Transforming growth factor β within fibrotic scleroderma lungs

doi:10.1016/0002-9343(92)90195-H

The American Journal of Medicine

Volume 93, Issue 6, December 1992, Pages 628-636

https://doi.org/10.1016/0002-9343(92)90195-H Get rights and content

Abstract

purpose, patients, and methods: Since transforming growth factor β (TGFβ) has been implicated as an important mediator of pulmonary fibrosis, we measured TGFβ protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGFβ protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms.

results: Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGFβ and immunoreacted with anti-TGFβ antibody. The normal population had a significantly higher average TGFβ concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGFβ gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGFβ concentration variability did not correlate with variations in BAL cellularity or TGFβ gene expression within the recovered mononuclear cells.

conclusions: It is concluded that both normal and fibrotic lungs have TGFβ present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGFβ protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGFβ protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGFβ protein concentration at the alveolar epithelial surface.

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Citation Excerpt :
In addition, several other cytokines, such as tumor necrosis factor (TNF)-α,40 keratinocyte growth factor,41 angiotensin II,42 and interleukin 1043 may exhibit profibrotic effects via the TGFβ pathway. An increased expression of TGFβ messenger RNA83235444546 and protein893233354446474849 as well as the type II receptor32 has been documented during the phase of tissue remodeling in several forms of acute as well as chronic lung disease and systemic diseases involving the lung (Table 1).505152535455565758596061 While most studies on human lung pathology focus on TGFβ1, which appears to be the predominant isoform involved in pulmonary fibrosis, increases in all three isoforms have been demonstrated.
Transforming growth factor (TGF) β plays an important role in normal pulmonary morphogenesis and function and in the pathogenesis of lung disease. The effect of TGFβ is regulated via a selective pathway of TGFβ synthesis and signaling that involves activation of latent TGFβ, specific TGFβ receptors, and intracellular signaling via Smad molecules. All three isoforms of TGFβ are expressed at high levels during normal lung development, being particularly important for branching morphogenesis and epithelial cell differentiation with maturation of surfactant synthesis. Small amounts of TGFβ are still present in the adult lung, and TGFβ is involved in normal tissue repair following lung injury. However, in a variety of forms of pulmonary pathology, the expression of TGFβ is increased. These include chronic lung disease of prematurity as well as several forms of acute and chronic adult lung disease. While TGFβ1 appears to be the predominant isoform involved, elevated levels of all three isoforms have been demonstrated. The increase in TGFβ precedes abnormalities in lung function and detectable lung pathology, but correlates with the severity of the disease. TGFβ plays a key role in mediating fibrotic tissue remodeling by increasing the production and decreasing the degradation of connective tissue via several mechanisms.
Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1
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Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-β (TGFβ). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFβ. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFβ in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFβ signaling but dependent at least in part on Sp1.
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CTGF and SMADs, Maintenance of Scleroderma Phenotype Is Independent of SMAD Signaling
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Citation Excerpt :
This is in contrast to CTGF whose expression patterns and levels are highly correlated with the severity of fibrosis (44, 58). Similarly, other studies that examined normal and scleroderma fibroblasts failed to see elevated TGFβ levels, enhanced binding of TGFβ to cells, or enhanced responsiveness to TGFβ in scleroderma fibroblasts (10, 11,12, 52). Combined with our data in this report, these results suggest that although the initial expansion of the sclerotic lesion may require TGFβ action, the maintenance of the sclerotic lesion does not result from TGFβ action per se.
In normal adult fibroblasts, transforming growth factor-β (TGFβ) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGFβ. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGFβ. Induction of transcription by TGFβ requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGFβ-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in theCTGF promoter. TGFβ-induction of CTGFis dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGFβRE in the CTGFpromoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGFpromoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGFβ signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.
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Pulmonary manifestations of systemic sclerosis (SSc, scleroderma) are manifold (Table 1) and often have a profound influence on the course of the disease. A discussion of all the pulmonary complications of SSc is beyond the scope of this article but is presented in a recent review.¹⁰ The two major pulmonary conditions occurring among SSc patients, pulmonary hypertension and interstitial fibrosis (Table 2), will be discussed in detail. Isolated pulmonary hypertension occurs primarily in patients with limited cutaneous SSc (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia [CREST] variant) in the absence of significant pulmonary fibrosis, whereas interstitial fibrosis occurs in either limited or diffuse cutaneous SSc.
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1995, Chest
Fibrotic process affecting the lung and other tissues is characterized by stimulation of fibroblast proliferation and connective tissue deposition. Conventional therapy consisting of glucocorticoids or cytotoxic agents is usually ineffective in blocking progression of disease. Potential new therapies have emerged from the use of animal models of pulmonary fibrosis and recent advances in the cellular and molecular biology of inflammatory reactions. Such therapies involve the use of substances directed against the action of certain growth factors, cytokines, or oxidants that are elaborated during the fibrotic reaction. In this article, we review possible therapeutic applications of these advances.

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^☆: This work was supported by Scleroderma Foundation Grant 1004 and National Institutes of Health Clinical Research Center Grant RR-32.

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Transforming growth factor β within fibrotic scleroderma lungs☆

Abstract

Am J Med

Am J Med

J Biol Chem

Survival with systemic sclerosis (scleroderma)

Ann Intern Med

Structural features of interstitial lung disease in systemic sclerosis

Am Rev Respir Dis

Pathobiology of pulmonary fibrosis

Am J Physiol (Lung Cell Mol Physiol)

Lung inflammation in scleroderma: clinical, radiographic, physiologic and cytopathological features

J Rheumatol

Subclinical pulmonary involvement in collagen-vascular diseases assessed by bronchoalveolar lavage

Am Rev Respir Dis

The transforming growth factor-β family

Annu Rev Cell Biol

Distribution and modulation of the cellular receptor for transforming growth factor-beta

J Cell Biol

Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-β precede bleomycin-induced pulmonary fibrosis in mice

J Pharmacol Exp Ther

Coordinate regulation of transforming growth factor β gene expression and cell proliferation in hamster lungs undergoing bleomycin-induced pulmonary fibrosis

J Clin Invest

Lung cytokine production in bleomycin-induced pulmonary fibrosis

Exp Lung Res

Stimulation of rat endothelial cell transforming growth factor-β production by bleomycin

J Clin Invest