David C. Rishikof

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.

Phenylbutyrate decreases type I collagen production in human lung fibroblasts

Fibrotic lung diseases are characterized by excess extracellular matrix production, in particular type I collagen. Phenylbutyrate (PB) is a non‐toxic pharmacological compound that functions as a weak histone deacetylase inhibitor. In hepatic stellate cells, the synthesis of type I collagen expression is decreased by inhibiting histone acetylation. Our studies examined the regulation of type I collagen by PB in human lung fibroblasts. We found that PB decreases basal and transforming growth factor‐β‐stimulated α1(I) collagen mRNA and protein levels. Northern blot analyses demonstrated that PB decreases steady‐state α1(I) collagen mRNA levels by 78% without significantly changing the stability of the mRNA transcript. PB stimulates cAMP production and increases the acetylation of histone H4, but does not affect the activity of two transforming growth factor‐β (TGF‐β)‐responsive luciferase reporter constructs. These data suggest that PB regulates type I collagen expression in human lung fibroblasts by mechanisms that include cAMP production and histone acetylation. PB may have therapeutic use in fibrotic lung diseases.

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 effect of prostaglandin E2 on cystine uptake and glutathione synthesis by human lung fibroblasts

Prostaglandin E2 (PGE2) is an inflammatory mediator capable of regulating fibroblast cell proliferation, matrix protein production, and system A amino acid transport. System x-c amino acid transport is regulated by electrophilic agents and oxygen. The effect of PGE2 on the x-c system transport of cystine and the synthesis of glutathione by human lung fibroblasts was examined. Preincubation of fibroblast cultures with PGE2 decreased cystine uptake by 42%. Kinetic studies revealed a 42% decrease in the Vmax of the x-c system transporter in PGE2-treated fibroblasts; however, the apparent Km was not affected. The glutathione content of PGE2-treated fibroblasts was decreased by up to 25% of control. These results demonstrate that system x-c transport of cystine is regulated by PGE2 and suggest that the limited availability of intracellular cysteine inhibited glutathione synthesis.

Amino acid availability regulates type I procollagen accumulation in human lung fibroblasts

Fibrotic lung diseases are characterized by excessive deposition of type I collagen. Amino acid availability regulates type I collagen mRNA levels in quiescent human lung fibroblasts. In these studies, the effect of amino acid availability on type I collagen protein accumulation in quiescent human lung fibroblasts was examined. Following amino acid deprivation, α1(I) procollagen protein levels were not detected by Western blot analysis in either the intracellular or the extracellular compartments. Fibronectin levels and total protein levels were not affected. Amino acid deprivation resulted in a more pronounced decrease in α1(I) procollagen protein levels than in α1(I) procollagen mRNA levels, suggesting that post‐transcriptional events were responsible for the further decrease inα1(I) procollagen protein levels. The addition of transforming growth factor‐β to amino acid deprived fibroblasts increased α1(I) procollagen mRNA levels without affecting α1(I) procollagen protein levels, confirming a post‐transcriptional site for regulatory control by amino acid deprivation. In the absence of ascorbic acid, α1(I) procollagen protein levels increased in amino acid deprived fibroblasts, but α1(I) procollagen mRNA levels were not affected. The absence of ascorbic acid likely resulted in the accumulation of nonhelical procollagen in the endoplasmic reticulum, indicating that translational mechanisms for α1(I) procollagen were intact. The addition of chloroquine, an inhibitor of lysosomal degradation of proteins, increased α1(I) procollagen protein levels in amino acid deprived fibroblasts. These data suggest that following amino acid deprivation of quiescent fibroblasts, newly synthesized type I collagen was degraded intracellularly, primarily by a process that involved lysosomal proteinases. J. Cell. Biochem. 75:130–137, 1999.

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.