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Elastin synthesis is essential for lung development and postnatal maturation as well as for repair following injury. Using human embryonic lung fibroblasts that express undetectable levels of elastin as assessed by Northern analyses, we found that treatment with exogenous transforming growth factor-[beta] (TGF-[beta]) induced rapid and transient increases in levels of elastin heterogeneous nuclear RNA (hnRNA) followed by increases of elastin mRNA and protein expression. In fibroblasts derived from transgenic mice, TGF-[beta] induced increases in the expression of a human elastin gene promoter fragment driving a chloramphenicol acetyl transferase reporter gene. The induction of elastin hnRNA and mRNA expression by TGF-[beta] was abolished by pretreatments with TGF-[beta] receptor I inhibitor, global transcription inhibitor actinomycin D, and partially blocked by addition of protein synthesis inhibitor cycloheximide, but was not affected by the p44/42 MAPK inhibitor U0126. Pretreatment with the p38 MAPK inhibitor SB-203580 also partially attenuated the levels of TGF-[beta]-induced elastin mRNA but not its hnRNA. Western analysis indicated that TGF-[beta] stimulated Akt phosphorylation. Inhibition of phosphatidylinositol 3-kinase and Akt phosphorylation by LY-294002 abolished TGF-[beta]-induced increases in elastin hnRNA and mRNA expression. Treatment of lung fibroblasts with interleukin-1[beta] or the histone deacetylase inhibitor trichostatin A inhibited TGF-[beta]-induced elastin mRNA and hnRNA expression by a mechanism that involved inhibition of Akt phosphorylation. Down-regulation of Akt2 but not Akt1 expression employing small interfering RNA duplexes blocked TGF-[beta]-induced increases of elastin hnRNA and mRNA levels. Together, our results demonstrated that TGF-[beta] activates elastin transcription that is dependent on phosphatidylinositol 3-kinase/Akt activity. heterogeneous nuclear RNA; emphysema; small interfering RNA; phosphatidylinositol 3-kinase/Akt

Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor-[beta] (TGF-[beta]) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steadystate level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-[beta] markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF-[beta] induction. Electrophoretic gel mobility shift assay revealed that TGF-[beta] strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3-dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF-[beta] was blocked by pretreatment with TGF-[beta] receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, and actinomycin D. Basal and TGF-[beta]-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-[beta]-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-[beta]-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-[beta]/Smad signaling and requires the PI3-kinase/Akt pathway. elastogenesis; emphysema; transcription; Smads; Akt

Macrophage and neutrophil proteinases damage lung elastin, disrupting alveolar epithelium and filling alveoli with inflammatory exudate. Alveolar collapse and regional hypoxia occur. Whether low oxygen tension alters fibroblast-mediated lung repair is unknown. To determine the effect of chronic hypoxia on repair of enzyme-induced elastin disruption, primary rat lung fibroblasts produced elastin matrix for 5 wk before treatment with porcine pancreatic elastase (PPE). After exposure to PPE or saline, cultures recovered for 2 wk in normoxia (21% [O.sub.2]) or hypoxia (3% [O.sub.2]). Hypoxia suppressed regeneration of hot alkali-resistant elastin, achieving only 49% of the repair achieved in normoxic cultures. Vascular smooth muscle cells and lung fibroblasts repair elastin by two pathways: de novo synthesis and salvage repair. Although both pathways were affected, hypoxia predominantly inhibited de novo synthesis, decreasing formation of new elastin matrix by 63% while inhibiting salvage repair by only 36%. Prolonged hypoxia alone downregulated steady-state levels of elastin mRNA by 45%, whereas PPE had no significant effect on elastin gene expression. Electron microscopy documented preservation of intracellular organelles and intact nuclei. Together, these data suggest that regional hypoxia limits lung elastin repair following protease injury at least in part by inhibiting elastin gene expression. tropoelastin; elastase; lung injury; elastic fiber; extracellular matrix

Elastin, a major extracellular matrix protein and the core component of elastic fiber, is essential to maintain lung structural integrity and normal physiological function. We previously found that the downregulation of elastin gene transcription by IL-1[beta] is mediated via activation of NF-[kappa]B and CCAAT/enhancer binding protein (C/EBP)[beta], both targets of the ubiquitin-proteasome pathway. To further investigate the molecular mechanisms that underlie the control of elastin gene expression, we disrupted the ubiquitin-proteasome pathway with specific proteasome inhibitors. We found that specific proteasome inhibitors decreased the steady-state level of elastin mRNA in a dose-responsive manner. Run-on assay and promoter reporter study indicated that the proteasome inhibitor MG-132 repressed the rate of elastin transcription. MG-132 did not affect mRNA levels of NF-[kappa]B and C/EBP[beta], or the nuclear presence of NF-[kappa]B, but markedly increased C/EBP[beta] isoforms, including liver-enriched transcriptional activating protein and liver-enriched transcriptional inhibitory protein. Addition of cyclo-heximide blocked these increases and the downregulation of elastin mRNA by MG-132. The MG-132-induced downregulation of elastin transcription was dependent on C/EBP[beta] expression as assessed with small interfering RNA. These results indicate that the ubiquitin-proteasome pathway plays an essential role in maintaining elastin gene expression in lung fibroblasts. Disruption of this pathway results in the downregulation of tropoelastin transcription via posttranscriptionally induced C/EBP[beta] isoforms. Interleukin-1[beta]; lung; fibroblasts; nuclear factor-[kappa]B; CCAAT/enhancer-binding protein [beta]

We previously showed that interleukin (IL)-1[beta] decreases elastin gene transcription through activation of the NF-[kappa]B subunit p65 in neonatal rat lung fibroblasts. The present study was undertaken to further explore the molecular mechanisms responsible for the inhibitory effect of IL-1[beta] on elastin gene transcription. We found that cycloheximide blocked IL-1[beta]-induced downregulation of elastin mRNA but did not inhibit IL-1[beta]-induced translocation of p65 into the nucleus. IL-1[beta] treatment increased CCAAT/ enhancer-binding protein (C/EBP)[beta] mRNA and protein levels including liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP), which was cycloheximide sensitive. C/EBP[beta] isoforms bound a GCAAT-containing sequence in the proximal elastin promoter as determined by electrophoretic gel shift studies and confirmed by using specific anti-C/EBP[beta] antibodies and by competition studies with oligonucleotides. Transient transfection of LIP expression vectors strongly decreased the transcriptional activity of the cotransfected elastin promoter and decreased levels of endogenous elastin mRNA. We demonstrated that IL-1[beta]-induced downregulation of elastin mRNA is dependent on NF-[kappa]B activation and C/EBP[beta] expression. These results indicate that IL-1[beta] treatment activates NF-[kappa]B, which subsequently induces LIP expression and inhibition of elastin gene transcription. lung; fibroblasts; emphysema; nuclear factor-[kappa]B

Kuang, Ping-Ping, Ronald H. Goldstein, Yue Liu, David C. Rishikof, Jyh-Chang Jean, and Martin Joyce-Brady. Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair. Am J Physiol Lung Cell Mol Physiol 285: L1147-L1152, 2003. First published August 8, 2003; 1,0.1152/ajplung.00098.2003.Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins [[alpha].sub.v][[beta].sub.3], [[alpha].sub.v][[beta].sup.5], and [[alpha.sup.9][[beta].sup.1]. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1[beta] abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair. developmental arteries and neural crest epidermal growth factor-like; elastase; interferon-[gamma]; interleukin-l[beta]

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis. interstitial lung disease; computer-assisted morphometry; C57BL/6J; transgenic mice

NF-[kappa]B induced by IL-1[beta] inhibits elastin transcription and myofibroblast phenotype. Am J Physiol Cell Physiol 283: C58-C65, 2002; 10.1152/ ajpcell.00314.2001.--Interleukin (IL)-1[beta] released after lung injury regulates the production of extracellular matrix components. We found that IL-1[beta] treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibro-blasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at -118 to -102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-[kappa]B. This element mediated IL-1[beta]-induced inhibition of the elastin promoter. IL-1[beta] treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-[kappa]B. Over-expression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression p65 or treatment with IL-1[beta] revealed downregulation of [alpha]-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1[beta] activates the nuclear localization of NF-[kappa]B that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype. nuclear factor-[kappa]B; interleukin-1[beta]; Sp1

The effects of interleukin-1beta (IL-1beta) on protein phosphorylation and regulation of elastin gene expression were analyzed in cultured rat lung fibroblast by utilizing okaidic acid (OA). The formation of tropoelastin and the steady-state gene expression of elastin by cultured rat fibroblasts was downregulated by OA. The selective serine/threonine protein phosphatase inhibitor regulated the amount of fibroblast-secreted extracellular matrix in the interstitium of the lung.

Phosphatidylinositol 3-kinase-dependent stabilization of [[Alpha].sub.1](I) collagen mRNA in human lung fibroblasts. Am J Physiol Cell Physiol 281: C99-C105, 2001.--We investigated the role of phosphatidylinositol 3-kinase (PI3K) in the expression of [[Alpha].sub.1](I) collagen mRNA. We report that the basal level of [[Alpha].sub.1](I) collagen mRNA was reduced when PI3K activity was inhibited by either LY-294002 or wortmannin. These PI3K inhibitors also blocked increases of [[Alpha].sub.1](I) collagen mRNA levels after the addition of transforming growth factor-[Beta]. The effect of PI3K inhibition was abolished by the removal of the inhibitor or by the addition of cycloheximide. Inhibition of PI3K activity decreased the stability of the [[Alpha].sub.1](I) collagen mRNA with no change in the rate of transcription of the [[Alpha].sub.1](I) collagen gene as assessed by Northern blotting with actinomycin D-treated fibroblasts and nuclear run-on assays. Expression of a truncated [[Alpha].sub.1](I) collagen minigene driven by a cytomegalovirus promoter in murine fibroblasts was decreased by LY-294002 treatment. These data indicate that PI3K activation results in increased stabilization of [[Alpha].sub.1](I) collagen mRNA. In vivo, the PI3K activity in fibroblasts may regulate basal levels of [[Alpha].sub.1](I) collagen mRNA expression. mRNA stability; LY-294002; wortmannin; actinomycin D

Ricupero, Dennis A., David C. Rishikof, Ping-Ping Kuang, Christine F. Poliks, and Ronald H. Goldstein. Regulation of connective tissue growth factor expression by prostaglandin [E.sub.2]. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1165-L1171, 1999.--Transforming growth factor-[Beta] (TGF-[Beta]) stimulates [[Alpha.sub.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.sub.2] ([PGE.sub.2])-treated fibroblasts. [PGE.sub.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.sub.2] and insulin regulate [[Alpha].sub.1]l(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of [[Alpha].sub.1](I) collagen mRNA levels by TGF-[Beta] and [PGE.sub.2] may function through both CTGF-dependent and CTGF-independent mechanisms. transforming growth factor-p; insulin; amino acid deficiency; type I collagen; human lung fibroblast

Berk, John L., Nima Massoomi, Christine Hatch, and Ronald H. Goldstein. Hypoxia downregulates tropoelastin gene expression in rat lung fibroblasts by pretranslational mechanisms. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L566-L572, 1999.--Elastolytic lung injury disrupts cell barriers, flooding alveoli and producing regional hypoxia. Abnormal [O.sub.2] tensions may alter repair of damaged elastin fibers. To determine the effect of hypoxia on extravascular elastin formation, we isolated rat lung fibroblasts and cultured them under a variety of [O.sub.2] conditions. Hypoxia down-regulated tropoelastin mRNA in a dose- and time-related fashion while upregulating glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The changes in tropoelastin gene expression were not due to cell toxicity as measured by chromium release and cell proliferation studies. Neither cycloheximide nor actinomycin D abrogated this effect. Hypoxia induced early decreases in tropoelastin mRNA stability; minor suppression of gene transcription occurred later. When returned to 21% [O.sub.2], tropoelastin mRNA recovered to control levels in part by upregulating tropoelastin gene transcription. Taken together, these data indicate that hypoxia regulates tropoelastin gene expression and may alter repair of acutely injured lung. extracellular matrix; elastin; lung injury