Analysis of the transforming growth factor-beta1 (TGF-beta1) codon 25 gene polymorphism by LightCycler-analysis in patients with chronic hepatitis C infection

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.     

Serum tumour necrosis factor-alpha and transforming growth factor-beta levels in chronic hepatitis C patients are immunomodulated by therapy

Our aims were: (i) to characterize serum levels of tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in non-cirrhotics with hepatitis C; (ii) to correlate levels of theses cytokines with degree of disease at baseline; (iii) to characterize the immunomodulatory effects of therapy with response and (iv) to compare profiles of cytokines in patients treated with pegylated-interferon alpha-2b monotherapy (PMT) vs its combination with ribavirin (PCT1-low dose ribavirin and PCT2-high dose ribavirin). We studied 56 patients that were part of two randomized, controlled, clinical trials. At baseline, high TNF-alpha levels paralleled the degree of inflammation as determined by histology. In PCT2, a significant reduction was seen in levels of TNF-alpha, TGF-beta and fibrosis scores when comparing baseline with follow-up. In sustained responders, regardless of therapy, the histological activity scores were lower at follow-up as compared to baseline. In conclusion, PCT2 is able to constantly reduce and sustain TNF-alpha levels, which is responsible for the sustained decline in liver inflammation as shown by the histological activity index and it is also able to reduce fibrosis as judged both by TGF-beta levels and fibrosis scores.     

Adenoviral supply of active transforming growth factor-beta1 (TGF-beta1) did not prevent lethality in transforming growth factor-beta1-knockout embryos.

In TGF-beta1-knockout mice, TGF-beta1-null conceptuses die during embryonic development with a penetrance of lethality that depends on the mouse genetic background. Studies have suggested that transplacental passage of maternal TGF-beta1 could account for the rescue of some TGF-beta1-null embryos. Herein, we have used an adenovirus-based gene delivery system and a strain of mice where most TGF-beta1-null conceptuses die prior to parturition, to investigate whether an increase in maternal TGF-beta1 during pregnancy would rescue TGF-beta1-null embryos. A single intravenous injection of an adenovirus containing a modified version of TGF-beta1 cDNA (Ad-TGF-beta1S223/S225), coding for a biologically active form of the cytokine, induced a 20-fold increase in plasma TGF-beta1 (active and latent forms) levels for up to 3 months in adult mice. Similar levels of TGF-beta1 were detected in 13-day post co?tum (dpc) embryos from Ad-TGF-beta1-treated mothers, demonstrating an efficient maternal/fetal transfer of the cytokine. However, no increase in the frequencies of TGF-beta1-null neonates nor in day 11.5 dpc TGF-beta1-null conceptuses was observed despite elevated levels of TGF-beta1 delivered throughout gestation. In addition, we show that the high levels of TGF-beta1-titrated in the plasma from Ad-TGF-beta1S223/S225-treated mice were partly the consequence of a stimulation of an autocrine production by exogenous bioactive TGF-beta1. These results indicate that transplacental passage of TGF-beta1 was not effective in rescuing TGF-beta1-null conceptuses from embryonic lethality.     

Immunohistochemical localization of transforming growth factor-beta (TGF-beta) in smooth muscle tissue

Anti-TGF-beta antibodies were raised in rabbits against a C-terminal synthetic peptide whose amino acid sequence was chosen after human TGF-beta 1 primary sequence antigenic study by continuous epitope prediction. Immunopurified antibodies were used for an immunohistochemical localization in normal and pathological human tissues. Smooth muscular type tissue seems to be an elective target.     

Purification of the transforming growth factor-beta (TGF-beta) binding proteoglycan betaglycan.

We report the purification of betaglycan, a low-abundance membrane proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). Betaglycan solubilized from rat embryo membrane preparations was purified to near-homogeneity by sequential chromatography through DEAE-Trisacryl, wheat germ lectin-Sepharose, and TGF-beta 1-agarose. Purified betaglycan has properties similar to betaglycan affinity-labeled in intact cells: it binds TGF-beta 1 and TGF-beta 2 with KD approximately 0.2 nM, contains heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains and N-linked glycans attached to a 110-kDa core protein, and can spontaneously associate with phosphatidylcholine liposomes. The betaglycan core obtained by enzymatic removal of the GAG chains has high affinity for TGF-beta and associates with artificial liposomes, indicating that the core protein binds TGF-beta and anchors to membranes independently of the GAG chains present on the native protein or of any ancillary protein.     

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.     

Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites.

Transforming growth factor-beta (TGF-beta) is a bifunctional, dose-dependent regulator of endothelial cell proliferation induced in vitro by heparin-binding growth factor 1 (HBGF-1, acidic FGF). Here we have examined the relationship between endothelial cell growth and the expression of cell surface binding sites for TGF-beta and HBGF-1. Fetal bovine heart endothelial cell (FBHEC) growth was stimulated by low concentrations of TGF-beta and inhibited by high concentrations of TGF-beta while expressing two distinct classes of TGF-beta binding sites with binding constants of 24 pM (6300 sites/cell) and 900 pM (12,000 sites/cell). In contrast, human umbilical vein endothelial cells (HUVEC), whose growth was slightly promoted by TGF-beta, exhibited a single class of high-affinity TGF-beta binding sites (Kd = 45 pM, 4500 sites/cell). Affinity crosslinking using [125I]TGF-beta showed that FBHEC expressed two distinct low molecular weight TGF-beta binding sites (Mr 85,000 and 58,000), while HUVEC expressed a single type of low molecular weight TGF-beta binding site (Mr 85,000). As detected by binding of [125I]HBGF-1, preincubation of FBHEC with high concentrations of TGF-beta transmodulated the expression of high-affinity HBGF-1 receptors. In contrast, no transmodulation of HBGF-1 receptors occurred in FBHEC during preincubation with low concentrations of TGF-beta. Furthermore, preincubation of HUVEC with TGF-beta did not transmodulate the expression of HBGF-1 receptors. The data suggest that the ability of TGF-beta to stimulate or inhibit endothelial cell proliferation in a dose-dependent manner correlated with the expression of specific TGF-beta binding site subtypes and involved the transmodulation of HBGF-1 receptors.     

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.     

Differential expression of transforming growth factor-beta 1 (TGF-beta 1) receptors in murine myeloid cell lines transformed with oncogenes. Correlation with differential growth inhibition by TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic polypeptide hormone known to play an important role as a modulator of hematopoietic processes in human and murine cells. One of the characteristics of TGF-beta 1 is the ability to inhibit the growth of several cell types, including cells of the myeloid lineage. To study the mechanism by which TGF-beta 1 inhibits the growth of myeloid cells, we have used three murine myeloid cell lines, the parental interleukin-3-dependent 32D-123 cell line and two retrovirally infected interleukin-3-independent cell lines (32D-abl, 32D-src), all of which are growth inhibited by TGF-beta 1. Each of these oncogene-transfected cells expresses a greater number of TGF-beta 1 receptors than the parental cell line and responds to TGF-beta 1 with increased sensitivity; 32D and 32D-src cells are 2- and 58-fold more sensitive to TGF-beta 1 inhibition than the parental cell line (ED50 = 35 pM). Both 32D-abl- and 32D-src-transformed cell lines expressed higher levels of the 65- and 85-kDa TGF-beta 1 receptor species than did the parental cells. We observed a correlation between the greater sensitivity of 32D-src cells to TGF-beta 1 and the more rapid down-modulation and reappearance of cell surface TGF-beta 1 receptors on 32D-src cells. Thus, the level of TGF-beta 1 receptor expression and rate of reexpression both have a crucial regulatory effect on the functional activity of the TGF-beta 1 ligand.     

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.     

The role of transforming growth factor-beta (TGF-beta) during ovarian follicular development in sheep

Background  

Recently, several members of the transforming growth factor-beta (TGF-beta) superfamily have been shown to be essential for regulating the growth and differentiation of ovarian follicles and thus fertility.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Serum endothelin-1 and transforming growth factor-beta levels in the newborns with respiratory distress

The purpose of this present study was to evaluate the serum levels of ET-1 and TGF-beta in the newborns with respiratory distress. In this study, newborns with respiratory distress hospitalized into the Newborn Intensive Care Unit were included. The highest values of ET-1 and TGF-beta were obtained from newborns with diagnosis as meconium aspiration syndrome (5.70 +/- 5.87 pg/mL and 3.75 +/- 1.94 pg/mL, resp) in the sample obtained in the first six hours after birth, and these are statistically different from control group (P < .05). Also, same results were obtained for newborns with respiratory distress syndrome (3.37 +/- 1.59 pg/mL and 2.05 +/- 0.98 pg/mL, resp). After oxygen treatment, ET-1 values obtained in the first six hours of life were decreased regularly in the following days (P < .05). In the differentiating diagnosis of the respiratory distress of newborns, the investigation of ET-1 and TGF-beta levels is meaningful. The ET-1 levels investigated in the first six hours is more useful in determining the prognosis, and repeating ET-1 levels in the following days is more meaningful to determine clinical response.     

NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.