may be an MVP causing gene Funding Acknowledgement Type of funding sources: Public hospital(s).

PKM2 promotes pulmonary fibrosis by stabilizing TGF-β1 receptor I and enhancing TGF-β1 signaling.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease, and the molecular mechanisms remain poorly understood. Our findings demonstrated that pyruvate kinase M2 (PKM2) promoted fibrosis progression by directly interacting with Smad7 and reinforcing transforming growth factor–β1 (TGF-β1) signaling. Total PKM2 expression and the portion of the tetrameric form elevated in lungs and fibroblasts were derived from mice with bleomycin (BLM)–induced pulmonary fibrosis. Pkm2 deletion markedly alleviated BLM-induced fibrosis progression, myofibroblast differentiation, and TGF-β1 signaling activation. Further study showed that PKM2 tetramer enhanced TGF-β1 signaling by directly binding with Smad7 on its MH2 domain, and thus interfered with the interaction between Smad7 and TGF-β type I receptor (TβR1), decreased TβR1 ubiquitination, and stabilized TβR1. Pharmacologically enhanced PKM2 tetramer by TEPP-46 promoted BLM-induced pulmonary fibrosis, while tetramer disruption by compound 3k alleviated fibrosis progression. Our results demonstrate how PKM2 regulates TGF-β1 signaling and is a key factor in fibrosis progression.

Thrombospondin-1 overexpression stimulates loss of Smad4 and accelerates malignant behavior via TGF-β signal activation in pancreatic ductal adenocarcinoma

IntroductionPancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma and cancer-associated fibroblasts (CAFs) provide a favorable tumor microenvironment. Smad4 is known as tumor suppressor in several types of cancers including PDAC, and loss of Smad4 triggers accelerated cell invasiveness and metastatic potential. The thrombospondin-1 (TSP-1) can act as a major activator of latent transforming growth factor-β (TGF-β) in vivo. However, the roles of TSP-1 and the mediator of Smad4 loss and TGF-β signal activation during PDAC progression have not yet been addressed. The aim is to elucidate the biological role of TSP-1 in PDAC progression. Methods and resultsHigh substrate stiffness stimulated TSP-1 expression in CAFs, and TSP-1 knockdown inhibited cell proliferation with suppressed profibrogenic and activated stroma-related gene expressions in CAFs. Paracrine TSP-1 treatment for PDAC cells promoted cell proliferation and epithelial mesenchymal transition (EMT) with activated TGF-β signals such as phosphorylated Akt and Smad2/3 expressions. Surprisingly, knockdown of DPC4 (Smad4 gene) induced TSP-1 overexpression with TGF-β signal activation in PDAC cells. Interestingly, TSP-1 overexpression also induced downregulation of Smad4 expression and enhanced cell proliferation in vitro and in vivo. Treatment with LSKL peptide, which antagonizes TSP-1-mediated latent TGF-β activation, attenuated cell proliferation, migration and chemoresistance with enhanced apoptosis in PDAC cells. ConclusionsTSP-1 derived from CAFs stimulates loss of Smad4 expression in cancer cells and accelerates malignant behavior by TGF-β signal activation in PDAC. TSP-1 could be a novel therapeutic target, not only for CAFs in stiff stroma, but also for cancer cells in the PDAC microenvironment.

Rosa davurica inhibits skin photoaging via regulating MAPK/AP-1, NF-κB, and Nrf2/HO-1 signaling in UVB-irradiated HaCaTs.

Rosa davurica is widely used to treat various kinds of diseases because of its high antioxidant, antiviral and anti-inflammatory activities. This use of plant-based materials as medicine is called phytomedicine and has been widely practiced since time immemorial. However, the pharmacological mechanism of R. davurica in skin photoaging is not yet fully understood. Therefore, this study was carried out to evaluate the recovery effects of R. davurica leaf extracts (RDE) in UVB-irradiated human skin keratinocytes (HaCaTs) and investigate whether RDE is a potential therapeutic agent against skin photoaging. The expression of aging-related markers including mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1), nuclear factor-κB (NF-κB), and nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) was evaluated using Western blot analysis. The reactive oxygen species (ROS) was also used by FACS in HaCaTs. Findings indicated that RDE is efficient in scavenging free radicals and dose-dependently reducing ROS generation. Furthermore, RDE notably decreased UVB-induced matrix metalloproteinase-1 (MMP-1) expression through inhibition of MAPK/AP-1 and NF-κB signaling pathways as well as induced blocking of extracellular matrix (ECM) degradation in UVB-irradiated HaCaTs. In addition, RDE improved Nrf2/HO-1 signaling that increases oxidative defense capacity and enhances transforming growth factor-beta (TGF-β) signaling activation to promote procollagen type I synthesis, relieving UVB-induced skin cell damage. In conclusion, the protective effects of RDE on skin cellular components suggest that it has a high biological potential for skin protection from UVB-induced skin photoaging and is a good candidate for drug and cosmetic application.

Regulation of transforming growth factor-β signaling as a therapeutic approach to treating colorectal cancer.

According to data from 2020, Slovakia has long been among the top five countries with the highest incidence rate of colorectal cancer (CRC) worldwide, and the rate is continuing to rise every year. In approximately 80% of CRC cases, allelic loss (loss of heterozygosity, LOH) occurs in the long arm of chromosome 18q. The most important genes that can be silenced by 18q LOH or mutations are small mothers against decapentaplegic homolog (SMAD) 2 and SMAD4, which are intracellular mediators of transforming growth factor (TGF)-β superfamily signals. TGF-β plays an important role in the pro-oncogenic processes, including such properties as invasion, epithelial-mesenchymal transition (commonly known as EMT), promotion of angiogenesis, and immunomodulatory effects. Several recent studies have reported that activation of TGF-β signaling is related to drug resistance in CRC. Because the mechanisms of drug resistance are different between patients in different stages of CRC, personalized treatment is more effective. Therefore, knowledge of the activation and inhibition of factors that affect the TGF-β signaling pathway is very important.

CGRP induces myofibroblast differentiation and the production of extracellular matrix in MRC5s via autocrine and paracrine signalings.

There are contradictory views on which calcitonin gene-related peptide (CGRP) causes pulmonary fibrosis. Fibrotic potency of CGRP was tested and compared to that of transforming growth factor-β (TGF-β). Myofibroblast differentiation, cell proliferation, and activations of TGF-β and Wnt pathways were examined for 24, 48, and 72 h in A549 and MRC5 cell lines stimulated with CGRP and TGF-β. CGRP-induced cell proliferation in MRC5s early on while cell proliferation in A549 occurred progressively. CGRP promoted fibroblast-myofibroblast differentiation by inducing the transcription of ACTA2, COL1A1, SMAD2/3, and SMAD4 genes, the production of collagen, fibronectin, α-smooth muscle actin, and activation of TGF-β signaling starting from 24 h. Additionally, TGF-β signaling induced by CGRP decreased theDKK1 level and activated the Wnt signaling in MRC5s. After CGRP stimulation, Wnt7a levels were increased from 24to 72 h, while Wnt5a levels were elevated at 72 h in MRC5s. CGRP did not induce epithelial-mesenchymal transition in A549s, unlike TGF-β.A comparison of fibrotic potency of CGRP and TGF-β showed that TGF-β is a powerful profibrotic molecule and induces earlier myofibroblast differentiation. Even so, CGRP promotes myofibroblast differentiation and extracellular matrix production by inducing Smad-dependent-TGF-β and Wnt signalings via autocrine and paracrine signalings in MRC5s.

Effective delivery of osteopontin small interference RNA using exosomes suppresses liver fibrosis via TGF-β1 signaling

Objective and aims: Osteopontin (OPN), an oxidant stress sensitive cytokine, plays a central role in liver fibrosis. While OPN expression can be reduced by small interfering RNA (siRNA), the challenge to deliver siRNA safely and effectively into liver remains unresolved. Exosomes are promising natural nanocarriers for drug delivery that are able to enter cells with different biological barriers efficiently. In this study, we used exosomes as a delivery vehicle to target OPN in liver fibrosis. Methods: Exosomes selectively home to fibrotic liver according to small animal imaging system. Electroporation technique was used to engineer exosomes to carry siRNA targeting OPN (ExosiRNA−OPN). Primary hepatic stellate cells (HSCs) were isolated and treated with ExosiRNA−OPN to assess the effect on activated HSCs (aHSCs). Immunofluorescence for α−SMA, an aHSCs marker, and sirius red staining were performed to assess ECM deposition. Finally, plasma OPN from patients with liver fibrosis was identified by ELISA assay. Results: Exosome-mediated siRNA delivery systems show high uptake and low toxicity. Besides, ExosiRNA−OPN suppressed HSCs activation and ECM deposition and more efficiently improved liver function when compared to naked siRNA-OPN. Moreover, ExosiRNA−OPN was assumed inhibiting TGF-β1 signaling activation, along with other fibrotic-related genes based on a GEO datasheet of liver fibrosis samples for correlation analyzes. ExosiRNA−OPN inhibited TGF-β1 signaling by decreasing high-mobility group box-1 (HMGB1). Plasma proteins from chronic HBV-induced fibrosis patients were identified that patients with high OPN expression correlates with more advanced fibrosis progression. Discussion: This study shows that exosome-mediated siRNA-OPN delivery may be an effective option for the treatment of liver fibrosis.

Ahnak depletion accelerates liver regeneration by modulating the TGF-β/Smad signaling pathway

Ahnak, a large protein first identified as an inhibitor of TGF-β signaling in human neuroblastoma, was recently shown to promote TGF-β in some cancers. The TGF-β signaling pathway regulates cell growth, various biological functions, and cancer growth and metastasis. In this study, we used Ahnak knockout (KO) mice that underwent a 70% partial hepatectomy (PH) to investigate the function of Ahnak in TGF-β signaling during liver regeneration. At the indicated time points after PH, we analyzed the mRNA and protein expression of the TGF -β/Smad signaling pathway and cell cycle-related factors, evaluated the cell cycle through proliferating cell nuclear antigen (PCNA) immunostai-ning, analyzed the mitotic index by hematoxylin and eosin staining. We also measured the ratio of liver tissue weight to body weight. Activation of TGF-β signaling was confirmed by analyzing the levels of phospho-Smad 2 and 3 in the liver at the indicated time points after PH and was lower in Ahnak KO mice than in WT mice. The expression levels of cyclin B1, D1, and E1; proteins in the Rb/E2F transcriptional pathway, which regulates the cell cycle; and the numbers of PCNA-positive cells were increased in Ahnak KO mice and showed tendencies opposite that of TGF-β expression. During postoperative regeneration, the liver weight to body weight ratio tended to increase faster in Ahnak KO mice. However, 7 days after PH, both groups of mice showed similar rates of regeneration, following which their active regeneration stopped. Analysis of hepatocytes undergoing mitosis showed that there were more mitotic cells in Ahnak KO mice, consistent with the weight ratio. Our findings suggest that Ahnak enhances TGF-β signaling during postoperative liver regeneration, resulting in cell cycle disruption; this highlights a novel role of Ahnak in liver regeneration. These results provide new insight into liver regeneration and potential treatment targets for liver diseases that require surgical treatment.

Macrophage-Derived TGF-β and VEGF Promote the Progression of Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is a pathological bone formation process caused by musculoskeletal trauma. HO is characterized by aberrant endochondral ossification and angiogenesis. Our previous studies have indicated that macrophage inflammation is involved in traumatic HO formation. In this study, we found that macrophage infiltration and TGF-βsignaling activation are presented in human HO. Depletion of macrophages effectively suppressed traumatic HO formation in a HO mice model, and macrophage depletion significantly inhibited the activation of TGF-β/Smad2/3 signaling. In addition, the TGF-β blockade created by a neutralizing antibody impeded ectopic bone formation in vivo. Notably, endochondral ossification and angiogenesis are attenuated following macrophage depletion or TGF-β inhibition. Furthermore, our observations on macrophage polarization revealed that M2 macrophages, rather than M1 macrophages, play a critical role in supporting HO development by enhancing the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Our findings on ectopic bone formation in HO patients and the mice model indicate that M2 macrophages are an important contributor for HO development, and that inhibition of M2 polarization or TGF-β activity may be a potential method of therapy for traumatic HO.

Effect of losartan treatment on Smad signaling and recovery from hindlimb unloading-induced soleus muscle atrophy in female rats

Losartan, an angiotensin II type 1 receptor blocker, exerts protective effect on soleus muscle atrophy in female rats. Thus, we aimed to examine the effect of losartan treatment on the recovery of atrophied soleus muscles. Female Wistar rats were subjected to hindlimb unloading for 7 d and then reloading for 7 d with either phosphate-buffered saline (PBS; n = 9) or losartan (40 mg/kg/day; n = 9). The soleus muscles were removed at rest (sedentary control [SED]; n = 9), after 7 d of hindlimb unloading (HU; n = 9), and after 7 d of reloading (HUR-PBS or HUR-LOS; n = 9 each). The absolute and relative weights, and fiber cross-sectional area (CSA) of the soleus muscles of rats in the HU group were significantly reduced as compared to those of the rats in the SED group at 7 d post-hindlimb unloading. Seven days of reloading significantly increased the muscle weights of rats in the HUR-PBS and HUR-LOS groups, with the recovery rate of the absolute muscle weight and type I fiber CSA being significantly higher in the HUR-LOS group (6.1% and 10.1%, respectively) than in the HUR-PBS group (4.7% and 5.2%, respectively) (p < 0.05). Moreover, the absolute and relative muscle weight in HUR-PBS were lower than SED; however, no significant difference was observed between the SED and HUR-LOS groups. CSAs of type I and IIa fiber were significantly higher in the HUR-LOS group than in the HU group. Losartan administration during reloading resulted in increased Smad1/5/8 and mTOR signaling and decreased Smad2/3 signaling and protein ubiquitination, facilitating the recovery of atrophied soleus muscle. Therefore, losartan administration-induced muscle recovery may partially be attributed to enhanced Smad1/5/8 and mTOR signaling activation, and reduced activation of canonical TGF-β signaling (Smad2/3) in the soleus muscle.

Abstract P1012: Tbx18 Drives Nodal-like Self-assembly Of Pacemaker Myocytes And Non-myocytes

Background: We have demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes (CMs) to induced pacemaker cells. Cell-cell interactions and cell-extracellular matrix (ECM) are important regulators of microtissue organization and assembly. Here, we sought to gain a finer understanding of self-organization and assembly of pacemaker microtissue with myocytes and non-myocytes driven by TBX18. Methods: 2D monolayer of neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno- GFP and Adeno-TBX18 in vitro model. AAV9-TBX18 was delivered via tail vein injection of Hcn4 (+/eGFP) transgenic mice to track the induced pacemaker myocytes. Result: TBX18-NRVMs showed more compact nodal-like aggregates with a higher density of CMs and surrounded by nonmyocytes. The number of alpha smooth muscle actin (αSMA) + myofibroblasts producing specific ECM and facilitating cell motility was increased by 190% in TBX18-NRVMs as well. Systemic delivery of AAV9-TBX18 via tail vein of mice created aggregation of de novo Hcn4+ induced pacemaker myocytes (iPMs) surrounded by αSMA+ myofibroblasts in vivo. Then, we treated pre-plated neonatal rat ventricular cells (NRVCs, including CMs and nonmyocytes) to explore the potential role of cell-cell communication and cell-ECM interaction in the formation of nodal-like aggregates through scRNA-seq. The data indicated that TBX18+ CM (28.6% % of all CMs) and TBX18-negative CMs (71.4% of all CMs) presented highly consistent transcriptomic profiles at day 3. No significant difference in Hcn4 transcript levels and the ratio of iPMs (defined as Hcn4+, Gja1 low , Nkx2.5 low , Tnni3 high , Actn2 high CM) was observed between them, as well as other pacemaker genes, suggesting cross-communication between them. Despite of TBX18+ fibroblasts (FBs) accounting for only 5% of all FBs, a higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control. Enrichment analysis revealed that TBX18-CMs lost ventricular specific electrical coupling and sarcomere organization (down-regulated Gja1, Cdh2, Irx3 ), but reconstructed hemidesmosome assembly and cell-ECM interaction enriched in sinoatrial node myocytes (Upregulated Lamc1, Itgb1, Plec, Des, Actn1, and Acta2 ) and activated Tgfβ signaling. Furthermore, treatment with gap junction inhibitor palmitoleic acid as well as Tgfβ receptor inhibitor A83-01 significantly suppressed the activation of quiescent fibroblasts to αSMA+ myofibroblasts and the formation of nodal-like aggregation of TBX18-NRVMs. Conclusion: TBX18 drives self-organization and nodal-like assembly of pacemaker microtissue with myocytes and non-myocytes through cell-cell communication of gap junction and Tgfβ signal.

TGFβ signaling activation correlates with immune-inflamed tumor microenvironment across human cancers and predicts response to immunotherapy

ABSTRACT Considering the determining role of TGFβ signaling in the tumor microenvironment (TME) on immune evasion, the inhibition of signaling is expected to enhance the therapeutic efficacy of immunotherapies, especially immune checkpoint blockade (ICB), which is confirmed in preclinical data. However, successive failures in clinical translation occur at the initial stage. To provide a better understanding of TGFβ signaling within the TME and its relation to the individual immunological status, we performed a pan-cancer analysis comparing the activation of TGFβ pathway among different TMEs based on multi-omics data. Compared with non-inflamed tumors, increased TGFβ signaling activity appeared in four non-cancer cell types within TME in inflamed tumors. Significant correlations were revealed between TGFβ signaling and reliable biomarkers for ICB therapy, as well as between TGFβ signaling and HPV status. Our findings contribute to explain the inconsistency between preclinical and clinical research, and are crucial to optimizing upcoming clinical trial design and improving patient stratification for personalized prediction.

A Phase Ib Dose-finding Study of Panobinostat and Ruxolitinib in Myelofibrosis.

A Phase Ib Dose-finding Study of Panobinostat and Ruxolitinib in Myelofibrosis.

Notch4 affects the proliferation and differentiation of deer antler chondrocytes through the Smad3/lncRNA27785.1 axis

Long non-coding RNA play an importantr role in the differentiation of chondrocytes. This study aims to explore the role of long non-coding RNA in the transcriptional regulation of Notch4. In previous studies, it has been found that Notch signal can be used as the downstream of TGF-β signal to affect the proliferation and differentiation of deer antler chondrocytes, but the specific mechanism remains unclear. Here we found that lncRNA27785.1 was involved in the regulation of TGF-β/ Smad3 signal and Notch4 gene. The overexpression lncRNA27785.1 can negatively regulate the expression of Notch4 to inhibit cell proliferation and differentiation, while interference with lncRNA27785.1 can promote the expression of Notch4 gene to promote the proliferation and differentiation of chondrocytes. Subsequently, through luciferase experiment and CHIP experiment, we found that lncRNA27785.1 is regulated by Smad3 transcription, and Smad3 inhibited the expression of lncRNA27785.1. In addition, activated TGF-β signaling can reduce the inhibitory effect of lncRNA27785.1 on Notch4 signaling. In summary, we found that lncRNA27785.1 and TGF-β/Smad3 play an important role in Notch4 signaling. Our findings provided evidence to explain how TGF-β signaling regulate the Notch signaling pathway to influence chondrocyte proliferation and differentiation by a specific lncRNA27785.1.

Extracellular HSP90α promotes cellular senescence by modulating TGF-β signaling in pulmonary fibrosis.

Recent findings suggest that extracellular heat shock protein 90α (eHSP90α) promotes pulmonary fibrosis, but the underlying mechanisms are not well understood. Aging, especially cellular senescence, is a critical risk factor for idiopathic pulmonary fibrosis (IPF). Here, we aim to investigate the role of eHSP90α on cellular senescence in IPF. Our results found that eHSP90α was upregulated in bleomycin (BLM)-induced mice, which correlated with the expression of senescence markers. This increase in eHSP90α mediated fibroblast senescence and facilitated mitochondrial dysfunction. eHSP90α activated TGF-β signaling through the phosphorylation of the SMAD complex. The SMAD complex binding to p53 and p21 promoters triggered their transcription. In vivo, the blockade of eHSP90α with 1G6-D7, a specific eHSP90α antibody, in old mice attenuated the BLM-induced lung fibrosis. Our findings elucidate a crucial mechanism underlying eHSP90α-induced cellular senescence, providing a framework for aging-related fibrosis interventions.