Autoantibody response against a novel testicular antigen protein highly expressed in testis (PHET) in SSc patients

Systemic sclerosis (SSc) is characterized by excessive fibrosis and autoantibody production. However, the pathogenesis of SSc is still under investigation. We have demonstrated that a novel testicular antigen, protein highly expressed in testis (PHET), is overexpressed in SSc dermal fibroblasts and targeted by autoantibodies. PHET was identified by screening of HepG2 cDNA library using an SSc serum and was found to belong to UniGene cluster of sperm associated antigen 9 (SPAG9) from its nucleotide sequence. PHET mRNA expression was examined by RT-PCR using mRNA panels of human tissues, and PHET mRNA was highly expressed only in testis in normal tissues. Anti-PHET antibodies were detected in 8.4% of sera of SSc patients by immunoblotting, and associated with diffuse scleroderma and lung involvement. Expression of PHET mRNA and protein was increased in cultured SSc dermal fibroblasts compared with control fibroblasts. These results suggest that ectopic expression of PHET in dermal fibroblasts induces autoantibody production against PHET in patients with SSc.     

The role of TGF-beta signaling in the pathogenesis of fibrosis in scleroderma

Excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that transforming growth factor (TGF)-beta is a key mediator of tissue fibrosis as a consequence of ECM accumulation in pathological states such as systemic sclerosis. TGF-beta regulates diverse biological activities including cell growth, cell death or apoptosis, cell differentiation, and ECM synthesis. TGF-beta is known to induce the expression of ECM proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. This review focuses on the possible role of TGF-beta in the pathogenesis of fibrosis in SSc.     

Activation of the activin A-ALK-Smad pathway in systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of unknown etiology that is characterized by multiple tissue fibrosis. Transforming Growth Factor-beta (TGF-β) is thought to be the most important mediator that induces fibrosis. However, the molecular mechanisms by which fibrosis is induced have not been fully elucidated. In this study, the role of activin, a member of the TGF-β superfamily, was investigated in the pathogenesis of fibrosis in SSc. Serum activin A levels in patients with SSc were measured by ELISA, and the expression of the activin receptor type IB (ACVRIB/ALK4) and the activity of the signaling pathway via ACVRIB/ALK4 were investigated using western blotting. To evaluate a potential therapeutic strategy for SSc, we also attenuated the ACVRIB/ALK4 pathway using an inhibitor. Serum activin A levels were significantly higher in SSc patients than in normal controls. Activin A and ACVRIB/ALK4 expression were also higher in cultured SSc fibroblasts. Activin A stimulation induced phosphorylation of Smad2/3 and CTGF expression in SSc fibroblasts. Procollagen production and Col1α mRNA also increased upon stimulation by activin A. The basal level of Smad2/3 phosphorylation was higher in cultured SSc fibroblasts than in control cells, and treatment with the ALK4/5 inhibitor SB431542 prevented phosphorylation of Smad2/3 and CTGF expression. Furthermore, production of collagen was also induced by activin A. Activin A-ACVRIB/ALK4-Smad-dependent collagen production was augmented in SSc fibroblasts, suggesting the involvement of this signaling mechanism in SSc. Inhibition of the activin A-ACVRIB/ALK4-Smad pathway would be a new approach for the treatment of SSc.     

In vivo investigations on anti-fibrotic potential of proteasome inhibition in lung and skin fibrosis

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.     

Pulmonary and activation-regulated chemokine stimulates collagen production in lung fibroblasts

Levels of pulmonary and activation-regulated chemokine (PARC) mRNA and protein are increased in the lungs of patients with pulmonary fibrosis. The purpose of this study was to establish whether PARC could be directly involved in development of pulmonary fibrosis by stimulating collagen production in lung fibroblasts. Exposure to PARC increased production of collagen mRNA and protein by 3- to 4-fold in normal adult lung and dermal fibroblast cells. Collagen mRNA transiently increased after 3-6 h of activation with PARC, with an increase in collagen protein detected after 24 h of activation. At the same time, PARC had less pronounced effect on fibroblast proliferation, not exceeding 50% increase over control nonstimulated cells. PARC intracellular signaling led to activation of ERK1/2, but not p38, in fibroblasts; pharmacologic inhibition of ERK, but not p38, also blocked PARC's effect on collagen production. Inhibition experiments with pertussis toxin suggested that PARC receptor is G protein-coupled. Thus, PARC is a member of the CC chemokine family that acts directly as a profibrotic factor.     

Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

OBJECTIVE:

To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice.

METHODS:

Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson''s trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR.

RESULTS:

Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3).

CONCLUSION:

Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.     

iNOS gene upregulation is associated with the early proliferative response of human lung fibroblasts to cytokine stimulation

Increased release of oxidants has been implicated in the pathogenesis of pulmonary fibrosis. Previous work in the rat showed that formation of the early fibrotic lesion is associated with increased expression of inducible nitric oxide synthase (iNOS) in pulmonary fibroblasts. The aim of this study was to test the hypothesis that NO is involved in the activation of pulmonary fibroblasts. The effects of endogenous and exogenous NO on proliferation of human pulmonary fibroblasts were investigated by administration of cytomix or SNAP, respectively. At low concentrations, both treatments increased cell numbers, an effect attenuated by iNOS inhibitor or NO scavenger. Induction of iNOS was confirmed by measurement of nitrate/nitrite production and by immunodetection. Quantitative RT-PCR showed an increase in iNOS mRNA as early as 3 h after stimulation. These results support the hypothesis and show that upregulation of the iNOS gene is an early event in the proliferative response of human lung fibroblasts to inflammatory stimuli.     

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type I collagen expression in scleroderma fibroblasts

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.     

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.     

MicroRNA-21 and microRNA-29a modulate the expression of collagen in dermal fibroblasts of patients with systemic sclerosis

MicroRNAs (miRNAs) are well-known candidates for modulating the dysregulated signaling pathways during fibrosis. In this study, we investigated the expression pattern of 16 miRNAs, which have previously been confirmed or predicted to target genes involved in extracellular matrix (ECM) homeostasis. Primary culture of dermal fibroblasts was obtained from skin biopsies of diffused cutaneous SSc (dcSSc) patients and healthy controls. Expression of let-7a, miR-1, miR-15a, miR-17, miR-19a, miR-20a, miR-21, miR-27b, miR-26a, miR-29a, miR-29b, miR29c, miR-141, miR-125a-5p, miR-193a-3p, and miR-200a were quantified by Real-time PCR. Functional analysis of microRNAs was performed using synthetic oligonucleotides. To further confirm the pro- or anti-fibrotic effects of miRNAs, normal fibroblasts were treated with 10 ng/mL of transforming growth factor (TGF)-β to generate an in vitro model of dermal fibrosis. miR-21 and miR-29a were upregulated and downregulated, respectively, in both dcSSc and TGF-β-treated fibroblasts. We observed that restoration of miR-29a expression or blockade of miR-21 function negatively affected collagen production. COL1A1 expression in SSc fibroblasts is more sensitive to changes of miR-29a and miR-21 expression in compare to normal fibroblasts. miR-29a alone was effective to decrease TGF-β-induced collagen production in dermal fibroblasts. miR-21 and TGF-β had synergistic effects on induction of collagen production. However, neither miR-21 nor miR-29a affected alpha smooth muscle actin (α-SMA) expression in the presence or absence of TGF-β in dermal fibroblasts. miR-21 and miR-29a as pro- and anti-fibrotic miRNAs modulate collagen production in an opposing manner. Focusing on miR-21 and miR-29s as therapeutic targets would be effective in patients with SSc or other fibrotic diseases which show aberrant expression of collagen expression.     

Heterogeneous response of nasal and lung fibroblasts to transforming growth factor-β1

Background Nasal polyposis is characterized by marked oedema, sparse extracellular matrix (ECM) and proliferating blood vessels. Pulmonary fibrosis is characterized by inflammatory cells accumulation, considerable ECM deposition and vascular abnormalities. Although lung fibrosis is not only and necessarily an inflammatory disorder, we hypothesized that the difference between nasal polyposis and pulmonary fibrosis may, in part, be due to the heterogeneity between nasal and lung fibroblasts. Fibroblasts participate in the inflammatory response by releasing ECM proteins and cytokines. TGF‐β is thought to participate in chronic inflammation and fibrosis. Myofibroblasts are the activated form of fibroblasts. A phenotypic hallmark of myofibroblasts is the expression of smooth muscle α‐actin (SMA). Objective We examined whether there is any heterogeneity between nasal and lung fibroblasts upon stimulation with TGF‐β₁ with regard to the synthesis of SMA, pro‐collagen type I and vascular endothelial growth factor (VEGF) as well as translocation of Smad proteins. Methods Fibroblasts lines were established from human biopsy tissue. The expression of SMA, pro‐collagen type I, VEGF mRNA was evaluated by reverse transciptase RT‐PCR. The amount of pro‐collagen type I and VEGF was measured by ELISA. By immunocytochemistry, we analysed the expression of SMA and Smad2, 3, 4 in cultured fibroblasts. Results TGF‐β₁ induced SMA and pro‐collagen type I synthesis in lung, but not in nasal fibroblasts. By contrast, TGF‐β₁ induced VEGF synthesis in both lung and nasal fibroblasts. After stimulation with TGF‐β₁, Smad2, 3, 4 were translocated from the cytoplasm to the nucleus in lung fibroblasts, whereas only Smad3 was translocated in nasal fibroblasts. Conclusion These results establish the heterogeneous responsiveness of fibroblast populations in the airways to TGF‐β₁ and that such a heterogeneity may contribute, at least in part, to the different pathological outcomes of inflammation in the upper and lower airways.     

Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF-β signaling in mice

Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor-β (TGF-β) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF-β signaling. In order to further evaluate TGF-β signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased α-smooth muscle actin-positive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-β1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-β1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 – a TGF-βRI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF-β signaling.     

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.     

Low molecular weight hyaluronan from stretched lung enhances interleukin-8 expression

Mechanical ventilation has been shown to cause ventilator-induced lung injury (VILI), probably by overdistending or stretching the lung. Hyaluronan (HA), a component of the extracellular matrix, in low molecular weight (LMW) forms has been shown to induce cytokine production. LMW HA is produced by hyaluronan synthase 3 (HAS 3). We found that HAS 3 mRNA expression was upregulated and that LMW HA accumulated in an animal model of VILI. We hypothesized that stretch-induced LMW HA production that causes cytokine release in VILI was dependent on HAS 3 mRNA expression. We explored this hypothesis with in vitro lung cell stretch. Cell stretch induced HAS 3 mRNA expression and LMW HA in fibroblasts. Nonspecific inhibitors of HAS 3 (cyclohexamide and dexamethasone), a nonspecific inhibitor of protein tyrosine kinases (genistein), and a janus kinase 2 inhibitor (AG490) blocked stretch-induced HAS 3 expression and synthesis of LMW HA. Stretch-induced LMW HA from fibroblasts caused a significant dose-dependent increase in interleukin-8 production both in static and stretched epithelial cells. These results indicated that de novo synthesis of LMW HA was induced in lung fibroblasts by stretch via tyrosine kinase signaling pathways, and may play a role in augmenting induction of proinflammatory cytokines in VILI.     

Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.     

Role of osteopontin in the pathogenesis of bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is initiated by migration, adhesion, and proliferation of fibroblasts. Osteopontin (OPN) is one of the cytokines produced by activated macrophages and mediates various functions, including cell attachment and migration, by interacting with alphav integrin. In this study, we have investigated the role of OPN in the pathogenesis of pulmonary fibrosis. We developed a mouse model for pulmonary fibrosis by intratracheal instillation of bleomycin (BLM). OPN was strongly expressed in alveolar macrophages accumulating in the fibrotic area of the lung. OPN messenger RNA (mRNA) in the lung was notably induced by BLM instillation, and the development of the fibrotic process was associated with an increase in the expression of OPN mRNA and protein. In vitro, recombinant OPN enhanced migration, adhesion, and platelet-derived growth factor (PDGF)-mediated DNA synthesis of murine fibroblast cell line NIH3T3. These effects of OPN on fibroblasts were significantly suppressed by addition of antimouse alphav integrin monoclonal antibody (RMV-7). Furthermore, treatment of mice with RMV-7 repressed the extent of pulmonary fibrosis in this model. Conclusively, these data suggest that OPN produced by alveolar macrophages functions as a fibrogenic cytokine that promotes migration, adhesion, and proliferation of fibroblasts in the development of BLM-induced pulmonary fibrosis.     

MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis

Three major MAP kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase (p38 MAPK), are involved in the regulation of lung inflammation and injury. This study investigated whether MAPKs are activated and associated with lung injury in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The expression of the active ERK, JNK, and p38 MAPK was examined using western blot analysis and immunohistochemistry and apoptosis was also examined by the TUNEL method, in lung tissues from ten patients with IPF obtained by thoracoscopic biopsy and in eight normal lung parenchyma specimens obtained by lobectomy for lung cancer. Activated MAPKs are significantly increased in lung homogenates from patients with IPF compared with controls. Activated ERK in epithelial and endothelial cells, but not in fibroblasts or smooth muscle cells, was decreased, accompanied by the progression of fibrosis. Activated JNK in epithelial and endothelial cells, but not in fibroblasts, was increased, accompanied by the progression of fibrosis. Activated p38 MAPK in epithelial, endothelial, smooth muscle cells, and fibroblasts was increased at the intermediate stage of fibrosis, in which the TUNEL-positive cells were predominantly detected. This is the first study to suggest that MAPKs may be associated with the regulation of inflammation and lung injury in IPF.     

Expression of transforming growth factor-beta type I and type II receptors is altered in rat lungs undergoing bleomycin-induced pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) is a family of autocrine/paracrine/endocrine cytokines involved in controlling cell growth and extracellular matrix metabolism. TGF-beta exerts its biological effects via binding to type I (TbetaRI) and type II (TbetaRII) receptors. To gain insight into the possible role of TGF-beta receptors in the pathogenesis of pulmonary fibrosis, we investigated the expression of TGF-beta receptors and their ligands in a bleomycin-induced model of pulmonary fibrosis. We found that the expression of both TbetaRI and TbetaRII was altered in rat lungs during pulmonary fibrosis induced by bleomycin. The increase in TbetaRI mRNA level was evident after 3 days of bleomycin administration, and TbetaRI mRNA continually increased for over 12 days after bleomycin instillation, whereas TbetaRII mRNA declined at day 3 post bleomycin instillation and then increased during the reparative phase of lung injury (days 8 and 12). The immunoreactivity for both TbetaRI and TbetaRII was detected in the cells of the interstitium, the epithelium, and the blood vessels of normal rat lungs. In bleomycin-induced pulmonary fibrosis, an extensive immunostaining for TbetaRI and TbetaRII was present in the cells at the sites of injury and active fibrosis. These results demonstrate that the expression of TGF-beta type I and type II receptors was altered during pulmonary fibrosis, suggesting that the TGF-beta signal transduction pathway may be involved in the pathogenesis of lung fibrosis.