Effects of interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) on neutrophil elastase release

The proinflammatory cytokine interleukin-6 (IL-6) and its potential opponent, transforming growth factor- (TGF- ₁), have been discussed as being involved in the regulation of inflammatory processes following trauma and infections. The aim of this study was to investigate the effect of these cytokines on the regulation of neutrophil degranulation. The posttraumatic time courses of the plasma concentrations of IL-6, and the elastase- ₁-proteinase-inhibitor complex as marker of degranulation in patients undergoing severe trauma were found to be highly correlated, whereas TGF- ₁ levels were determined to be not significantly altered. The close temporal correlation of IL-6 and elastase levels could be confirmed by investigation of exudates derived from the surgical area. To prove these in vivo findings, the effect of IL-6 and TGF- ₁ on the degranulation of isolated neutrophils of healthy donors was investigated in vitro. Pathological high IL-6 concentrations were found to be capable of inducing a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation was unaffected by TGF- ₁. In conclusion, these data suggest an involvement of IL-6 in the regulation of neutrophil degranulation under pathological conditions. However, TGF- ₁ seems to have no direct regulatory effect besides its described chemotactic function on neutrophils.     

Roles of transforming growth factor-β in graft-versus-host and graft-versus-tumor effects

Transforming growth factor (TGF)-β is a pleiotropic cytokine with widespread and profound effects on immune cells. Consequently, it has generated considerable interest in relation to the immunologic outcomes after allogeneic hematopoietic cell transplantation. The TGF-β pathway has been shown to be an important modulator of alloimmunity, with direct consequences on graft-versus-host disease pathophysiology and graft-versus-tumor response. The TGF-β-related effects can be both beneficial and detrimental to the host, underscoring the complexity of TGF-β biology. This article reviews the evidence linking TGF-β to alloimmune responses in allogeneic hematopoietic cell transplantation and highlights foreseeable strategies that?would maximize the beneficial effects of TGF-β pathway modulation on both graft-versus-host disease pathophysiology and the graft-versus-tumor effect.     

The Cain and Abl of epithelial-mesenchymal transition and transforming growth factor-β in mammary epithelial cells

Transforming growth factor-β (TGF-β) normally inhibits breast cancer development by preventing mammary epithelial cell (MEC) proliferation, by inducing MEC apoptosis, and by creating cell microenvironments that maintain MEC homeostasis and prevent their uncontrolled growth and motility. Mammary tumorigenesis elicits dramatic alterations in MEC architecture and microenvironment integrity, which collectively counteract the tumor-suppressing activities of TGF-β and enable its stimulation of breast cancer invasion and metastasis. How malignant MECs overcome the cytostatic actions imposed by normal microenvironments and TGF-β, and how abnormal microenvironments conspire with TGF-β to stimulate the development and progression of mammary tumors remains largely undefined. These knowledge gaps have prevented science and medicine from implementing treatments effective in simultaneously targeting abnormal cellular microenvironments, and in antagonizing the oncogenic activities of TGF-β in developing and progressing breast cancers. c-Abl is a ubiquitously expressed nonreceptor protein tyrosine kinase that essentially oversees all aspects of cell physiology, including the regulation of cell proliferation, migration and adhesion, as well as that of cell survival. Thus, the biological functions of c-Abl are highly reminiscent of those attributed to TGF-β, including the ability to function as either a suppressor or promoter of tumorigenesis. Interestingly, while dysregulated Abl activity clearly promotes tumorigenesis in hematopoietic cells, an analogous role for c-Abl in regulating solid tumor development, including those of the breast, remains controversial. Here, we review the functions of c-Abl in regulating breast cancer development and progression, and in alleviating the oncogenic activities of TGF-β and its stimulation of epithelial-mesenchymal transition during mammary tumorigenesis.     

Latent TGF-β binding proteins (LTBPs) 1 and 3 differentially regulate transforming growth factor-β activity in malignant mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura with poor prognosis. There is a need to identify new biomarkers and therapeutic targets for this invasive and fatal disease. Transforming growth factor β (TGF-β) can promote mesothelioma tumorigenesis through multiple mechanisms. Latent TGF-β binding proteins (LTBPs) regulate TGF-β activation by targeting the growth factor into the extracellular matrix from where it can be released and activated. We investigated here the expression patterns of different LTBP isoforms in malignant mesothelioma tissues and in 2 established malignant mesothelioma cell lines. All LTBPs were expressed, but LTBP-3 was the main isoform in healthy pleura and in cultured nonmalignant mesothelial cells. We observed down-regulation of LTBP-3 expression in malignant mesothelioma, which was associated with high P-Smad2 levels indicative of TGF-β activity specifically in the tumor tissue. Small interfering RNA-mediated suppression of LTBP-3 expression in mesothelioma cells increased the secretion of TGF-β activity. Immunoreactivity of LTBP-1, on the other hand, was markedly strong in the tumor stroma, which showed significantly lower levels of P-Smad2. A strong negative correlation between LTBP-1 and P-Smad2 immunoreactivity was found, implying that LTBP-1 is not likely to contribute directly to the increased levels of TGF-β activity in malignant mesothelioma. Current results suggest that LTBPs 1 and 3 may have specific roles in malignant mesothelioma pathogenesis through the regulation of TGF-β activation in the tumor tissue and the structure of the tumor stroma.     

In vitro effects of bone- and platelet-derived transforming growth factor-β on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor- (TGF-), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF- produced a dose-dependent growth response in Walker cells with an ED₅₀ equal to 0·05 ng/ml. Bone-derived TGF- activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-1 neutralizing antibody. We conclude that TGF- is an important growth stimulatory component from fetal rat calvaria.     

Dynamic expression patterns of transforming growth factor-β<Subscript>2</Subscript> and transforming growth factor-β receptors in experimental glomerulonephritis

Numerous studies have demonstrated the involvement of the transforming growth factor (TGF) isoform beta(1) in the pathogenesis of renal fibroproliferative diseases. Although in vitro studies suggest that TGF-beta(2) is equally potent to TGF-beta(1) in terms of its antimitogenic and fibrogenic effects, much less is known about the regulation of TGF-beta(2) in renal diseases associated with glomerular cell hyperplasia and matrix expansion. Here we investigated the glomerular expression patterns of TGF-beta(2) and of the TGF-beta receptors I, II, and III during the course of rat anti-Thy1.1 nephritis (days 2, 6, 12, and 56), a model characterized by transient mesangial hypercellularity and extracellular matrix accumulation. TGF-beta(2) exhibited dynamic changes in expression. Immunohistochemical double-staining of renal sections revealed that most TGF-beta(2)-positive cells in control glomeruli were podocytes with few TGF-beta(2)-positive mesangial cells. This staining pattern could also be observed in human kidney. On day 6 of anti-Thy1.1 nephritis both TGF-beta(2) positive podocytes and mesangial cells were more abundant. By western blot analysis of isolated glomeruli from nephritic rats, protein expression of TGF-beta(2) was upregulated tenfold over control glomeruli, peaking on day 6 of the disease. In cultured rat mesangial cells we found that the TGF-beta(2) and TGF-beta(1) isoforms were equally potent in terms of nuclear accumulation of phosphorylated Smad 2/3, inhibition of DNA synthesis, and induction of beta(1)-integrin and type I collagen protein synthesis. Protein expression of the TGF-beta receptor I was not detected by immunohistochemistry in control glomeruli but was markedly induced in the mesangium on day 6 of nephritis. Mesangial staining for TGF-beta receptors II and III was detected in normal kidneys. Expression of TGF-beta receptor II was strongly enhanced on days 6 and 12 of disease, while TGF-beta receptor III was upregulated only on day 6. In summary, we report marked yet transient upregulation of TGF-beta(2) protein and of TGF-beta receptors I, II, and III in glomerular cells during anti-Thy1.1 nephritis. These results are in keeping with the notion that TGF-beta(2) and its receptors participate in the pathogenesis and/or resolution of this transient form of glomerulonephritis.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

Objective To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Methods Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. Results The cultured HMC treated with TGF-$l showed increased expression of AR and FN,the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR. Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-$l ,and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3. 6-fold after treatment with TGF-pl (P <0. 05) , and the induction effect on FN expression was suppressed by G(O)6983 (42%) in HMCs (P < 0. 05) . The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs(P <0. 01) , and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P <0. 01). Conclusions AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.     

Effect of ibuprofen and diclofenac on the chemotaxis induced by substance P and transforming growth factor-β on human monocytes and polymorphonuclear cells

The neuropeptide substance P and the cytokine transforming growth factor-β are potent chemotactic factors for monocytes or polymorphonuclear cells. They are present in synovial fluid of arthritic patients, and participate in the pathogenesis of arthritis. We investigated, in vitro, the effect of two non-steroidal anti-inflammatory drugs, ibuprofen and diclofenac, on the chemotactic effect of substance P and transforming growth factor-β at concentrations that can be present in the synovial fluid of arthritic patients. Both drugs decrease the chemotaxis induced by substance P and transforming growth factor-β, at concentrations that can be easily reached in the synovial fluid during therapy. This event could be involved in the effect of some non-steroidal anti-inflammatory drugs on the development and progress of arthritic disease.     

Retinoic acid induced repair in the lung of adult hyperoxic mice,reducing transforming growth factor-β1 (TGF-β1) mediated abnormal alterations

The aim of the study was to determine the effects of retinoic acid on lung alveolar repair in adult hyperoxic mice and to investigate the relationship between TGF-β1 and retinoic acid during the repair processes. Adult mice were divided into 4 groups. Two groups were given daily intraperitoneal injections of peanut oil/dimethylsulfoxide mixture and retinoic acid (50 mg/kg body weight, 50 μl of volume) dissolved in peanut oil/dimethylsulfoxide mixture for 12 days with a 2-day break on days 6 and 7. Following hyperoxia (100% oxygen) for 72 h the remaining two groups were treated in the same manner as already described: peanut oil/dimethylsulfoxide mixture and retinoic acid. Lung structure was investigated by light microscopy. TGF-β1 and Smad protein expressions in the lung were assayed by biochemical methods. Hyperoxic mice exhibited damage to the alveolar walls, increased cell proliferation and induced Smad3/TGF-β1 signaling. Smad2 and phospho-Smad2 protein expressions were unchanged in all groups. Retinoic acid administration improved the degenerative alterations caused by hyperoxia and helped in alveolar repair. This positive effect of retinoic acid resulted from the inhibition of Smad3/TGF-β1 signaling via reduced Smad4 mRNA and increased Smad7 protein expression. Retinoic acid also induced alveolarization and restricted Smad3/TGF-β1 signaling by decreasing Smad4 mRNA in healthy mice. Thus, retinoic acid helped repair Smad3/TGF-β1-induced lung damage in hyperoxic mice.     

Polymorphisms of transforming growth factor-β signaling pathway and Kawasaki disease in the Taiwanese population

Kawasaki disease (KD) is a systemic vasculitis associated with cardiovascular symptom. A previous study in the European descent has indicated that genetic variants of the transforming growth factor-β (TGF-β) pathway are involved in the KD susceptibility and clinical status. This study was conducted to investigate if polymorphisms in TGF-β signaling pathway are associated with KD susceptibility, and the coronary artery lesion formation. A total of 950 subjects (381 KD patients and 569 controls) were investigated to identify 12 single-nucleotide polymorphisms in the TGF-β signaling pathway (rs2796817, rs10482751, rs2027567, rs12029576, rs11466480, rs4776338, rs12901071, rs7162912, rs1438386, rs6494633, rs12910698 and rs4776339) by using TaqMan Allelic Discrimination assay. Our results indicated that rs1438386 in the SMAD3 is significantly associated with the susceptibility of KD. Additionally, both haplotypes of TGFβ2 and SMAD3 were also associated with the risk of KD. This study showed that genetic polymorphisms in TGF-β signaling pathway are associated with KD susceptibility, but not coronary artery lesions formation, or intravenous immunoglobulin treatment response in the Taiwanese population.     

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-β1 (TGF-β1)

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-β1 (TGF-β1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-β1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.     

Transforming growth factor-β (TGF-β) pathway abnormalities in tenascin-X deficiency associated with CAH-X syndrome

Patients with congenital adrenal hyperplasia (CAH) with tenascin-X deficiency (CAH-X syndrome) have both endocrine imbalances and characteristic Ehlers Danlos syndrome phenotypes. Unlike other subtypes, tenascin-X-related Ehlers Danlos syndrome is caused by an extracellular matrix protein deficiency rather than a defect in fibrillar collagen or a collagen-modifying enzyme, and the understanding of the disease mechanisms is limited. We hypothesized that transforming growth factor-β pathway dysregulation may, in part, be responsible for connective tissue phenotypes observed in CAH-X, due to this pathway's known role in connective tissue disorders.Fibroblasts and direct tissue from human skin biopsies from CAH-X probands and age- and sex-matched controls were screened for transforming growth factor-β biomarkers known to be dysregulated in other hereditary disorders of connective tissue. In CAH-X fibroblast lines and dermal tissue, pSmad1/5/8 was significantly upregulated compared to controls, suggesting involvement of the bone morphogenetic protein pathway. Additionally, CAH-X samples compared to controls exhibited significant increases in fibroblast-secreted TGF-β3, a cytokine important in secondary palatal development, and in plasma TGF-β2, a cytokine involved in cardiac function and development, as well as palatogenesis. Finally, MMP-13, a matrix metalloproteinase important in secondary palate formation and tissue remodeling, had significantly increased mRNA and protein expression in CAH-X fibroblasts and direct tissue.Collectively, these results demonstrate that patients with CAH-X syndrome exhibit increased expression of several transforming growth factor-β biomarkers and provide a novel link between this signaling pathway and the connective tissue dysplasia phenotypes associated with tenascin-X deficiency.     

Insulin-like growth factor-2 (IGF-2) activates estrogen receptor-α and -β via the IGF-1 and the insulin receptors in breast cancer cells

The estrogen receptor (ER) is a primary target for breast cancer (BC) treatment. As BC progresses to estrogen-independent growth, the insulin-like growth factor-1 receptor (IGF-1R) and the ER interact in synergistic cross-talk mechanisms, which result in enhanced activation of both receptors' signaling cascades. Insulin-like growth factor-2 (IGF-2) is critical in BC progression and its actions are mediated by the IGF-1R. Our previous studies showed that IGF-2 regulates survival genes that protect the mitochondria and promote chemoresistance. In this study, we analyzed BC cells by subcellular fractionation, Western-Blot, qRT-PCR, and siRNA analysis. Our results demonstrate that IGF-2 activates ER-α and ER-β, and modulates their translocation to the nucleus, membrane organelles, and the mitochondria. IGF-2 actions are mediated by the IGF-1R and the insulin receptor. This novel mechanism of IGF-2 synergistic cross-talk signaling with ER-α and ER-β can promote estrogen-independent BC progression and provide new therapeutic targets for the treatment of BC patients.     

Insulin-like growth factor-2 (IGF-2) activates estrogen receptor-α and -β via the IGF-1 and the insulin receptors in breast cancer cells

The estrogen receptor (ER) is a primary target for breast cancer (BC) treatment. As BC progresses to estrogen-independent growth, the insulin-like growth factor-1 receptor (IGF-1R) and the ER interact in synergistic cross-talk mechanisms, which result in enhanced activation of both receptors' signaling cascades. Insulin-like growth factor-2 (IGF-2) is critical in BC progression and its actions are mediated by the IGF-1R. Our previous studies showed that IGF-2 regulates survival genes that protect the mitochondria and promote chemoresistance. In this study, we analyzed BC cells by subcellular fractionation, Western-Blot, qRT-PCR, and siRNA analysis. Our results demonstrate that IGF-2 activates ER-α and ER-β, and modulates their translocation to the nucleus, membrane organelles, and the mitochondria. IGF-2 actions are mediated by the IGF-1R and the insulin receptor. This novel mechanism of IGF-2 synergistic cross-talk signaling with ER-α and ER-β can promote estrogen-independent BC progression and provide new therapeutic targets for the treatment of BC patients.     

Insulin-like growth factor-1 and 17β-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WT1 regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17β-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 μM ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 μM LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 μM SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.     

Effects of transforming growth factor-β2 on myocilin expression and secretion in human primary cultured trabecular meshwork cells

High intraocular pressure (IOP) is a risk factor for primary open-angle glaucoma (POAG). The trabecular meshwork (TM), a reticular tissue in the outflow passage of the aqueous humor (AH), is a major contributor to intraocular outflow resistance. High levels of myocilin (MYOC), which is expressed in the TM, are associated with high IOP. Furthermore, transforming growth factor-β2 (TGF-β2) concentrations in human AH are significantly elevated in POAG patients. This study was designed to investigate the effects of TGF-β2 on MYOC expression and secretion in human primary cultured TM cells. Primary cultured human TM cells were treated with 0 (control group), 1, 10, and 100 ng/mL TGF-β2 for 12, 24, or 48 h. MYOC mRNA and protein expressions in TM cells and protein secretion in conditioned media were analyzed by semi-quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays (ELISA), respectively. TM cells treated with 1, 10, and, 100 ng/mL TGF-β2 for 48 h showed higher MYOC mRNA and protein expressions than those in the control group (0 ng/mL TGF-β2) (all P < 0.05). Treatment with TGF-β2 for 48 h also induced MYOC secretion in conditioned media in a dose-dependent manner (0 ng/mL: 7.107±1.163 pg/ml; 1 ng/mL: 7.879±1.894 pg/ml; 10 ng/mL: 8.063±1.181 pg/ml; 100 ng/mL: 8.902±0.699 pg/ml; all P < 0.05). In Conclusion, TGF-β2 induced MYOC expression and secretion in human primary cultured TM cells. Further investigations are required to confirm the involvement of these two factors in POAG pathogenesis.     

Role of elevated monocyte transforming growth factorβ (TGFβ) production in posttrauma immunosuppression

We previously reported that increased production of prostaglandin E₂ by monocytes is a pivotal mechanism in posttrauma immunopathology. Here we characterize monocyte levels of transforming growth factor and examine the effects of elevated transforming growth factor on prostaglandin E₂ release by patients' monocytes. Trauma patients' and normals' monocyte supernates (± stimulation with muramyl dipeptide) were acid treated and assayed for transforming growth factor using the mink lung-cell bioassay. Alternatively, human transforming growth factor was added to patients' and normals' monocytes and prostaglandin E₂ production assayed. Significantly elevated transforming growth factor levels (median=181.7 pmol/10⁶ monocytes) were detected in immunosuppressed patients' monocytes but not immuno-competent trauma patients' (median=32.0 pM) or normals' (median=20.4 pM) monocytes. Adding transforming growth factor to monocytes resulted in a significant elevation of prostaglandin E₂ levels. Elevated monocyte transforming growth factor levels in trauma patients could be both suppressing T-lymphocyte functions and maintaining elevated monocyte prostaglandin E₂ synthesis.