Transforming Growth Factor Beta-induced Protein Research Articles

Cathepsin D promotes polarization of tumor-associated macrophages and metastasis through TGFBI-CCL20 signaling

M2-like tumor-associated macrophages (TAMs) are risk factors for cancer progression and metastasis. However, the mechanisms underlying their polarization are still not fully understood. Although cathepsin D (Cat D) has been reported as a procarcinogenic factor, little is known about the functional role of Cat D in the tumor microenvironment (TME). This study aimed to explore the effect and molecular mechanisms of Cat D in the TME. Cat D knockout (KO) altered the cytokine secretion pattern and induced TAM reprogramming from the M2 to M1 subtype, thereby preventing epithelial-mesenchymal transition and tumor metastasis. Mechanistically, we identified transforming growth factor beta-induced protein (TGFBI) as a Cat D target protein that is specifically associated with TAM polarization. Elevated TGFBI expression in Cat D KO cancer cells resulted in a decline in M2-like TAM polarization. Our RNA-sequencing results indicated that the cancer cell-secreted chemokine CCL20 is a major secretory chemokine for Cat D-TGFBI-mediated TAM polarization. In contrast, Cat D overexpression accelerated TAM polarization into M2-like cells by suppressing TGFBI expression. In addition, the double Cat D and TGFBI KO rescued the inhibitory effects of Cat D KO on tumor metastasis by controlling TAM and T-cell activation. These findings indicated that Cat D contributes to cancer metastasis through TGFBI-mediated TAM reprogramming. Cat D deletion inhibits M2-like TAM polarization through TGFBI-mediated CCL20 expression, reprogramming the immunosuppressive TME. Our results open a potential new avenue for therapy focused on eliminating tumor metastasis.

Hypoxia-induced TGFBI maintains glioma stem cells by stabilizing EphA2.

Rationale: Glioma stem cells (GSCs) have emerged as pivotal drivers of tumor malignancy, sustained by various microenvironmental factors, including immune molecules and hypoxia. In our previous study, we elucidated the significant role of transforming growth factor beta-induced protein (TGFBI), a protein secreted by M2-like tumor-associated macrophages, in promoting the malignant behavior of glioblastoma (GBM) under normoxic conditions. Building upon these findings, the objective of this study was to comprehensively explore the crucial role and underlying mechanisms of autocrine TGFBI in GSCs under hypoxic conditions. Methods: We quantified TGFBI expression in glioma specimens and datasets. In vitro and in vivo assays were employed to investigate the effects of TGFBI on sustaining self-renewal and tumorigenesis of GSCs under hypoxia. RNA-seq and LC-MS/MS were conducted to explore TGFBI signaling mechanisms. Results: TGFBI is preferentially expressed in GSCs under hypoxic conditions. Targeting TGFBI impair GSCs self-renewal and tumorigenesis. Mechanistically, TGFBI was upregulated by HIF1α in GSCs and predominantly activates the AKT-c-MYC signaling pathway in GSCs by stabilizing the EphA2 protein through preventing its degradation. Conclusion: TGFBI plays a crucial role in maintaining the stem cell properties of GSCs in the hypoxic microenvironment. Targeting the TGFBI/EphA2 axis emerges as a promising and innovative strategy for GBM treatment, with the potential to improve the clinical outcomes of patients.

A novel role of TGFBI in macrophage polarization and macrophage-induced pancreatic cancer growth and therapeutic resistance

Tumor-associated macrophages (TAMs), as a major and essential component of tumor microenvironment (TME), play a critical role in orchestrating pancreatic cancer (PaC) tumorigenesis from initiation to angiogenesis, growth, and systemic dissemination, as well as immunosuppression and resistance to chemotherapy and immunotherapy; however, the critical intrinsic factors responsible for TAMs reprograming and function remain to be identified. By performing single-cell RNA sequencing, transforming growth factor-beta-induced protein (TGFBI) was identified as TAM-producing factor in murine PaC tumors. TAMs express TGFBI in human PaC and TGFBI expression is positively related with human PaC growth. By inducing TGFBI loss-of-function in macrophage (MΦs) in vitro with siRNA and in vivo with Cre-Lox strategy in our developed TGFBI-floxed mice, we demonstrated disruption of TGFBI not only inhibited MΦ polarization to M2 phenotype and MΦ-mediated stimulation on PaC growth, but also significantly improved anti-tumor immunity, sensitizing PaC to chemotherapy in association with regulation of fibronectin 1, Cxcl10, and Ccl5. Our studies suggest that targeting TGFBI in MΦ can develop an effective therapeutic intervention for highly lethal PaC.

The TGFBI gene and protein expression in topotecan resistant ovarian cancer cell lines

PurposeThe primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer, is drug resistance. The mechanisms of drug resistance of cancer cells during chemotherapy may include compounds of the extracellular matrix, such as the transforming growth factor-beta-induced protein (TGFBI). In this study, we aimed to analyze the TGFBI gene and protein expression in different sensitive and drug-resistant ovarian cancer cell lines, as well as test if TGFBI can be involved in the response to topotecan (TOP) at the very early stages of treatment. Materials and methodsIn this study, we conducted a detailed analysis of TGFBI expression in different ovarian cancer cell lines (A2780, A2780TR1, A2780TR2, W1, W1TR, SKOV-3, PEA1, PEA2 and PEO23). The level of TGFBI mRNA (QPCR), intracellular and extracellular protein (Western blot analysis) were assessed in this study. ResultsWe observed upregulation of TGFBI mRNA in drug-resistant cell lines and estrogen-receptor positive cell lines, which was supported by overexpression of both intracellular and extracellular TGFBI protein. We also showed the TGFBI expression after a short period of treatment of sensitive ovarian cancer cell lines with TOP. ConclusionThe expression of TGFBI in ovarian cancer cell lines suggests its role in the development of drug resistance.

Type II Diabetes Mellitus Causes Extracellular Matrix Alterations in the Posterior Cornea That Increase Graft Thickness and Rigidity.

There is a pressing need to investigate the impact of type II diabetes mellitus on the posterior cornea in donor tissues given its increasing prevalence and potential impact on endothelial keratoplasty surgical outcomes. Immortalized human cultured corneal endothelial cells (CECs; HCEC-B4G12) were grown in hyperglycemic media for 2 weeks. Extracellular matrix (ECM) adhesive glycoprotein expression and advanced glycation end products (AGEs) in cultured cells and corneoscleral donor tissues, as well as the elastic modulus for the Descemet membrane (DMs) and CECs of diabetic and nondiabetic donor corneas, were measured. In CEC cultures, increasing hyperglycemia resulted in increased transforming growth factor beta-induced (TGFBI) protein expression and colocalization with AGEs in the ECM. In donor corneas, the thicknesses of the DM and the interfacial matrix (IFM) between the DM and stroma both increased from 8.42 ± 1.35 µm and 0.504 ± 0.13 µm in normal corneas, respectively, to 11.13 ± 2.91 µm (DM) and 0.681 ± 0.24 µm (IFM) in non-advanced diabetes (P = 0.013 and P = 0.075, respectively) and 11.31 ± 1.76 µm (DM) and 0.744 ± 0.18 µm (IFM) in advanced diabetes (AD; P = 0.0002 and P = 0.003, respectively). Immunofluorescence in AD tissues versus controls showed increased AGEs (P < 0.001) and markedly increased labeling intensity for adhesive glycoproteins, including TGFBI, that colocalized with AGEs. The elastic modulus significantly increased between AD and control tissues for the DMs (P < 0.0001) and CECs (P < 0.0001). Diabetes and hyperglycemia alter human CEC ECM structure and composition, likely contributing to previously documented complications of endothelial keratoplasty using diabetic donor tissue, including tearing during graft preparation and reduced graft survival. AGE accumulation in the DM and IFM may be a useful biomarker for determining diabetic impact on posterior corneal tissue.

Identification of the Potential Molecular Mechanism of TGFBI Gene in Persistent Atrial Fibrillation.

Transforming growth factor beta-induced protein (TGFBI, encoded by TGFBI gene), is an extracellular matrix protein, widely expressed in variety of tissues. It binds to collagens type I, II, and IV and plays important roles in the interactions of cell with cell, collagen, and matrix. It has been reported to be associated with myocardial fibrosis, and the latter is an important pathophysiologyical basis of atrial fibrillation (AF). However, the mechanism of TGFBI in AF remains unclear. We aimed to detect the potential mechanism of TGFBI in AF via bioinformatics analysis. The microarray dataset of GSE115574 was examined to detect the genes coexpressed with TGFBI from 14 left atrial tissue samples of AF patients. TGFBI coexpression genes were then screened using the R package. Using online analytical tools, we determined the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Ontology (GO) annotation, and protein-protein interaction (PPI) network of TGFBI and its coexpression genes. The modules and hub genes of the PPI-network were then identified. Another dataset, GSE79768 was examined to verify the hub genes. DrugBank was used to detect the potential target drugs. In GSE115574 dataset, a total of 1818 coexpression genes (769 positive and 1049 negative) were identified, enriched in 120 biological processes (BP), 38 cellular components (CC), 36 molecular functions (MF), and 39 KEGG pathways. A PPI-network with average 12.2-degree nodes was constructed. The genes clustered in the top module constructed from this network mainly play a role in PI3K-Akt signaling pathway, viral myocarditis, inflammatory bowel disease, and platelet activation. CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 were identified and finally verified as the hub genes, mainly enriched in pathways like leukocyte transendothelial migration, PI3K-Akt signaling pathway, viral myocarditis, rheumatoid arthritis, and platelet activation. Pegcetacoplan, ocriplasmin, and carvedilol were the potential target drugs. We used microdataset to identify the potential functions and mechanisms of the TGFBI and its coexpression genes in AF patients. Our findings suggest that CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 may be the hub genes.

Niclosamide Suppresses Migration and Invasion of Human Osteosarcoma Cells by Repressing TGFBI Expression via the ERK Signaling Pathway.

Osteosarcoma is a highly common malignant bone tumor. Its highly metastatic properties are the leading cause of mortality for cancer. Niclosamide, a salicylanilide derivative, is an oral antihelminthic drug of known anticancer potential. However, the effect of niclosamide on osteosarcoma cell migration, invasion and the mechanisms underlying have not been fully clarified. Therefore, this study investigated niclosamide’s underlying pathways and antimetastatic effects on osteosarcoma. In this study, U2OS and HOS osteosarcoma cell lines were treated with niclosamide and then subjected to assays for determining cell migration ability. The results indicated that niclosamide, at concentrations of up to 200 nM, inhibited the migration and invasion of human osteosarcoma U2OS and HOS cells and repressed the transforming growth factor beta-induced protein (TGFBI) expression of U2OS cells, without cytotoxicity. After TGFBI knockdown occurred, cellular migration and invasion behaviors of U2OS cells were significantly reduced. Moreover, niclosamide significantly decreased the phosphorylation of ERK1/2 in U2OS cells and the combination treatment of the MEK inhibitor (U0126) and niclosamide resulted in the intensive inhibition of the TGFBI expression and the migratory ability in U2OS cells. Therefore, TGFBI derived from osteosarcoma cells via the ERK pathway contributed to cellular migration and invasion and niclosamide inhibited these processes. These findings indicate that niclosamide may be a powerful preventive agent against the development and metastasis of osteosarcoma.

Identification of novel hub genes associated with lymph node metastasis of head and neck squamous cell carcinoma by completive bioinformatics analysis.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is one of the most serious diseases affecting populations worldwide and lymph node metastasis is a key pathological feature of HNSCC which predicts poor survival. However, the molecular mechanisms associated with the development of lymph node metastasis in HNSCC have not been fully elucidated.MethodsDifferentially expressed genes (DEGs) were identified in two HNSCC datasets (GES6631 and GES58911). Functional annotation analysis was constructed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Meanwhile, the protein-protein interaction (PPI) network and module analysis using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape were carried out to identify the hub genes. The expression differences, overall survival (OS), and disease-free survival (DFS) of hub genes were analyzed by Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and verified by immunohistochemistry (IHC) from Human Protein Atlas (HPA). Moreover, receiver operating characteristic (ROC) curve analysis was conducted to judge whether those hub genes had good diagnostic and prognostic ability, and the web tool Tumor Immune Estimation Resource (TIMER) was used to analyze the correlation of hub genes’ expression and immune infiltration.ResultsIn total, 913 DEGs including 476 upregulated and 437 downregulated genes were identified. The genes Aurora kinase A (AURKA), CyclinB1 (CCNB1), Cyclin-dependent kinase regulatory subunit 1B (CKS1B), Serpin Family H Member 1(SERPINH1), and Transforming growth factor-beta-induced protein (TGFBI) were screened out as hub genes and were associated with lymph node metastasis, showing notably abnormal expression in HNSCC samples, and the high expression of all the hub genes in HNSCC patients was related to worse overall survival.ConclusionsThe genes AURKA, CCNB1, CKS1B, SERPINH1, and TGFBI may be involved in the lymph node metastasis of HNSCC and reveal the potential to serve as molecular biomarkers in the diagnosis of HNSCC. This study may help to elucidate the molecular mechanisms of the development of lymph node metastasis and facilitate the selection of targets for the treatment and diagnosis of HNSCC.

Pan-Cancer Analysis of the Associations of TGFBI Expression With Prognosis and Immune Characteristics.

Transforming growth factor-beta-induced (TGFBI) protein has important roles in tumor growth, metastasis, and immunity. However, there is currently no pan-cancer evidence regarding TGFBI. In this study, we conducted a pan-cancer analysis of TGFBI mRNA and protein expression and prognoses of various cancer types using public databases. We also investigated the associations of TGFBI expression with tumor microenvironment (TME) components, immune cell infiltration, tumor mutational burden (TMB), and microsatellite instability (MSI), along with the TGFBI genetic alteration types. The results showed that TGFBI expression varied among different cancer types, and it was positively or negatively related to prognosis in various cancers. TGFBI expression was also significantly correlated with TME components, TMB, MSI, immune cell infiltration, and immunoinhibitory and immunostimulatory gene subsets. These findings indicate that TGFBI participates in various immune responses and it may function as a prognostic marker in various cancers. The findings may be useful for developing immunotherapies that target TGFBI.

Platelet-Released Growth Factors Induce Genes Involved in Extracellular Matrix Formation in Human Fibroblasts.

Platelet concentrate products are increasingly used in many medical disciplines due to their regenerative properties. As they contain a variety of chemokines, cytokines, and growth factors, they are used to support the healing of chronic or complicated wounds. To date, underlying cellular mechanisms have been insufficiently investigated. Therefore, we analyzed the influence of Platelet-Released Growth Factors (PRGF) on human dermal fibroblasts. Whole transcriptome sequencing and gene ontology (GO) enrichment analysis of PRGF-treated fibroblasts revealed an induction of several genes involved in the formation of the extracellular matrix (ECM). Real-time PCR analyses of PRGF-treated fibroblasts and skin explants confirmed the induction of ECM-related genes, in particular transforming growth factor beta-induced protein (TGFBI), fibronectin 1 (FN1), matrix metalloproteinase-9 (MMP-9), transglutaminase 2 (TGM2), fermitin family member 1 (FERMT1), collagen type I alpha 1 (COL1A1), a disintegrin and metalloproteinase 19 (ADAM19), serpin family E member 1 (SERPINE1) and lysyl oxidase-like 3 (LOXL3). The induction of these genes was time-dependent and in part influenced by the epidermal growth factor receptor (EGFR). Moreover, PRGF induced migration and proliferation of the fibroblasts. Taken together, the observed effects of PRGF on human fibroblasts may contribute to the underlying mechanisms that support the beneficial wound-healing effects of thrombocyte concentrate products.

Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy

ABSTRACT Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD. Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type

Pan-Cancer Analysis of the Associations of TGFBI Expression with Prognosis and Immune Characteristics

Transforming growth factor-beta-induced (TGFBI) protein has important roles in tumor growth, metastasis, and immunity. However, there is currently no pan-cancer evidence regarding TGFBI. Here, we conducted a pan-cancer analysis of TGFBI mRNA and protein expression and prognoses of various cancer types using public databases. We also explored the associations of TGFBI expression with tumor microenvironment (TME) components, tumor mutational burden (TMB), microsatellite instability (MSI), and immune cell infiltration, along with the TGFBI genetic alteration types. The results showed that TGFBI expression varied among different cancer types, and it was positively or negatively related to prognosis in various cancers. TGFBI expression was also significantly correlated with TME components, TMB, MSI, immune cell infiltration, and immunoinhibitory and immunostimulatory gene subsets. These findings indicate that TGFBI participates in various immune responses and it may function as a prognostic marker in various cancers. The findings may be useful for developing immunotherapies that target TGFBI.

Endothelial angiogenic activity and adipose angiogenesis is controlled by extracellular matrix protein TGFBI

Several studies have suggested that extracellular matrix (ECM) remodeling and the microenvironment are tightly associated with adipogenesis and adipose angiogenesis. In the present study, we demonstrated that transforming growth factor-beta induced (TGFBI) suppresses angiogenesis stimulated by adipocyte-conditioned medium (Ad-CM), both in vitro and in vivo. TGFBI knockout (KO) mice exhibited increased numbers of blood vessels in adipose tissue, and blood vessels from these mice showed enhanced infiltration into Matrigel containing Ad-CM. The treatment of Ad-CM-stimulated SVEC-10 endothelial cells with TGFBI protein reduced migration and tube-forming activity. TGFBI protein suppressed the activation of the Src and extracellular signaling-related kinase signaling pathways of these SVEC-10 endothelial cells. Our findings indicated that TGFBI inhibited adipose angiogenesis by suppressing the activation of Src and ERK signaling pathways, possibly because of the stimulation of the angiogenic activity of endothelial cells.

A Miniature Bio-Photonics Companion Diagnostics Platform for Reliable Cancer Treatment Monitoring in Blood Fluids

In this paper, we present the development of a photonic biosensor device for cancer treatment monitoring as a complementary diagnostics tool. The proposed device combines multidisciplinary concepts from the photonic, nano-biochemical, micro-fluidic and reader/packaging platforms aiming to overcome limitations related to detection reliability, sensitivity, specificity, compactness and cost issues. The photonic sensor is based on an array of six asymmetric Mach Zender Interferometer (aMZI) waveguides on silicon nitride substrates and the sensing is performed by measuring the phase shift of the output signal, caused by the binding of the analyte on the functionalized aMZI surface. According to the morphological design of the waveguides, an improved sensitivity is achieved in comparison to the current technologies (<5000 nm/RIU). This platform is combined with a novel biofunctionalization methodology that involves material-selective surface chemistries and the high-resolution laser printing of biomaterials resulting in the development of an integrated photonics biosensor device that employs disposable microfluidics cartridges. The device is tested with cancer patient blood serum samples. The detection of periostin (POSTN) and transforming growth factor beta-induced protein (TGFBI), two circulating biomarkers overexpressed by cancer stem cells, is achieved in cancer patient serum with the use of the device.

Evaluation of the Genetic Variation Spectrum Related to Corneal Dystrophy in a Large Cohort.

AimsTo characterize the genetic landscape and mutation spectrum of patients with corneal dystrophies (CDs) in a large Han ethnic Chinese Cohort with inherited eye diseases (IEDs).MethodsRetrospective study. A large IED cohort was recruited in this study, including 69 clinically diagnosed CD patients, as well as other types of eye diseases patients and healthy family members as controls. The 792 genes on the Target_Eye_792_V2 chip were used to screen all common IEDs in our studies, including 22 CD-related genes.ResultsWe identified 2334 distinct high-quality variants on 22 CD-related genes in a large IEDs cohort. A total of 21 distinct pathogenic or likely pathogenic mutations were identified, and the remaining 2313 variants in our IED cohort had no evidence of CD-related pathogenicity. Overall, 81.16% (n = 56/69) of CD patients received definite molecular diagnoses, and transforming growth factor-beta-induced protein (TGFBI), CHTS6, and SLC4A11 genes covered 91.07, 7.14, and 1.79% of the diagnosed cases, respectively. Twelve distinct disease-associated mutations in the TGFBI gene were identified, 11 of which were previously reported and one is novel. Four of these TGFBI mutations (p.D123H, p.M502V, p.P501T, and p.P501A) were redefined as likely benign in our Han ethnic Chinese IED cohort after performing clinical variant interpretation. These four TGFBI mutations were detected in asymptomatic individuals but not in CD patients, especially the previously reported disease-causing mutation p.P501T. Among 56 CD patients with positive detected mutations, the recurrent TGFBI mutations were p.R124H, p.R555W, p.R124C, p.R555Q, and p.R124L, and the proportions were 32.14, 19.64, 14.29, 10.71, and 3.57%, respectively. Twelve distinct pathogenic or likely pathogenic mutations of CHTS6 were detected in 28 individuals. The recurrent mutations were p.Y358H, p.R140X, and p.R205W, and the proportions were 25.00, 21.43, and 14.29%, respectively. All individuals associated with TGFBI were missense mutations; 74.19% associated with CHTS6 mutations were missense mutations, and 25.81% were non-sense mutations. Hot regions were located in exons 4 and 12 of TGFBI individuals and located in exon 3 of CHTS6 individuals. No de novo mutations were identified.ConclusionFor the first time, our large cohort study systematically described the variation spectrum of 22 CD-related genes and evaluated the frequency and pathogenicity of all 2334 distinct high-quality variants in our IED cohort. Our research will provide East Asia and other populations with baseline data from a Han ethnic population-specific level.

Comprehensively investigating the expression levels and the prognostic role of transforming growth factor beta-induced (TGFBI) in glioblastoma multiforme.

BackgroundTransforming growth factor beta-induced (TGFBI) protein has been found expressed in several cancer types, and expression levels of TGFBI can affect the cancer patients’ outcomes, but the role of TGFBI in glioblastoma multiforme (GBM) remains obscure.MethodsThe TGFBI expression levels in GBM were performed via Gene Expression Profiling Interactive Analysis (GEPIA) and UALCAN databases. Further, the mutations types of TGFBI were analyzed by using the cBioportal dataset. LinkedOmics selected correlated genes, kinases, and microRNA (miRNA) targets of TGFBI. GEPIA conducted the prognostic value of TGFBI and correlated genes. Then, the relationship between TGFBI and immune infiltrates was performed by Tumor Immune Estimation Resource (Timer). We compared the TGFBI protein expression levels in GBM and control samples through the Human Protein Atlas (HPA). Finally, the GSCAlite was used to achieve the drugs, and molecules target the TGFBI and significantly correlated genes.ResultsTGFBI is significantly overexpressed in GBM, but the clinical features do not have considerable influence on TGFBI expression levels. Overexpression of TGFBI acts as an adverse biomarker of GBM. The enrichment function of TGFBI showed that the main biological functions, including extracellular matrix (ECM) organization, angiogenesis, leukocyte migration, T cell activation, cell cycle G2/M phase transition, and growth factor binding. About the significant correlated genes, overexpression of mitogen-activated protein kinase 13 (MAPK13) [Log-rank P=0.08 HR (high) =1.4], myosin IG (MYO1G) [Log-rank P=0.06 HR (high) =1.4], plasminogen activator urokinase receptor (PLAUR) [Log-rank P=0.03 HR (high) =1.5], thrombomodulin (THBD) [Log-rank P=0.028 HR (high) =1.5] indicated the poor prognosis of GBM. Further, TGFBI had a significant association with dendritic cell (DC) infiltrates (cor =0.516, P=9.00e−30). The higher the DC infiltration, the shorter survival of GBM. TGFBI protein expression levels were not significantly different in GBM and normal tissue. Finally, TGFBI is associated with resistance to belinostat, LAQ824, CAY10603, CUDC-101, methotrexate, 5-fluorouracil, and navitoclax.ConclusionsIn the present study, we showed TGFBI was overexpressed in GBM, and TGFBI is associated with DC cell infiltrates. Overexpression of TGFBI and high DC infiltration might be an adverse biomarker of GBM. Finally, TGFBI is associated with tumor multi-drug resistance.

Platelet-Released Growth Factors and Platelet-Rich Fibrin Induce Expression of Factors Involved in Extracellular Matrix Organization in Human Keratinocytes

Platelet-released growth factor (PRGF) is a thrombocyte concentrate lysate which, like its clinically equivalent variations (e.g., Vivostat PRF® (platelet-rich fibrin)), is known to support the healing of chronic and hard-to-heal wounds. However, studies on the effect of PRGF on keratinocytes remain scarce. This study aims to identify genes in keratinocytes that are significantly influenced by PRGF. Therefore, we performed a whole transcriptome and gene ontology (GO) enrichment analysis of PRGF-stimulated human primary keratinocytes. This revealed an increased expression of genes involved in extracellular matrix (ECM) organization. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed the PRGF-mediated induction of selected ECM-related factors such as transforming growth factor beta-induced protein, fibronectin 1, matrix metalloproteinase-9, transglutaminase 2, fermitin family member 1, collagen type I alpha 1 and collagen type XXII alpha 1. PRGF-induced expression of the above factors was influenced by blockade of the epidermal growth factor receptor (EGFR), a receptor playing a crucial role in wound healing. A differential induction of the investigated factors was also detected in skin explants exposed to PRGF and in experimentally generated in vivo wounds treated with Vivostat PRF®. Together, our study indicates that the induction of ECM-related factors may contribute to the beneficial wound-healing effects of PRGF-based formulations.

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of TGFBI-corneal dystrophy

Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman’s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Klotho Ameliorates Renal Fibrosis in a Murine Model of ADTKD‐UMOD by Enhancing UMOD secretion and Downregulation of TGFBI

Heterozygous Uromodulin (UMOD) mutations cause autosomal dominant tubulo‐interstitial kidney disease (ADTKD). ADTKD‐UMOD is a model for renal fibrosis.Currently, no therapies are available for ADTKD‐UMOD. The antiaging hormone Klotho improves glomerular disease, hypertension, uremic cardiomyopathy, and acute kidney injury (AKI). We tested if Klotho ameliorates renal fibrosis and tubular disease in a murine model of ADTKD‐UMOD.To generate a more pronounced phenotype we used homozygous UmodC93F/C93F mice. Mice were crossed with mice overexpressing Klotho (TgKl). We compared the phenotype with wild‐type (WT), TgKl, and UmodC93F/C93F mice (8 animals per group). We studied blood pressure, urine and blood samples at ages of 6 and 13 months. In different animals of the four groups, we harvested kidneys at 3 months to perform quantitative proteome analysis via LC‐MS/MS on renal outer medulla.Compared to UmodC93F/C93F mice, TgKl/UmodC93F/C93F had better renal function as indicated by significantly lower BUN, creatinine, and cystatin C values, and less renal fibrosis. PTH and FGF23 improved significantly in TgKl/UmodC93F/C93F vs. UmodC93F/C93F mice. Mutant UMOD is retained in the endoplasmic reticulum. We found that relative urinary UMOD secretion was higher in TgKl/UmodC93F/C93F vs. UmodC93F/C93F. No major serum electrolyte differences were found among the four groups. Urinary studies in TgKl/UmodC93F/C93F mice showed less urinary wasting of Na+, Ca2+, Mg2+ and Phosphorus than in UmodC93F/C93F mice. Compared to UmodC93F/C93F, Klotho‐overexpressing animals had significantly lower systolic and diastolic blood pressures. Markers of cardiac hypertrophy such as ANP, BNP, and MYH7 mRNA were significantly lower in TgKl/UmodC93F/C93F than in UmodC93F/C93F animals. We confirmed impaired unfolded protein response (UPR) and autophagy in UmodC93F/C93F compared to WT mice. However, improved renal function and fibrosis in Klotho‐overexpressing mice were not due to changes in UPR or autophagy. Therefore, we performed an unbiased proteomics approach. We identified 262 downregulated and 179 upregulated proteins in kidneys. Analyzing proteins by signaling pathways, we identified in TgKl/UmodC93F/C93F vs. UmodC93F/C93F animals downregulation of Transforming growth factor‐beta‐induced protein (TGFBI) together with collagens Col1a1, Col4a3, Col4a4, Col4a5, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, prolargin, biglycan, and osteoglycin/mimecan. We confirmed lower Col1a1 mRNA expression with qPCR.We show that Klotho improves renal outcome in ADTKD‐UMOD by increasing urinary UMOD secretion and ameliorating renal fibrosis by downregulation of TGFBI and collagen. TGFBI is secreted by mesenchymal stromal cells and infiltrating macrophages and stimulates collagen synthesis. Infiltrating macrophages are an important part of AKI and ADTKD‐UMOD. Our data reveal a new specific target for treatment of renal fibrosis.Support or Funding InformationNIH R03 DK111776, NIH P30 DK079328, and CCRAC Senior Investigator Bridge Award, Children’s Health DallasAt 6 and 13 months TgKl/UmodC93F/C93F show significantly lower BUN, creatinine, and cystatin C values.Figure 1STRING analysis showing networks formed by downregulated proteins in renal outer medulla of TgKl/UmodC93F/C93F compared to UmodC93F/C93F mice. Red circle outlines downregulated TGFBI and collagens.Figure 2

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.