Cryo-EM Reveals Integrin-Mediated TGF-ß Activation without Release from Latent TGF-ß.

Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.

LRG1 loss effectively restrains glomerular TGF-ß signaling to attenuate diabetic kidney disease.

Transforming growth factor (TGF)-ß signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-ß signaling regulators can substantially influence TGF-ß's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-ß-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-ß-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-ß signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-ß signaling to attenuate DKD.

FBXO28 promotes cell proliferation, migration and invasion via upregulation of the TGF-beta1/SMAD2/3 signaling pathway in ovarian cancer.

BACKGROUND: Ovarian cancer is one of the most common gynecological malignancies due to the lack of early symptoms, early diagnosis and limited screening. Therefore, it is necessary to understand the molecular mechanism underlying the occurrence and progression of ovarian cancer and to identify a basic biomarker for the early diagnosis and clinical treatment of ovarian cancer. METHODS: The association between FBXO28 and ovarian cancer prognosis was analyzed using Kaplan‒Meier survival analysis. The difference in FBXO28 mRNA expression between normal ovarian tissues and ovarian tumor tissues was obtained from The Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) cohorts. The expression levels of the FBXO28 protein in ovarian cancer tissues and normal ovarian tissues were measured via immunohistochemical staining. Western blotting was used to determine the level of FBXO28 expression in ovarian cancer cells. The CCK-8, the colony formation, Transwell migration and invasion assays were performed to evaluate cell proliferation and motility. RESULTS: We found that a higher expression level of FBXO28 was associated with poor prognosis in ovarian cancer patients. Analysis of the TCGA and GTEx cohorts showed that the FBXO28 mRNA level was lower in normal ovarian tissue samples than in ovarian cancer tissue samples. Compared with that in normal ovarian tissues or cell lines, the expression of FBXO28 was greater in ovarian tumor tissues or tumor cells. The upregulation of FBXO28 promoted the viability, proliferation, migration and invasion of ovarian cancer cells. Finally, we demonstrated that FBXO28 activated the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer. CONCLUSIONS: In conclusion, FBXO28 enhanced oncogenic function via upregulation of the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

Phosphoinositide-binding activity of Smad2 is essential for its function in TGF-ß signaling.

As a central player in the canonical TGF-ß signaling pathway, Smad2 transmits the activation of TGF-ß receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-ß to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-ß receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-ß receptor I that transduces TGF-ß-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-ß receptor complex, leading to its binding to the TGF-ß receptor I. The PI(4,5)P2-binding activity of Smad2 is essential for the TGF-ß-stimulated phosphorylation, nuclear transport, and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-ß-mediated biological processes.

The Role of Local Inflammation and Hypoxia in the Formation of Hypertrophic Scars-A New Model in the Duroc Pig.

Hypertrophic scars continue to be a major burden, especially after burns. Persistent inflammation during wound healing appears to be the precipitating aspect in pathologic scarring. The lack of a standardized model hinders research from fully elucidating pathophysiology and therapy, as most therapeutic approaches have sparse evidence. The goal of this project was to investigate the mechanisms of scar formation after prolonged wound inflammation and to introduce a method for generating standardized hypertrophic scars by inducing prolonged inflammation. Four wound types were created in Duroc pigs: full-thickness wounds, burn wounds, and both of them with induced hyperinflammation by resiquimod. Clinical assessment (Vancouver Scar Scale), tissue oxygenation by hyperspectral imaging, histologic assessment, and gene expression analysis were performed at various time points during the following five months. Native burn wounds as well as resiquimod-induced full-thickness and burn wounds resulted in more hypertrophic scars than full-thickness wounds. The scar scale showed significantly higher scores in burn- and resiquimod-induced wounds compared with full-thickness wounds as of day 77. These three wound types also showed relative hypoxia compared with uninduced full-thickness wounds in hyperspectral imaging and increased expression of HIF1a levels. The highest number of inflammatory cells was detected in resiquimod-induced full-thickness wounds with histologic features of hypertrophic scars in burn and resiquimod-induced wounds. Gene expression analysis revealed increased inflammation with only moderately altered fibrosis markers. We successfully created hypertrophic scars in the Duroc pig by using different wound etiologies. Inflammation caused by burns or resiquimod induction led to scars similar to human hypertrophic scars. This model may allow for the further investigation of the exact mechanisms of pathological scars, the role of hypoxia and inflammation, and the testing of therapeutic approaches.

PDLIM5 inhibits STUB1-mediated degradation of SMAD3 and promotes the migration and invasion of lung cancer cells.

Transforming growth factor ß (TGFß) signaling plays an important role in regulating tumor malignancy, including in non-small cell lung cancer (NSCLC). The major biological responses of TGFß signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFß-SMAD signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDLIM5 (PDZ and LIM domain protein 5, ENH) critically promotes TGFß signaling by maintaining SMAD3 stability in NSCLC. First, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro and lung metastasis in vivo In addition, TGFß signaling and TGFß-induced epithelial-mesenchymal transition was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFß-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2 and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration, and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFß signaling and NSCLC progression.

Anti-inflammatory cytokines IL-27, IL-10, IL-1Ra and TGF-ß in subjects with increasing grades of glucose intolerence (DM-LTB-2).

Anti-inflammatory cytokines act as double edged swords- they can dampen inflammation but can also suppress immunity. The role played by these cytokines in latent TB infected (LTBI) subjects, with various grades of glucose intolerance was studied. Both serum levels and recall-secretion of IL-27, IL-10, IL-1Ra and TGF-ß in Normal Glucose Tolerance (NGT), Pre-Diabetes (PDM), Newly diagnosed Diabetes (NDM) and Known Diabetes (KDM) subjects, both with and without LTBI (n = 382), were quantified by ELISA. All the subjects were screened for LTBI by QuantiFERON-TB Gold test. Serum levels of IL-27, IL-10 and IL-1Ra were significantly elevated in the LTB-PDM, compared to LTB-NGT group. Increased IL-27 and IL-10 levels and decreased levels of TGF-ß were seen in the LTB-NDM, compared to LTB-NGT group. Decreased serum levels of IL-27 and increased levels of IL-1Ra and TGF-ß were seen in the LTB-KDM, compared to LTB-NGT group. TB antigens induced the secretion of IL-1Ra in LTB+ subjects in the NGT, PDM and NDM groups, but not in the KDM group. Co-morbidity with LTBI brought about (diabetic) stage-specific modulation, in these cytokine levels. Major defects in the circulating levels and recall secretion of anti-inflammatory cytokines, as seen in LTB+KDM subjects, could fuel DM-TB synergy.

The TGF-b/SOX4 axis and ROS-driven autophagy co-mediate CD39 expression in regulatory T-cells.

The ectonucleotidase CD39 on human regulatory T-cells (Treg) is an important immune regulator which is dysregulated in autoimmune diseases and cancer immunosuppression. We here define that CD39 expression on Treg is independent of the Treg-specific transcription factors FOXP3 and HELIOS and promoted by canonical TGF-b- and mTOR-signaling. Furthermore, the TGF-b mediated upregulation of CD39 is counteracted by reactive oxygen species (ROS)-driven autophagy. In line, CD39+ peripheral blood Treg constitute a distinct lineage with low autophagic flux and absent ROS production. Patients with rare genetic defects in autophagy show supraphysiological levels of CD39+ Treg, validating our observations in vivo. These biological processes rely on a distinct transcriptional program with CD39+ Treg expressing low levels of two genes with putative involvement in autophagy, NEFL and PLAC8. Furthermore, the TGF-b downstream transcription factor SOX4 is selectively upregulated in CD39+ Treg. Overexpression of SOX4 in Treg strongly increases CD39 expression, while Crispr/Cas9-mediated knockout of SOX4 in Treg has the opposing effect. Thus, we identify a crucial role of SOX4 in immune regulation and provide new insights involving the interplay of tolerogenic cues and autophagy in Treg.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells.

Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

A Promising Role of TGF-ß Pathway in Response to Regorafenib in Metastatic Colorectal Cancer: A Case Report.

Colorectal cancer (CRC) is one of the most common cancer types around the world. The prognosis of patients with advanced diseases is still poor in spite of currently available therapeutic options. Regorafenib is an oral tyrosine kinase inhibitor (TKI) approved to treat refractory metastatic colorectal cancer (mCRC). We investigated Somatic mutations in several genes involved in immunological response and cancer progression in both long/short responder mCRC patients who underwent third-line therapy with regorafenib to identify predictive biomarkers of response using Ion Torrent PGM sequencing and bioinformatic tools. We found Somatic mutations in TGFBR1, TGFBR2, and TGFBR3 genes in primary tumor and metastases samples of long-responder patients. Furthermore, our bioinformatic results show that they were mainly enriched in immune response, cell junction, and cell adhesion in long responder patients, particularly in primary tumor and metastatic sites. These data suggest that the TGF-b pattern could be the leading actor of a prolonged response to this drug.

CD44 inhibits α-SMA gene expression via a novel G-actin/MRTF-mediated pathway that intersects with TGFßR/p38MAPK signaling in murine skin fibroblasts.

Well-regulated differentiation of fibroblasts into myofibroblasts (MF) is critical for skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), an established marker for MF differentiation, is driven by TGFß receptor (TGFßR)-mediated signaling. Hyaluronan (HA) and its receptor CD44 may also participate in this process. To further understand this process, primary mouse skin fibroblasts were isolated and treated in vitro with recombinant TGF-ß1 (rTGF-ß1) to induce α-SMA expression. CD44 expression was also increased. Paradoxically, CD44 knockdown by RNA interference (RNAi) led to increased α-SMA expression and α-SMA-containing stress fibers. Removal of extracellular HA or inhibition of HA synthesis had no effect on α-SMA levels, suggesting a dispensable role for HA. Exploration of mechanisms linking CD44 knockdown to α-SMA induction, using RNAi and chemical inhibitors, revealed a requirement for noncanonical TGFßR signaling through p38MAPK. Decreased monomeric G-actin but increased filamentous F-actin following CD44 RNAi suggested a possible role for myocardin-related transcription factor (MRTF), a known regulator of α-SMA transcription and itself regulated by G-actin binding. CD44 RNAi promoted nuclear accumulation of MRTF and the binding to its transcriptional cofactor SRF. MRTF knockdown abrogated the increased α-SMA expression caused by CD44 RNAi, suggesting that MRTF is required for CD44-mediated regulation of α-SMA. Finally, chemical inhibition of p38MAPK reversed nuclear MRTF accumulation after rTGF-ß1 addition or CD44 RNAi, revealing a central involvement of p38MAPK in both cases. We concluded that CD44 regulates α-SMA gene expression through cooperation between two intersecting signaling pathways, one mediated by G-actin/MRTF and the other via TGFßR/p38MAPK.

The antiviral protein viperin regulates chondrogenic differentiation via CXCL10 protein secretion.

Viperin (also known as radical SAM domain-containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor ß (TGF-ß)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin-CXCL10-TGF-ß/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.

TGF-ß2 uses the concave surface of its extended finger region to bind betaglycan's ZP domain via three residues specific to TGF-ß and inhibin-α.

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-ß family that selectively binds transforming growth factor-ß (TGF-ß) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-ß2 with BG's C-terminal binding domain, BGZP-C, and surface plasmon resonance binding measurements with TGF-ß2 variants, we found that the BGZP-C-binding site on TGF-ß2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-ß isoforms and the InhA α-subunit, but they are unconserved in other TGF-ß family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-ß2 variants with the corresponding residues from BMP-2 bound BGZP-C more weakly than corresponding alanine variants. The BGZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-ß2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-ß2 and InhA bind BGZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BGZP-C-binding site on TGF-ß2 and shed light on the specificity of BG for select TGF-ß-type GFs and the mechanisms by which BG influences their signaling.

FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor ß1 signaling.

The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor ß1 (TGFß1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFß1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFß1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFß1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFß1 signaling.

MicroRNA-144-3p targets relaxin/insulin-like family peptide receptor 1 (RXFP1) expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis.

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). We have previously shown that a potential limitation to relaxin-based IPF therapy is decreased expression of a relaxin receptor, relaxin/insulin-like family peptide receptor 1 (RXFP1), in IPF fibroblasts. The mechanism that down-regulates RXFP1 in IPF remains unclear. To determine whether microRNAs (miRs) regulate RXFP1 gene expression, here we employed a bioinformatics approach to identify miRs predicted to target RXFP1 and identified a putative miR-144-3p target site in the RXFP1 mRNA. In situ hybridization of IPF lung biopsies revealed that miR-144-3p is expressed in fibroblastic foci. Furthermore, we found that miR-144-3p is up-regulated in IPF fibroblasts compared with lung fibroblasts from healthy donors. Transforming growth factor ß increased miR-144-3p expression in both healthy and IPF lung fibroblasts in a SMAD family 2/3 (SMAD2/3)-dependent manner, and Jun proto-oncogene AP-1 transcription factor subunit (AP-1) was required for constitutive miR-144-3p expression. Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced α-SMA expression. Of note, a lentiviral luciferase reporter carrying the WT 3' UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p-binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates RXFP1 in IPF lung fibroblasts by targeting its 3' UTR. We conclude that miR-144-3p directly represses RXFP1 mRNA and protein expression.

TGF-ß/PI3K/AKT/mTOR/NF-kB pathway. Clinicopathological features in prostate cancer.

Prostate cancer is one of the most common cancers in the male population. The objective of this investigation was to study the relationship of components of transforming growth factor-B (TGF-ß)/phosphoinositide-3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR)/nuclear factor kappa B (NF-kB) transduction pathway with clinical-pathological markers. By immunohistochemical methods, we determined the expression of several factors [TGF-ß, Transforming Growth Factor B Receptor I (TGFBRI), TGFBRII, PI3K, AKT-Ser, AKT-Thr, mTOR, p-mTOR, inhibitor kB kinase (IKK), pIKK, inhibitor kB (IkB), pIkB, NF-kBp50, and NF-kBp65]. To know their relationship with established classical markers (Preoperative serum prostate specific antigen, pathological tumor stage, clinical tumor stage, Gleason score, perineural invasion, node involvement, positive surgical margins, biochemical progression, and survival) and their importance in the prognosis of biochemical progression, Spearman test, survival analysis, Log-rang test, Kaplan-Meier curves, univariate and multivariate Cox proportional Hazard regression analyses were performed. Spearman analysis showed that there was at least one correlation between TGF-ß, TGFBRI, PI3K, pAKT-Thr, p-mTOR, NF-kBp50, and classical markers. Cox multivariate analysis between the prognostic variables (pathological tumor stage, Gleason score, and node involvement) and inmunohistochemical parameters confirmed TGFBR1 and PI3K as a prognostic and independent marker of biochemical progression in prostate cancer. Our results suggest that TGFBR1 and PI3K could be used as useful biomarkers for early diagnosis and prognoses for biochemical recurrence in prostate cancer after radical prostatectomy.

Value of cord blood Treg population properties and function-associated characteristics for predicting allergy development in childhood.

Allergic diseases represent some of the most common immunological disorders with high clinical and economic impact. Despite intensive research, there are still few universally accepted and reliable biomarkers capable of predicting their development at an early age. There is therefore a pressing need for identification of potential predictive factors and validation of their prognostic value by correlating them with allergy development. Dysbalance of the branches of immune response, most often excessive Th2 polarization, is the principal cause of allergic diseases. Regulatory T cells (Treg) are a crucial population for the timely establishment of physiological immune polarization and induction and maintenance of tolerance against environmental antigens. This makes them a potentially promising candidate for an early marker predicting allergy development. In our study, we analysed samples of cord blood of children of allergic mothers and children of healthy mothers by flow cytometry and retrospectively correlated the data with clinical allergy status of the children at the age of 6 to 10 years. Studied parameters included cord blood Treg population proportions and functional properties - intracellular presence of IL-10 and TGF-b, MFI of FoxP3. We observed higher percentage of Tregs in cord blood of children who did not develop allergy compared with allergic children. Further, we found higher numbers of IL-10+ Tregs in cord blood of healthy children of healthy mothers than in cord blood of children of allergic mothers and decreased TGF-b+ cord blood Tregs in the group of allergic children of allergic mothers compared to all other groups.

Glutaminolysis is required for transforming growth factor-ß1-induced myofibroblast differentiation and activation.

Myofibroblasts participate in physiological wound healing and pathological fibrosis. Myofibroblast differentiation is characterized by the expression of α-smooth muscle actin and extracellular matrix proteins and is dependent on metabolic reprogramming. In this study, we explored the role of glutaminolysis and metabolites of TCA in supporting myofibroblast differentiation. Glutaminolysis converts Gln into α-ketoglutarate (α-KG), a critical intermediate in the TCA cycle. Increases in the steady-state concentrations of TCA cycle metabolites including α-KG, succinate, fumarate, malate, and citrate were observed in TGF-ß1-differentiated myofibroblasts. The concentration of glutamate was also increased in TGF-ß1-differentiated myofibroblasts compared with controls, whereas glutamine levels were decreased, suggesting enhanced glutaminolysis. This was associated with TGF-ß1-induced expression of the glutaminase (GLS) isoform, GLS1, which converts Gln into glutamate, at both the mRNA and protein levels. The stimulation of GLS1 expression by TGF-ß1 was dependent on both SMAD3 and p38 mitogen-activated protein kinase activation. Depletion of extracellular Gln prevented TGF-ß1-induced myofibroblast differentiation. The removal of extracellular Gln postmyofibroblast differentiation decreased the expression of the profibrotic markers fibronectin and hypoxia-inducible factor-1α and reversed TGF-ß1-induced metabolic reprogramming. Silencing of GLS1 expression, in the presence of Gln, abrogated TGF-ß1-induced expression of profibrotic markers. Treatment of GLS1-deficient myofibroblasts with exogenous glutamate or α-KG restored TGF-ß1-induced expression of profibrotic markers in GLS1-deficient myofibroblasts. Together, these data demonstrate that glutaminolysis is a critical component of myofibroblast metabolic reprogramming that regulates myofibroblast differentiation.

Interleukin-6-mediated trans-signaling inhibits transforming growth factor-ß signaling in trabecular meshwork cells.

Glaucoma is one of the major causes of blindness, and transforming growth factor-ß2 (TGF-ß2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-ß2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-ß in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-ß signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-ß2 stimulation. To elucidate the relationship between TGF-ß2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-ß-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-ß-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-ß-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-ß were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-ß-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-ß signaling in HTM cells.

TGF-ß1 modifies histone acetylation and acetyl-coenzyme A metabolism in renal myofibroblasts.

Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine TGF-b1 on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Fibroblasts propagated from rat kidneys after ureteric obstruction were treated with recombinant TGF-b1 or vehicle for 48 hours. TGF-b1 -induced myofibroblast activation was accompanied by a net decrease in total H3 acetylation, although changes in individual marks were variable. This was paralleled by a generalised reduction in histone acetyltransferases (HAT), and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally this was manifest in a reduction in total HAT activity and increase in HDAC activity. TGF-b1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate or alternative sources of acetyl-CoA, without ameliorating changes in HAT/HDAC activity. In conclusion, TGF-b1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetylation, increased deacetylation and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-b1.