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To study the intrahepatic expression of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in chronic hepatitis B patients with and without hepatocellular carcinoma.A total of 33 chronic hepatitis B patients (mean age of 40.3 ± 2.5 years), comprising of 14 HBeAg positive and 19 HBeAg negative patients; and 13 patients with hepatitis B virus related hepatocellular carcinoma (mean age of 49.6 ± 4.7 years), were included in our study. Immunohistochemical staining for HBcAg and HBsAg was done using standard streptavidin-biotin-immunoperoxidase technique on paraffin-embedded liver biopsies. The HBcAg and HBsAg staining distributions and patterns were described according to a modified classification system.Compared to the HBeAg negative patients, the HBeAg positive patients were younger, had higher mean HBV DNA and alanine transaminases levels. All the HBeAg positive patients had intrahepatic HBcAg staining; predominantly with "diffuse" distribution (79%) and "mixed cytoplasmic/nuclear" pattern (79%). In comparison, only 5% of the HBeAg-negative patients had intrahepatic HBcAg staining. However, the intrahepatic HBsAg staining has wider distribution among the HBeAg negative patients, namely; majority of the HBeAg negative cases had "patchy" HBsAg distribution compared to "rare" distribution among the HBeAg positive cases. All but one patient with HCC were HBeAg negative with either undetectable HBV DNA or very low level of viremia. Intrahepatic HBcAg and HBsAg were seen in 13 (100%) and 10 (77%) of the HCC patients respectively. Interestingly, among the 9 HCC patients on anti-viral therapy with suppressed HBV DNA, HBcAg and HBsAg were detected in tumor tissues but not the adjacent liver in 4 (44%) and 1 (11%) patient respectively.Isolated intrahepatic HBcAg and HBsAg can be present in tumors of patients with suppressed HBV DNA on antiviral therapy; that may predispose them to cancer development.

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.

Pulmonary fibrosis is a pathological condition in which lungs become scarred due to the excess extracellular matrix (ECM) deposition and structural alterations in the interstitium of lung parenchyma. Many patients with interstitial lung diseases (ILDs) caused by long-term exposure to toxic substances, chronic infections, or autoimmune responses develop fibrosis. Etiologies for many ILDs are unknown, such as idiopathic pulmonary fibrosis (IPF), a devastating, relentless form of pulmonary fibrosis with a median survival of 2–3 years. Despite several decades of research, factors that initiate and sustain the fibrotic response in lungs remain unclear and there is no effective treatment to block progression of fibrosis. Here we summarize recent findings on the antifibrotic activity of miR-29, a small noncoding regulatory RNA, in the pathogenesis of fibrosis by regulating ECM production and deposition, and epithelial–mesenchymal transition (EMT). We also describe interactions of miR-29 with multiple profibrotic and inflammatory pathways. Finally, we review the antifibrotic activity of miR-29 in animal models of fibrosis and highlight miR-29 as a promising therapeutic reagent or target for the treatment of pulmonary fibrosis.

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

Differentiation of lung vascular smooth muscle cells (vSMCs) is tightly regulated during development or in response to challenges in a vessel specific manner. Aberrant vSMCs specifically associated with distal pulmonary arteries have been implicated in the pathogenesis of respiratory diseases, such as pulmonary arterial hypertension (PAH), a progressive and fatal disease, with no effective treatment. Therefore, it is highly relevant to understand the underlying mechanisms of lung vSMC differentiation. miRNAs are known to play critical roles in vSMC maturation and function of systemic vessels; however, little is known regarding the role of miRNAs in lung vSMCs. Here, we report that miR-29 family members are the most abundant miRNAs in adult mouse lungs. Moreover, high levels of miR-29 expression are selectively associated with vSMCs of distal vessels in both mouse and human lungs. Furthermore, we have shown that disruption of miR-29 in vivo leads to immature/synthetic vSMC phenotype specifically associated with distal lung vasculature, at least partially due to the derepression of KLF4, components of the PDGF pathway and ECM-related genes associated with synthetic phenotype. Moreover, we found that expression of FBXO32 in vSMCs is significantly upregulated in the distal vasculature of miR-29 null lungs. This indicates a potential important role of miR-29 in smooth muscle cell function by regulating FBXO32 and SMC protein degradation. These results are strongly supported by findings of a cell autonomous role of endogenous miR-29 in promoting SMC differentiation in vitro. Together, our findings suggested a vessel specific role of miR-29 in vSMC differentiation and function by targeting several key negative regulators., Author Summary The pathogenesis of some vascular diseases, such as PAH is selectively associated with aberrant differentiation and proliferation of vSMCs of distal arteries. While significant progresses have been made in understanding the core mechanism of differentiation and proliferation of vSMCs, little is known regarding vessel specific regulations. By investigating the expression and function of miR-29 in vivo, we found a vessel selective enriched expression and function of miR-29 during mouse lung development. Interestingly, disruption of miR-29 results in defects in vSMCs differentiation of distal vessels, reminiscent of vSMC phenotype observed in the early stage of PAH in which immature/synthetic vSMCs of distal arteries failed to differentiate and were unable to tune down the expression of collagens and other extracellular-related genes. This is the first evidence that miR-29 selectively regulates vSMCs differentiation and vessel wall formation. Future implications are to study the expression and function of miR-29 in human pulmonary vascular diseases, which might lead to establishing miR-29 as a therapeutic target for disease intervention.

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 steady-state 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 LY-294002, 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.

Osteopontin is a multifunctional matricellular protein identified as one of the most upregulated genes in pulmonary fibrosis. Experimental animal models have identified early pro-fibrotic cytokines as essential to the pathogenesis of inflammation-induced pulmonary fibrosis. However, the principal sources of osteopontin in the fibroproliferative lung, and the factors responsible for its induction, have not been fully defined. We isolated primary rat lung fibroblasts in culture to examine the expression and regulation of lung fibroblast-derived osteopontin. Our results demonstrate a potent and dramatic increase in osteopontin expression induced by interleukin-1beta (IL-1beta), whereas tumor necrosis factor-alpha, transforming growth factor-beta, and angiotensin II had minimal effect. Stimulation with IL-1beta resulted in the secretion of soluble osteopontin protein. We found that osteopontin expression by IL-1beta was regulated via signaling primarily through the mitogen-activated protein kinase member ERK1/2, partially by p38 MAPK, but not at all by JNK. Finally, the mechanism of IL-1beta increase in osteopontin mRNA requires de novo transcription and translation. In conclusion, we find that osteopontin is expressed by primary lung fibroblasts and is potently upregulated by the early inflammatory and pro-fibrotic cytokine IL-1beta. Activated fibroblasts may be a significant source of osteopontin production during lung fibrogenesis.

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 alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. We also examined elastin and alpha1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, alpha-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced alpha-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and alpha1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that alpha1(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 alpha-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.

Interstitial fibroblasts are an integral component of the alveolar wall. These cells produce matrix proteins that maintain the extracellular scaffold of alveolar structures. Emphysema is characterized by airspace enlargement resulting from the loss of alveolar cellularity and matrix. In this study, we explored the endotracheal delivery of fibroblasts to the lung parenchyma as a means of repairing damaged alveolar structures directly or indirectly for the delivery of transgenes. Fibroblasts were isolated from the lungs of neonatal transgenic mice expressing GFP during the period of rapid alveolarization. These GFP+ cells maintained their myofibroblast phenotype in culture and expressed elastin and alpha-smooth muscle actin mRNA. We administered GFP+ fibroblasts to saline- and elastase-treated mice by endotracheal instillation. We detected more GFP+ fibroblasts in the alveolar walls and in the interstitial areas of elastase-injured lungs than in normal lungs as assessed by immunohistochemistry and fluorescent imaging. The presence of GFP+ fibroblasts in the interstitium demonstrated transepithelial migration of these cells. Expression of GFP+ fibroblasts in recipient lungs was maintained for at least 20 d after endotracheal administration. These cells synthesize matrix components including elastin in vitro and could contribute to restoring the structural integrity of the alveolar wall.

We previously showed that interleukin (IL)-1beta decreases elastin gene transcription through activation of the NF-kappaB 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-1beta on elastin gene transcription. We found that cycloheximide blocked IL-1beta-induced downregulation of elastin mRNA but did not inhibit IL-1beta-induced translocation of p65 into the nucleus. IL-1beta 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/EBPbeta 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/EBPbeta 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-1beta-induced downregulation of elastin mRNA is dependent on NF-kappaB activation and C/EBPbeta expression. These results indicate that IL-1beta treatment activates NF-kappaB, which subsequently induces LIP expression and inhibition of elastin gene transcription.

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. © 2002 Wiley-Liss, Inc.

A single intratracheal dose of porcine pancreatic elastase, which is cleared from the lung by 24 hours, was administered to wild-type, IL-1beta type 1 receptor-deficient, double TNF-alpha (type 1 and type 2) receptor-deficient, and combined TNF-alpha (type 1 receptor) plus IL-1beta receptor-deficient mice. The mean linear intercept (Lm) of saline-treated mice was 32(3) microm [mean(SE)]. For wild-type elastase-treated mice, Lm was 81(6) microm at 21 days versus 52(5) microm 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) microm in elastase-treated mice deficient in the IL-1beta receptor, double TNF-alpha 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-1beta, the double TNF-alpha, and the combined receptor-deficient mice. Our results indicate that inflammatory mediators exacerbate elastase-induced emphysema. We estimate that in the combined TNF-alpha + IL-1beta 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.

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 alpha1(I) collagen and alpha-smooth muscle actin mRNAs were markedly reduced. Apigenin also attenuated the transforming growth factor-beta-stimulated increases of alpha1(I) collagen and alpha-smooth muscle actin mRNAs. Characterization of the apigenin effects indicates that apigenin reduces both the stability of the alpha1(I) collagen mRNA and the rate of transcription of the alpha1(I) collagen gene through a cycloheximide-sensitive pathway. Western blot analyses indicate that Akt activity is reduced in apigenin-treated myofibroblasts.

We investigated the role of phosphatidylinositol 3-kinase (PI3K) in the expression of α1(I) collagen mRNA. We report that the basal level of α1(I) collagen mRNA was reduced when PI3K activity was inhibited by either LY-294002 or wortmannin. These PI3K inhibitors also blocked increases of α1(I) collagen mRNA levels after the addition of transforming growth factor-β. 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 α1(I) collagen mRNA with no change in the rate of transcription of the α1(I) collagen gene as assessed by Northern blotting with actinomycin D-treated fibroblasts and nuclear run-on assays. Expression of a truncated α1(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 α1(I) collagen mRNA. In vivo, the PI3K activity in fibroblasts may regulate basal levels of α1(I) collagen mRNA expression.

The steady state levels of alpha1(I) collagen mRNA are decreased by retinoic acid and prostaglandin E2. These effector substances decrease the uptake of A system amino acids. We examined the effect of amino acid deprivation on the steady state levels of alpha1(I) collagen in human lung fibroblasts. Maintenance of fibroblasts in amino acid-free medium decreased alpha1(I) collagen mRNA levels by 29% at 24 h and 78% at 72 h. Frequent refeeding of cultures with amino acid-free medium resulted in more rapid decreases in intracellular amino acids and in alpha1(I) collagen mRNA levels. The decrease in alpha1(I) collagen mRNA levels was mediated by decreases in mRNA stability as assessed by a half-life determination using actinomycin D and by decreases in the rate of transcription as assessed by nuclear run-on assay. Treatment of fibroblasts with medium containing amino acids resulted in rapid restoration of alpha1(I) collagen mRNA levels. This increase in alpha1(I) collagen mRNA expression required protein synthesis as determined by cycloheximide sensitivity and was inhibited by prostaglandin E2. These data indicate that alpha1(I) collagen mRNA levels are sensitive to alterations in the amount of intracellular amino acids and suggest a potential mechanism whereby alpha1(I) collagen accumulation may be regulated independent of inflammatory mediators following lung injury.

The effects and interaction of transforming growth factor-beta (TGF-beta) and insulin on collagen production in human fetal lung fibroblasts was examined. Fibroblasts were labeled with [3H]proline and collagen production was analyzed by polyacrylamide gel electrophoresis. The addition of insulin (2 micrograms/ml) increased collagen production 5 fold and TGF-beta (5 ng/ml) increased collagen production 6-fold. The combination of TGF-beta and insulin further increased type I collagen production (12 fold). We found that TGF-beta increased pro-alpha 1 (I) collagen mRNA levels 2-3 fold, insulin increased mRNA levels by less than 2 fold, and the combination stimulated a 3-4 fold increase. In a nuclear run-on assay, we found a 1.7 fold increase in the rate of transcription for the pro-alpha 1 (I) collagen gene in insulin-treated cultures and a 2-fold increase in TGF-treated cultures. In fibroblasts transfected with a plasmid containing 2.4 kb of the 5' flanking sequences of the human pro-alpha 1 (I) collagen gene, TGF-beta stimulated a 2.8 fold increase in promoter activity. In contrast, the addition of insulin stimulated a small increase (less than 2 fold) in the pro-alpha 1 (I) collagen promoter activity when administered alone or in combination with TGF-beta. Insulin prolonged the half-life of pro-alpha 1 (I) collagen mRNA from 9.1 h to 14.3 h as assessed by treatment with actinomycin D. The insulin-induced increase in pro-alpha 1 (I) collagen mRNA was blocked by the presence of cycloheximide indicating a requirement for new protein synthesis. These results show that the combination of TGF-beta and insulin stimulate large increases in type I collagen formation by acting at different sites in the collagen biosynthetic pathway.

MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix-associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)-β1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-β1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-β1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-β1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.

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-1beta 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. Downregulation 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.

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) 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-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta 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-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-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-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.

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.

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-1beta is mediated via activation of NF-kappaB 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-kappaB and C/EBPbeta, or the nuclear presence of NF-kappaB, but markedly increased C/EBPbeta isoforms, including liver-enriched transcriptional activating protein and liver-enriched transcriptional inhibitory protein. Addition of cycloheximide blocked these increases and the downregulation of elastin mRNA by MG-132. The MG-132-induced downregulation of elastin transcription was dependent on C/EBPbeta 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/EBPbeta isoforms.

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.

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 αvβ3, αvβ5, and α9β1. 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-1β 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.

Interleukin (IL)-1beta released after lung injury regulates the production of extracellular matrix components. We found that IL-1beta treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. 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)-kappaB. This element mediated IL-1beta-induced inhibition of the elastin promoter. IL-1beta treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-kappaB. Overexpression 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 of p65 or treatment with IL-1beta 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-1beta activates the nuclear localization of NF-kappaB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.

Nonalcoholic fatty liver disease (NAFLD) is an important cause of liver disease. The prevalence of hepatic steatosis in chronic hepatitis B (CHB) is not well understood. There are reports that low betaine level contributes to hepatic steatosis by increasing oxidative stress. Our aims are to determine the prevalence and impact of hepatic steatosis on CHB and the role of betaine in this disease state. In a single center, CHB patients with pretreatment liver biopsy between 1998 and 2013 were reviewed. The analysis excluded patients with alcoholism (>50g/day), HCV, HDV or HIV coinfection, other liver diseases and liver transplantation. To date, 231 patients (75% Asians) met the inclusion criteria. Serum betaine levels were measured in 40 randomly selected serum samples from patients with and without steatosis in both genders. Results: Total 127 (55%) pts had hepatic steatosis in biopsied tissue. Steatosis was more common in males (91/136, 67%) than females (36/95, 38%) [p=0.0001]. Among those with steatosis, males (mean 41.5 yrs) were younger than females (mean 48.5 yrs) [p=0.02]. For both genders, those with steatosis had higher rate of severe hepatic inflammation (M 63%, F 30%) compared to those without (M 5%, F 3%) [p=0.0001]. Advanced fibrosis (Stage 3+4) was more frequent in males (14%) than females (8%). Hepatic iron was present in 47 of 91 (52%) males with steatosis but in only 7 of 36 (19%) females with steatosis [p=0.0001]. Females with steatosis had higher rates of elevated LDL cholesterol (64% vs. 41%) [p=0.04], triglyceride (31% vs.13%) [p=0.02] and increased body weight (150lbs vs. 126lbs) [p=0.001] compared to those without. The lipid profiles and weight, however, were similar in males with and without steatosis. Interestingly, males with steatosis (N=10) had lower serum level of betaine compared those without steatosis (N=10) [28.3 vs. 43.3 umol/L; p=0.056]. In contrast, females with (N=10) or without steatosis (N=10) had similar betaine levels [42.9 vs. 39.3umol/L; p=0.3]. Viral factors such as serum HBV DNA levels, HBeAg status were not different between those with and without steatosis. Conclusions: High prevalence of hepatic steatosis is identified in this predominantly Asian cohort of patients with chronic hepatitis B. Steatosis is more common in males. For both genders, steatosis is associated with more severe hepatic inflammation. There appears to be a gender difference in predisposition to steatosis. In females, metabolic syndrome is a major contributing factor. In males, presence of hepatic iron and lower betaine levels correlate with steatosis and inflammation. It is possible that betaine attenuates iron-iduced oxidative stress in the liver. The gender differences in steatosis predisposition in CHB warrant further evaluation. Its understanding likely will contribute to better preventive and therapeutic strategies.

Transforming growth factor-β (TGF-β) stimulates α1(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-β stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-β-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-β-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-β increases the CTGF transcription rate. The TGF-β-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E2(PGE2)-treated fibroblasts. PGE2fails to attenuate luciferase activity induced by TGF-β in fibroblasts transfected with the TGF-β-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE2and insulin regulate α1(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of α1(I) collagen mRNA levels by TGF-β and PGE2may function through both CTGF-dependent and CTGF-independent mechanisms.

The steady-state level of alpha1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases alpha1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of alpha1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of alpha1(I) collagen mRNA. We found that re-expression of alpha1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of alpha1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased alpha1(I) collagen mRNA levels. The combination of glutamine and cystine increased alpha1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase alpha1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of alpha1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of alpha1(I) collagen mRNA.