Regulation Of Transforming Growth Factor Beta Research Articles

High expression of TBRG4 in relation to unfavorable outcome and cell ferroptosis in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of malignant liver tumor with poor prognosis. In this study, we investigated the expression of transforming growth factor beta regulator 4 (TBRG4) in HCC and its effects on the proliferation, invasion, and metastasis of HCC cells, and analyzed the possible molecular mechanisms.MethodDownloading the expression and clinical information of HCC samples in the TCGA database, analyzing the expression differences of TBRG4 by bioinformatics methods, analyzing the clinical relevance and prognostic significance. Performing GO, KEGG and GSEA enrichment analysis on the TBRG4-related gene set in patient HCC tissues. Applying cell counting, scratch test and Transwell experiment to study the biological function of TBRG4 in HCC. Mitochondrial membrane potential, apoptosis and ROS levels were evaluated to assess cell iron death. Western blot, RT-PCR, laser confocal microscopy and co-immunoprecipitation were used to detect and analyze the downstream signaling pathways and interacting molecules of TBRG4.ResultsBioinformatics analysis revealed that TBRG4 was abnormally highly expressed in HCC tumor tissues and was associated with poor prognosis and metastasis in HCC patients. GO and KEGG functional enrichment analysis showed that TBRG4 was related to oxidative stress and NADH dehydrogenase (ubiquinone) activity. GSEA enrichment analysis showed that TBRG4 was associated with Beta catenin independent wnt signaling and B cell receptor. Functional experiments confirmed that knocking down TBRG4 could inhibit the proliferation, migration, and invasion of HCC cells. Mechanistically, TBRG4 inhibited the function of HCC cells through the DDX56/p-AKT/GSK3β signaling pathway. In addition, interference with TBRG4 expression could reduce the mitochondrial membrane potential and accumulate ROS in HCC cells, leading to increased ferroptosis. Co-IP analysis showed that TBRG4 specifically bound to Beclin1.ConclusionTBRG4 is highly expressed in HCC tumor tissues and is associated with poor prognosis. It may regulate the proliferation, invasion, and metastasis of HCC cells through the DDX56/p-AKT/GSK3β signaling pathway. TBRG4 may interact with Beclin1 to regulate the ferroptosis of HCC cells.

Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-β. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs’ expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

Mitochondrial protein, TBRG4, modulates KSHV and EBV reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr (EBV) are gammaherpesviruses associated with multiple human malignancies. KSHV is the etiological agent of Kaposi's Sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). EBV is associated with Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). KSHV and EBV establish life-long latency in the human host with intermittent periods of lytic reactivation. Here, we identified a cellular factor named transforming growth factor-beta regulator 4 (TBRG4) that plays a role in the gammaherpesvirus lifecycle. We find that TBRG4, a protein that is localized to the mitochondria, can regulate lytic reactivation from latency of both KSHV and EBV. Knockdown of TBRG4 in cells latently infected with KSHV or EBV induced viral lytic gene transcription and replication. TBRG4 deficiency causes mitochondrial stress and increases reactive oxygen species (ROS) production. Treatment with a ROS scavenger decreased viral reactivation from latency in TBRG4-depleted cells. These data suggest that TBRG4 serves as a cellular repressor of KSHV and EBV reactivation through the regulation of ROS production.

Regulation of the plasminogen activator activity and inflammatory environment via transforming growth factor-beta regulation of sperm in porcine uterine epithelial cells

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

Knockdown of TBRG4 suppresses proliferation, invasion and promotes apoptosis of osteosarcoma cells by downregulating TGF-β1 expression and PI3K/AKT signaling pathway

Transforming growth factor beta regulator 4 (TBRG4) is a novel regulator in tumorigenic progression of several tumors. However, so far, the expression and functions of TBRG4 in osteosarcoma are unknown. The aim of this study was to investigate the potential biological functions of TBRG4 in osteosarcoma. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TBRG4 in osteosarcoma tissues and cell lines. The levels of TBRG4 protein in osteosarcoma tissues were assessed by immunohistochemistry. Lentivirus-mediated short hairpin (sh) RNA was employed to knock down TBRG4 in osteosarcoma cells, and the expressions of TBRG4 mRNA and protein were determined by qRT-PCR and Western blot assay, respectively. Subsequently, the proliferation, clonogenic ability, apoptosis and invasion of osteosarcoma cells were measured using high content screening analysis and CCK8 assay, tumor sphere formation assay, flow cytometry and Transwell invasion assays, respectively. Furthermore, the osteosarcoma cells growth and metastasis in vivo were detected, and the effect of TBRG4 on the transforming growth factor β1 (TGF-β1) and PI3K/AKT signaling pathway was explored by qRT-PCR and Western blot assay, respectively. The results showed the levels of TBRG4 were overexpressed in osteosarcoma tissues and cell lines, confirming that the high TBRG4 expression was related to advanced tumor stages, large tumor size, and lymph node metastasis. Functional assays showed knockdown of TBRG4 could inhibit proliferation, invasion and induce apoptosis of osteosarcoma cells in vitro, and could also suppress osteosarcoma growth and metastasis in vivo. By examining the expression levels of TGF-β1, p-PI3K, PI3K, p-AKT and AKT, it showed that the suppression of TBRG4 would reduce TGF-β1 expression and inactivate the PI3K/AKT signaling pathway. These results showed for the first time that TBRG4 knockdown could suppress osteosarcoma progression, suggesting TBRG4 might be a promising therapeutic target for osteosarcoma treatment.

Genome survey, high-resolution genetic linkage map construction, growth-related quantitative trait locus (QTL) identification and gene location in Scylla paramamosain

Scylla paramamosain is one of the most economically important crabs in China. In this study, the first genome survey sequencing of this crab was performed, and the results revealed that the estimated genome size was 1.21 Gb with high heterozygosity (1.3%). Then, RAD technology was used to construct a high-resolution linkage map for this species. A total of 24,444 single nucleotide polymorphism (SNP) makers were grouped into 47 linkage groups. The total length of the linkage groups was 3087.53 cM with a markers interval of 0.92 cM. With the aid of transcriptome and genome scaffold data, 4,271 markers were linked to genes, including several important growth-related genes such as transforming growth factor-beta regulator I, immune related-gene C-type lectin and ecdysone pathway gene broad-complex-like protein. Further, 442 markers, representing 279 QTLs, associated with 24 traits were identified, and of these markers, 78 were linked to genes. Some interesting genes, such as dedicator of cytokinesis protein 3, tenascin-X and DNA helicase MCM8, were believed to have important relationship with specific traits and merit further exploration. The results of this study will accelerate the genetic improvement and genome sequencing analysis of the mud crab.

Characterization of transforming growth factor beta regulator 1-like and association between its expression levels and growth of the giant tiger shrimp Penaeus monodon

Molecular markers that allow selection of juveniles and broodstock with improved growth performances are useful for the shrimp industry. Here, the full-length cDNA of transforming growth factor beta regulator 1-like (PmTbrg1-l) in the giant tiger shrimp Penaeus monodon was determined. It was 1184 bp in length and contained an open reading frame (ORF) of 975 bp corresponding to a deduced polypeptide of 324 amino acids. Successful RNA interference (RNAi) carried out using juveniles injected with PmTbrg1-l dsRNA revealed reduced levels of PmTbrg1-l and myostatin (PmMstm) in hemocytes when compared to shrimp injected with saline solution and GFP dsRNA (P < .05). Associations between single-strand conformational polymorphism (SSCP) patterns or single nucleotide polymorphism (SNP) patterns and growth-related parameters (average body weight and total length) were examined. Juveniles with pattern III (corresponding to A/A918; N = 37) showed a trend for greater average body weight and total length than those with patterns II (G/G918; N = 42) and IV (A/G918; N = 75). The expression level of PmTbrg1-l in the hepatopancreas of females was significantly higher than that in males (P < .05) in two sample sets of three-month-old domesticated juveniles (N = 59 and 50; P < .05). Moreover, its expression level in large-size juveniles was significantly higher than that in medium-size and small-size juveniles in both groups of samples (P < .05). Results indicated that PmTbrg1-l is functionally related with growth of P. monodon.

Knockdown of Transforming Growth Factor Beta Regulator 4 (Tbrg4) Promotes Apoptosis in Human Lung Cancer Cell Line H1299 via Regulating Ddit3, Cav1 and Rrm2

Abstract Transforming growth factor beta regulator 4 (TBRG4) gene is located on 7 p14-p13, a region correlated with many cancer types, such as hematologic malignancies, head and neck squamous cell carcinoma, and multiple myeloma. However, the role of TBRG4 in human lung cancer is largely unknown. Thus, in the present study, the TBRG4 mRNA level was evaluated in the lung cancer cell line H1299 by quantitative PCR (qPCR) after knocking down TBRG4 using lentivirus-mediated small interfering RNA (siRNA). The result showed that TBRG4 was significantly inhibited in H1299 by RNAi. Microarray analysis revealed 586 differentially-expressed genes after TBRG4 silencing. Analysis of the dataset with differentially-expressed genes using Ingenuity Pathway Analysis (IPA) software showed that infectious diseases, cancer, organismal injury and abnormalities were functions associated with the highest rated network. Further analysis of this network by IPA revealed that TBRG4 knockdown in H1299 cells changed the levels of downstream genes, including the upregulation of DDIT3 and downregulation of CAV1 and RRM2. These results have been validated by qPCR and Western blotting. Moreover, TBRG4 was shown to be highly expressed in carcinoma by immunohistochemstry compared with adjacent carcinomatous normal tissue. However, there were no associations between TBRG4 expression and clinicopathological characteristic, including sex, age, distant metastasis, TNM stage, and tumour grade. Taken together, TBRG4 plays an anti-apoptotic role in tumorigenesis of lung cancer via regulating DDIT3, CAV1 and RRM2 expression. The present findings are potentially significant to advance the understanding of TBRG4 as a candidate for the treatment of lung cancer.

Abstract 4782: Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression

Abstract Cancer stem cells (CSCs) are a subpopulation of tumor cells that are uniquely capable of initiating and sustaining tumorigenesis, and they have also been implicated in driving disease recurrence after cancer therapy. We have previously developed a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of cells with CSC properties. The construct consists of a tandemly-repeated composite SOX2-OCT4 response element (“SORE6”) driving expression of a destabilized fluorescent protein reporter (Tang et al. Stem Cell Reports, 2015). TGF-βs are pleiotropic regulatory factors with complex roles in tumorigenesis. We and others have hypothesized that TGF-β can regulate the CSC population in breast cancer with stimulatory or inhibitory effects depending the model and stage of cancer development. Using the MCF10Ca1h and MCF7 breast cancer models, which retain tumor suppressor responses to TGF-β, here we show that TGF-β treatment significantly reduces the size of the CSC population in vitro. Although TGF-β had relatively little effect on invasion and migration of the bulk population in these models, it clearly inhibited migration and invasion of the CSCs, suggesting that biological responses to TGF-β can vary depending on the position of the cell in the differentiation hierarchy. Combining our stem cell reporter with a TGF-β pathway reporter, we further show that CSCs in the MCF10Ca1h model have higher endogenous activation of the TGF-β pathway than does the bulk tumor cell population, and by time-lapse video microscopy we find that CSCs with active TGF-β signaling are relatively quiescent. Furthermore, neutralization of TGF-β in vivo leads to an increased representation of CSCs in MCF10Ca1h and MCF7 tumors. Thus our preliminary results suggest that in breast cancer models where TGF-β acts as a tumor suppressor, TGF-β signaling is preferentially activated in the CSC compartment and keeps a subpopulation of CSCs in a quiescent and stationary state. These findings have important implications for the clinical use of TGF-β pathway antagonists. In contrast, in the metastatic breast cancer MDA-MB231 model, in which TGF-β acts as a pro-progression factor, we show that TGF-β can increase the size of the CSC population and enhance invasion. In this model, the CSCs are enriched for the ability to form macroscopic lung metastases when compared with non-CSCs, and we show that this effect is due in part to a selective survival advantage of the CSCs in the first 24 hours after arrival at the metastatic site. The role of TGF-β in this process is currently under investigation. Citation Format: Binwu Tang, George Baker, Joshua Zent, Yuval Raviv, Lalage Wakefield. Transforming growth factor-beta regulation of cancer stem cell dynamics at different stages of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2017-4782

Global Gene Expression Profiling in Omental Adipose Tissue of Morbidly Obese Diabetic African Americans.

Adipose tissues play important role in the pathophysiology of obesity-related diseases including type 2 diabetes (T2D). To describe gene expression patterns and functional pathways in obesity-related T2D, we performed global transcript profiling of omental adipose tissue (OAT) in morbidly obese individuals with or without T2D. Twenty morbidly obese (mean BMI: about 54 kg/m2) subjects were studied, including 14 morbidly obese individuals with T2D (cases) and 6 morbidly obese individuals without T2D (reference group). Gene expression profiling was performed using the Affymetrix U133 Plus 2.0 human genome expression array. Analysis of covariance was performed to identify differentially expressed genes (DEGs). Bioinformatics tools including PANTHER and Ingenuity Pathway Analysis (IPA) were applied to the DEGs to determine biological functions, networks and canonical pathways that were overrepresented in these individuals. At an absolute fold-change threshold of 2 and false discovery rate (FDR) < 0.05, 68 DEGs were identified in cases compared to the reference group. Myosin X (MYO10) and transforming growth factor beta regulator 1 (TBRG1) were upregulated. MYO10 encodes for an actin-based motor protein that has been associated with T2D. Telomere extension by telomerase (HNRNPA1, TNKS2), D-myo-inositol (1, 4, 5)-trisphosphate biosynthesis (PIP5K1A, PIP4K2A), and regulation of actin-based motility by Rho (ARPC3) were the most significant canonical pathways and overlay with T2D signaling pathway. Upstream regulator analysis predicted 5 miRNAs (miR-320b, miR-381-3p, miR-3679-3p, miR-494-3p, and miR-141-3p,) as regulators of the expression changes identified. This study identified a number of transcripts and miRNAs in OAT as candidate novel players in the pathophysiology of T2D in African Americans.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

The structure of the FYR domain of transforming growth factor beta regulator 1

Many chromatin-associated proteins contain two sequence motifs rich in phenylalanine/tyrosine residues of unknown function. These so-called FYRN and FYRC motifs are also found in transforming growth factor beta regulator 1 (TBRG1)/nuclear interactor of ARF and MDM2 (NIAM), a growth inhibitory protein that also plays a role in maintaining chromosomal stability. We have solved the structure of a fragment of TBRG1, which encompasses both of these motifs. The FYRN and FYRC regions each form part of a single folded module (the FYR domain), which adopts a novel α + β fold. Proteins such as the histone H3K4 methyltransferases trithorax and mixed lineage leukemia (MLL), in which the FYRN and FYRC regions are separated by hundreds of amino acids, are expected to contain FYR domains with a large insertion between two of the strands of the β-sheet.

Interleukin-23 Restrains Regulatory T Cell Activity to Drive T Cell-Dependent Colitis

SummaryInterleukin-23 (IL-23) is an inflammatory cytokine that plays a key role in the pathogenesis of several autoimmune and inflammatory diseases. It orchestrates innate and T cell-mediated inflammatory pathways and can promote T helper 17 (Th17) cell responses. Utilizing a T cell transfer model, we showed that IL-23-dependent colitis did not require IL-17 secretion by T cells. Furthermore, IL-23-independent intestinal inflammation could develop if immunosuppressive pathways were reduced. The frequency of naive T cell-derived Foxp3+ cells in the colon increased in the absence of IL-23, indicating a role for IL-23 in controlling regulatory T cell induction. Foxp3-deficient T cells induced colitis when transferred into recipients lacking IL-23p19, showing that IL-23 was not essential for intestinal inflammation in the absence of Foxp3. Taken together, our data indicate that overriding immunosuppressive pathways is an important function of IL-23 in the intestine and could influence not only Th17 cell activity but also other types of immune responses.

Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes.

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

Transforming growth factor-beta regulation of retinoblastoma gene product and E2F transcription factor during cell cycle progression in mouse fibroblasts.

The mechanism by which transforming growth factor beta (TGF beta) exerts growth stimulatory effects was examined in C3H/10T1/2 mouse fibroblasts by study of cell cycle regulation of the retinoblastoma gene product (p110Rb) and transcriptional regulation of the p110Rb-associated transcription factor, E2F. Northern blotting analysis shows that TGF beta and/or epidermal growth factor (EGF) stimulate by three to sixfold the level of Rb mRNA which is also reflected by the increased levels of p110Rb. p110Rb becomes phosphorylated in mid-G1 and further phosphorylated at the G1/S transition. Hyperphosphorylation of p110Rb by TGF beta can be observed when cells are in S phase. TGF beta stimulates by three to fourfold the activity of cdk2 kinase consistent with the observed phosphorylation of p110Rb and also with the possibility that the kinase is involved in phosphorylating p110Rb close to the G1/S transition. Thus, TGF beta as a growth stimulator induces, as does EGF, the phosphorylation of p110Rb during cell cycle progression. Transient transfection of E2F promoter constructs was used to analyze the effect of TGF beta on the modulation of E2F-mediated transcription. The data revealed that TGF beta can stimulate wild-type adenoviral E2 promoter activity by 12-fold. Taken together, TGF beta-induced phosphorylation of p110Rb in mouse fibroblasts appears to exert a positive regulatory function upon genes that have a pivotal role in the G1/S transition of the cell cycle.

IL-1 and transforming growth factor-beta regulation of fibroblast-derived IL-11.

IL-11 and IL-6 are fibroblast-derived cytokines with overlapping biologic properties. To determine whether IL-11 and IL-6 are similarly regulated, we characterized the effects of rIL-1 and TGF-beta (beta 1 and beta 2) on human lung fibroblast IL-11 production and compared this regulation with that of IL-6. Unstimulated fibroblasts did not produce significant amounts of IL-11, whereas rIL-1 alpha and TGF-beta were dose-dependent stimulators of IL-11 protein production, mRNA accumulation, and gene transcription. rIL-1 alpha and TGF-beta also interacted in a synergistic fashion to further increase IL-11 protein production and mRNA accumulation. The effects of rIL-1 and TGF-beta individually were not altered by the cyclic nucleotide-dependent protein kinase inhibitor HA1004, protein kinase C (PKC) inhibition with staurosporine, or chronic phorbol ester preincubation, or the calmodulin antagonists W7 and TFP. The effects of rIL-1 alpha and TGF-beta in combination were also unaltered by HA1004, staurosporine, and chronic phorbol ester exposure. A23187, however, did induce IL-11 mRNA accumulation and W7 and TFP did reverse the synergistic stimulation caused by rIL-1 and TGF-beta in combination. In contrast with the regulation of IL-11, TGF-beta did not effectively stimulate IL-6 mRNA accumulation, rIL-1 alpha was a more potent stimulator of IL-6 than IL-11 production, and rIL-1-induced IL-6 mRNA accumulation was augmented by W7 and TFP. These studies demonstrate that: 1) rIL-1, TGF-beta, and agents that increase intracellular calcium stimulate lung fibroblast IL-11; 2) the IL-11 stimulatory effects of rIL-1 and TGF-beta are, at least partially, transcriptionally mediated and are the result of signal transduction pathways that are largely PKC, cyclic nucleotide, and calmodulin independent; and 3) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast IL-11 production and that this synergy is mediated by a largely PKC- and cyclic nucleotide-independent and calmodulin-dependent activation pathway. Importantly, they also demonstrate that rIL-1 and TGF-beta stimulate lung fibroblast IL-6 and IL-11 production via distinct and differentially regulatable activation pathways.

Transforming growth factor-beta regulation of IL-6 production by unstimulated and IL-1-stimulated human fibroblasts.

We characterized the ability of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) to regulate IL-6 production by unstimulated and rIL-1-stimulated lung fibroblasts. rTGF-beta 1-, purified TGF-beta 1-, and purified TGF-beta 2-stimulated fibroblasts produced IL-6 bioactivity as assessed with the B9 hybridoma proliferation assay. These TGF-beta moieties also bidirectionally regulated the IL-6 production of rIL-1-stimulated fibroblasts. The addition of TGF-beta to cultures in which fibroblasts were vigorously stimulated with rIL-1 resulted in an inhibition of fibroblast IL-6 production and mRNA accumulation. In contrast, the addition of TGF-beta to cultures in which fibroblasts were incubated with suboptimal concentrations of rIL-1 resulted in a synergistic increase in IL-6 production and mRNA accumulation [corrected]. Nuclear run-on analysis demonstrated that IL-6 gene [corrected] transcription was synergistically augmented when rTGF-beta 1 was combined with suboptimal concentrations of rIL-1. These studies demonstrate that TGF-beta stimulates fibroblast IL-6 production. They also show that TGF-beta can augment or inhibit the IL-6 production of IL-1-stimulated fibroblasts. Lastly, [corrected] they demonstrate that the stimulatory effects of TGF-beta are, at least partially, mediated by alterations in IL-6 gene transcription. TGF-beta may be an important regulator of IL-6 production. stimulated fibroblasts. Last, they demonstrate that the stimulatory effects of TGF-beta are, at least partially, mediated by alterations in IL-6 gene transcription. TGF-beta may be an important regulator of IL-6 production.

Transforming growth factor beta regulation of cell proliferation.

Two types of transforming growth factors (TGF) have been purified and well characterized, TGF alpha and TGF beta. TGF alpha is a 5.6 kD single chain molecule that shows sequence homology to epidermal growth factor (EGF), binds to the EGF receptor, and has biological effects very similar to those of EGF. TGF beta is different from TGF alpha in its molecular structure and biological activity, and has its own specific cell surface receptor. TGF beta is a 25 kD homodimer of 12.5 kD subunits that shows no sequence homology to TGF alpha. TGF beta is a highly ubiquitous molecule produced by a variety of cell types in an inactive form. Most cells have receptors for TGF beta, suggesting that a major regulatory step in TGF beta action is through activation of the inactive form. Growth stimulatory effects with TGF beta have been observed so far only in fibroblastic cells. In at least one circumstance, there is evidence that the stimulatory effects of TGF beta in fibroblastic cells is indirect through induction of c-sis and autocrine stimulation by platelet-derived growth factor (PDGF)-like material. TGF beta inhibits in vitro proliferation of most cell types tested, including normal epithelial cells. Thus TGF beta is primarily a growth inhibitor and not a classical growth factor. Increased autocrine stimulation by endogenous TGF beta in fibroblastic cells or decreased inhibitory effects in epithelial cells (or other cells normally inhibited by TGF beta) could lead to an increased proliferative potential and thereby contribute to the neoplastic phenotype.