TGF-beta1 regulates TGF-beta1 and FGF-2 mRNA expression during fibroblast wound healing.

AIMS: To evaluate the expression of transforming growth factor beta1 (TGF-beta1) and fibroblast growth factor 2 (FGF-2) mRNA in stromal cells in response to injury in the presence of either TGF-beta1 or FGF-2. It has been shown previously that heparan sulfate proteoglycans and FGF-2 are present transiently during wound repair in vivo and that an increase in TGF-beta1 mRNA is detected rapidly after injury. METHODS: Primary corneal fibroblasts were cultured to confluency, serum starved, and linear wound(s) were made in medium containing TGF-beta1 or FGF-2. TGF-beta1 and FGF-2 mRNA expression were evaluated using both northern blot analysis and in situ hybridisation. Both dose dependent and time course experiments were performed. Whole eye organ culture experiments were also carried out and growth factor expression was assessed. RESULTS: Injury and exogenous TGF-beta1 increased TGF-beta1 mRNA values. The increase in expression of FGF-2 mRNA was not detected until wound closure. In contrast, FGF-2 inhibited the expression of TGF-beta1. TGF-beta1 increased TGF-beta1 mRNA stability but did not alter that of FGF-2. Migration assay data demonstrated that unstimulated stromal cells could be activated to migrate to specific growth factors. CONCLUSIONS: TGF-beta1 specifically enhances cellular responsiveness, as shown by increased stability after injury and the acquisition of a migratory phenotype. These data suggest that there is an integral relation during wound repair between TGF-beta1 and FGF-2.     

Expression and regulation of latent TGF-beta binding protein-1 transcripts and their splice variants in human glomerular endothelial cells

Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H(2)O(2), and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1Sdelta53. TGF-beta1, but not high glucose, H(2)O(2) or VEGF, tended to increase LTBP-1Sdelta53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H(2)O(2), and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S 53. Modification of LTBP-1S 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.     

Characterization of transforming growth factor-beta 1 gene expression in porcine immune cells.

We have investigated the regulation of transforming growth factor beta 1 gene expression in a variety of porcine immune cell populations, including peripheral blood mononuclear cells (PBMC), peripheral blood monocytes, alveolar macrophages and lymphoid cells from various swine lymphoid tissues. Using porcine transforming growth factor beta 1 cDNA probes in Northern blot assays, messages of 2.5 and 3.5 kb TGF beta 1 mRNA were detected in the cells investigated. A variety of mitogenic and immunomodulatory substances were examined for their ability to induce TGF beta 1 mRNA expression. These include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS), dexamethasone (Dex), tumor necrosis factor (TNF) and interleukin (IL)-1 alpha. While low level constitutive expression of TGF beta 1 mRNA was detected from all cells investigated, PMA treatment of PBMC and alveolar macrophages resulted in a more than 10-fold increase in the steady-state level of TGF beta 1 mRNA within 2 hr of PMA addition. Also, the effect of opiate drugs, methadone (Md) and morphine (Mor), on TGF beta 1 gene expression was determined. Cells treated with opiates expressed the same levels of TGF beta 1 mRNA as untreated cells. Since TGF beta 1 biological activity can be induced by opiates, the regulation of TGF beta 1 gene expression likely involves mechanisms that do not cause changes in mRNA levels.     

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.     

Differential expression of TGF-beta isoforms during differentiation of HaCaT human keratinocyte cells: implication for the separate role in epidermal differentiation

The three mammalian isoforms of transforming growth factor-beta(TGF-beta1, beta2, beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell growth and differentiation induction of the human keratinocyte cell line, HaCaT by elevating the Ca(2+) concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta 1 mRNA were increased during calcium-induced differentiation. In contrast, major differences were seen for TGF-beta 2 and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta 2 and TGF-beta3 protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associated with the more differentiated state, and TGF-beta 2 and TGF-beta 3 may be associated the more proliferated state.     

Expression and synthesis of fibroblast growth factor-9 in human gammadelta T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-beta1/IL-15

Gammadelta T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether gammadelta T-lymphocytes produce fibroblast growth factor (FGF)-9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood gammadelta T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor-beta1 (TGF-beta1)/interleukin-15 (IL-15) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human gammadelta T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-beta1/IL-15 did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon-gamma mRNA. Western blot analysis of gammadelta T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage gammadelta T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.     

TGF—β,TNF—α和LIFmRNA在白血病细胞中的表达

用斑点杂交半定量法对6株人、1株大鼠和5株小鼠白血病细胞以及部分正常对照细胞进行了TGF-β、TNF-α和LIF三种抑制因子mRNA表达水平的检测。得出以下结论:(1)615小鼠白血病相关抑制因子(LAI-615)属于TGF-β类因子。(2)白血病细胞与正常对照细胞相比,三种负性因子的表达存在差异。(3)LIF的表达与否,可能与白血病细胞类型有关。(4)白血病细胞中负性因子的表达强度高于正常对照细胞。(5)TGF-β较TNF-α和LIF更普遍存在并参与正常和白血病造血细胞增殖和分化的调控。     

Effect of antisense TGF-beta1 oligodeoxynucleotides in streptozotocin- induced diabetic rat kidney

Transforming growth factor (TGF)-beta1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of circular antisense TGF-beta1 oligodeoxynucleotides (ODNs) on the TGF-beta1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats. Circular antisense TGF-beta1 ODNs were found to be stable in rat serum, significantly decreasing TGF-beta1 mRNA expression compared with linear antisense ODNs in the rat mesangial cell culture. Circular antisense TGF-beta1 ODNs were introduced into the tail vein of normal rats using hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method and were confirmed to be delivered effectively into the kidney, liver, lungs, and spleen. To inhibit the overexpression of TGF-beta1 in diabetic kidneys, we introduced circular antisense TGF-beta1 ODNs into the STZ-induced diabetic rats. On day 13 after circular antisense TGF-beta1 ODNs injection, TGF-beta1 mRNA and protein expression markedly decreased and urinary TGF-beta1 excretion rate also dropped in the circular antisense TGF-beta1 ODNs-treated diabetic rats. These results suggest that circular antisense TGF-beta1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.     

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

Gene expression of bone-associated cytokines in MG63 osteoblast-like cells incubated with acrylic bone cement extracts in minimum essential medium

This study examined the effects of in vitro challenge with four polymerized acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the expression of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-beta1) mRNAs in the osteoblast-like cell line MG63. The extracts of the cements in minimal essential medium (MEM) were tested following 1-h and 7-day curing. A semi-quantitative analysis of the cytokine-specific mRNAs was carried out by agarose gel densitometry and expression was compared with the GAPDH housekeeping gene. The ratio between cytokine gene expression and GAPDH expression was calculated. The mRNA specific for the bone-resorbing cytokines IL-1beta and IL-6 was low in basal conditions. IL-1beta mRNA increased only after incubation with the extract of CMW 1 following 1-h curing. The mRNA specific for the bone-resorbing cytokine IL-6 also increased after contact with CMW 1 at both curing times. Sulfix-60 and CMW 3 following 7-day curing, but not after 1 h, induced higher levels of IL-6 mRNA than the control. CMW 2 after 1-h curing constantly determined the expression of IL-6 mRNA, but at low levels. The mRNA specific for TGF-beta1 was also expressed by the MG63 osteoblast-like cells in basal conditions. The levels increased after contact with Sulfix-60 after 7-day curing and with CMW 1 after 1-h curing. CMW 2 after 7-day curing decreased TGF-beta1 mRNA. In conclusion, the highest expression of the cytokines IL-1beta, IL-6, and TGF-beta1 mRNA was determined by CMW 1. If the results are confirmed in vivo, the increased expression of the osteolytic cytokines induced by the bone cement might result in loosening of the prosthesis, even with all the restrictions of an in vitro study on continuous cell lines.     

Regulation of matrix metalloproteinases (MMPs) and their tissue inhibitors in human myometrial smooth muscle cells by TGF-beta1.

The objective of the present study was to determine whether transforming growth factor beta (TGF-beta) regulates the expression of matrix metalloproteinases (MMP) and the tissue inhibitor of MMP (TIMP) in myometrial smooth muscle cells. Using primary cultures of human myometrial smooth muscle cells we found that these cells express MMP-1, MMP-3, TIMP-1 and TIMP-2 mRNA and protein, with significantly higher values of TIMP than MMP. We also found that TGF-beta1 (1 ng/ml) increased the expression of TIMP-1 mRNA, while it reduced the expression of MMP-1 and MMP-3 mRNA, compared with untreated controls. In addition, TGF-beta1 slightly increased the production of TIMP-1, but not TIMP-2. Production of MMP-1 and MMP-3 was reduced by treatment with TGF-beta1, compared with the untreated control. A major portion of MMP-1 released into the culture-conditioned media was in complex with TIMP-1, and the levels of this complex were reduced by treatment with TGF-beta1. In conclusion, the data indicate that myometrial smooth muscle cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by TGF-beta1. Such a differential regulation of MMP and TIMP by TGF-beta may influence the rate of extracellular matrix (ECM) turnover following tissue injury, induced during myomectomy and Caesarean section, or in leiomyomas during growth.     

A novel truncated TGF-beta receptor II downregulates collagen synthesis and TGF-beta I secretion of keloid fibroblasts

Hypertrophic scars and keloid are dermal proliferative disorders in wound healing. Transforming growth factor beta (TGF-beta) has been implicated in scar formation through the activation of fibroblasts and the acceleration of collagen deposition. Our study aimed to design a novel truncated (27-123 residues) type II TGF-beta receptor (tTGFbetaRII) and to determine its effects on the proliferation of keloid fibroblasts and the collagen synthesis as well as TGF-beta I expression of the cells. The coding sequences of TGF-beta I and tTGFbetaRII were amplified using RT-PCR and then cloned into pGBKT7 and pGADT7 vectors. A yeast two-hybrid experiment and a glutathione S-transferase (GST)-pull down assay were performed to verify the affinity of tTGFbetaRII to TGF-beta I. Our results indicated that treatment with tTGFbetaRII inhibited the growth of keloid fibroblasts and suppressed the synthesis of type I collagen in keloid fibroblasts in a concentration-dependent manner. Moreover, northern and western blot analysis revealed a decline of the TGF-beta I expression at both mRNA and protein levels after exposure to 5, 10 or 20 mug/ml of tTGFbetaRII. Together, our data suggested that the exogenous tTGFbetaRII can efficiently trap TGF-beta I from access to wild-type receptors and can suppress TGF-beta I triggered signals. Thus it may potentially be clinically applied to scar therapy.     

IL-10 and TGF-beta differ in their regulation of aminopeptidase N/CD13 expression in monocytes

Human monocytic cells express considerable amounts of aminopeptidase N (APN)/CD13, a transmembrane protein proposed to play a role in the modulation of kinins, neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Previous studies have shown that APN/CD13 participates in antigen processing and presentation, trimming peptides protruding out of MHC class II molecules. In several inflammatory processes, macrophages have been shown to express especially high amounts of MHC class II molecules and of this peptidase. To learn more about the regulation of APN/CD13 on monocytes we investigated its expression under the influence of cytokines. Here, we report a dose- and time-dependent up-regulation of APN/CD13 mRNA and protein expression by transforming growth factor (TGF)-beta on human monocytes. To the contrary, we found IL-10 down-regulating the expression of APN/CD13 mRNA and protein. Both the regulation of the APN/CD13 protein assessed by immunofluorescence and the gene expression assessed by real-time PCR were highly correlated. Using the Dual-Luciferase reporter assay, we demonstrate that TGF-beta treatment of monocytes results in a higher activity of the APN/CD13 myeloid promoter. Our results implicate differences in the expression of the membrane peptidase APN/CD13 and therefore in the peptide-modulating ability of monocytes after exposure to these two immunosuppressive cytokines, TGF-beta and IL-10.