Ping-Ping Kuang

Severity of Elastase-Induced Emphysema Is Decreased in Tumor Necrosis Factor-α and Interleukin-1β Receptor-Deficient Mice

A single intratracheal dose of porcine pancreatic elastase, which is cleared from the lung by 24 hours, was administered to wild-type, IL-1β type 1 receptor-deficient, double TNF-α (type 1 and type 2) receptor-deficient, and combined TNF-α (type 1 receptor) plus IL-1β receptor-deficient mice. The mean linear intercept (Lm) of saline-treated mice was 32(3) μm [mean(SE)]. For wild-type elastase-treated mice, Lm was 81(6) μm at 21 days versus 52(5) μm at 5 days after treatment, indicating that alveolar wall remodeling occurs long after the elastase injury. At 21 days, Lm values were 67(10), 62(3), and 39(5) μm in elastase-treated mice deficient in the IL-1β receptor, double TNF-α receptors, and combined receptors, respectively. The level of apoptosis assessed by a terminal deoxynucleotidyl transferase-catalyzed in situ nick end-labeling assay was increased at 5 days after elastase treatment and was markedly and similarly attenuated in the IL-1β, the double TNF-α, and the combined receptor-deficient mice. Our results indicate that inflammatory mediators exacerbate elastase-induced emphysema. We estimate that in the combined TNF-α + IL-1β receptor-deficient mice, inflammation accounts for about 80% of the emphysema that develops after elastase treatment; decreased apoptosis of lung cells likely contributes to decreased severity of emphysema.

Regulation of connective tissue growth factor expression by prostaglandin E2

Transforming growth factor-beta (TGF-beta) stimulates alpha(1)(I) collagen mRNA synthesis in human lung fibroblasts through a mechanism that is partially sensitive to cycloheximide and that may involve synthesis of connective tissue growth factor (CTGF). Northern blot analyses indicate that TGF-beta stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-beta-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-beta-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-beta increases the CTGF transcription rate. The TGF-beta-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E(2) (PGE(2))-treated fibroblasts. PGE(2) fails to attenuate luciferase activity induced by TGF-beta in fibroblasts transfected with the TGF-beta-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE(2) and insulin regulate alpha(1)(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of alpha(1)(I) collagen mRNA levels by TGF-beta and PGE(2) may function through both CTGF-dependent and CTGF-independent mechanisms.

Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-β receptor : regulation by TGF-β

Abstract The type I transforming growth factor-β (TGF-β) receptors are serine/threonine kinases that are essential for the action of TGF-β. In this paper, we describe the molecular cloning and expression of the R4/ALK5 human type I TGF-β receptor promoter. DNA sequence analysis indicates that the promoter lacks a TATA and CAAT box but is highly GC-rich and contains putative Spl binding sites. The transcriptional start site is approx. 232 base pairs upstream of the AUG start codon. In human lung fibroblasts, TGF-β induced a 3-fold increase in steady-state level for type I receptor mRNA. Exposure of cells transfected with a 618 by promoter fragment to TGF-β1 up-regulated transcriptional activity indicating that a TGF-β response element is contained within this region.

INTERLEUKIN-4 REGULATES CONNECTIVE TISSUE GROWTH FACTOR EXPRESSION IN HUMAN LUNG FIBROBLASTS

Transforming growth factor‐β (TGF‐β) and interleukin‐4 (IL‐4) have fibrogenic properties and induce extracellular matrix production in a variety of lung diseases. Connective tissue growth factor (CTGF) is a matrix signaling molecule stimulated by TGF‐β that in part mediates α1(I) collagen mRNA expression. In these studies, the regulation of CTGF expression by IL‐4 in human lung fibroblasts was examined. Following 6 h of stimulation with IL‐4, basal CTGF mRNA levels were unchanged as assessed by Northern blot analysis. However, IL‐4 attenuated the TGF‐β‐stimulated induction of CTGF mRNA expression by 50%. This effect was selective because IL‐4 did not affect fibronectin or α1(I) collagen mRNA expression induced by TGF‐β. Experiments employing the transcriptional inhibitor actinomycin D suggest that IL‐4 did not affect the stability of the CTGF mRNA. Transient transfection assays with 3TP‐Lux, a luciferase gene controlled by a TGF‐β inducible promoter, and with a CTGF promoter construct indicate that IL‐4 interfered with the TGF‐β‐induced transcriptional activation of the CTGF gene. J. Cell. Biochem. 85: 496–504, 2002.

Apigenin decreases expression of the myofibroblast phenotype

We investigated the effect of the dietary flavonoid apigenin on myofibroblast function. We report that in myofibroblasts treated with apigenin, proliferation and basal levels of α1(I) collagen and α‐smooth muscle actin mRNAs were markedly reduced. Apigenin also attenuated the transforming growth factor‐β‐stimulated increases of α1(I) collagen and α‐smooth muscle actin mRNAs. Characterization of the apigenin effects indicates that apigenin reduces both the stability of the α1(I) collagen mRNA and the rate of transcription of the α1(I) collagen gene through a cycloheximide‐sensitive pathway. Western blot analyses indicate that Akt activity is reduced in apigenin‐treated myofibroblasts.

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of α-smooth muscle actin (α-SMA) by immunohistochemistry. We also examined elastin and α1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, α-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced α-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and α1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that α1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased α-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.

The Roles of MicroRNAs and Protein Components of the MicroRNA Pathway in Lung Development and Diseases

Decades of studies have shown evolutionarily conserved molecular networks consisting of transcriptional factors, diffusing growth factors, and signaling pathways that regulate proper lung development. Recently, microRNAs (miRNAs), small, noncoding regulatory RNAs, have been integrated into these networks. Significant advances have been made in characterizing the developmental stage- or cell type-specific miRNAs during lung development by using approaches such as genome-wide profiling and in situ hybridization. Results from gain- or loss-of-function studies revealed pivotal roles of protein components of the miRNA pathway and individual miRNAs in regulating proliferation, apoptosis, differentiation, and morphogenesis during lung development. Aberrant expression or functions of these components have been associated with pulmonary disorders, suggesting their involvement in pathogenesis of these diseases. Moreover, genetically modified mice generated in these studies have become useful models of human lung diseases. Challenges in this field include characterization of collective function and responsible targets of miRNAs specifically expressed during lung development, and translation of these basic findings into clinically relevant information for better understanding of human diseases. The goal of this review is to discuss the recent progress on the understanding of how the miRNA pathway regulates lung development, how dysregulation of miRNA activities contributes to pathogenesis of related pulmonary diseases, and to identify relevant questions and future directions.

Modulation of amino acid uptake by TGF‐β in lung myofibroblasts

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor‐β (TGF‐β) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3‐kinase (PI3‐kinase)‐dependent pathway. We examine the effect and interaction of TGF‐β, insulin, and PI3‐kinase activity on amino acid uptake in human lung myofibroblasts. TGF‐β treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho‐Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho‐Akt levels. LY294002, a PI3‐kinase inhibitor, blocked the TGF‐β‐induced amino acid uptake only partially, but completely blocked TGF‐β‐induced Akt phosphorylation. Moreover, the level of phospho‐Smad3 was found to be high even when LY294002 blocked TGF‐β‐induced phospho‐Akt levels. Inhibition of PI3‐kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3‐kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF‐β induced Smad‐3 and amino acid uptake through a PI3‐kinase independent pathway.

Methods for Measuring Type I Collagen Synthesis In Vitro

The excess accumulation of type I collagen within tissues leads to organ dysfunction and occurs as a result of an imbalance between synthesis and degradation. This chapter outlines several methods to assess the in vitro production of type I collagen that are employed in our laboratory. We describe Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides. The measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction is then outlined. Finally, methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay are described.