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 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.

Phosphatidylinositol 3-kinase-dependent stabilization of alpha1(I) collagen mRNA in human lung fibroblasts.

We investigated the role of phosphatidylinositol 3-kinase (PI3K) in the expression of alpha1(I) collagen mRNA. We report that the basal level of alpha1(I) collagen mRNA was reduced when PI3K activity was inhibited by either LY-294002 or wortmannin. These PI3K inhibitors also blocked increases of alpha1(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 alpha1(I) collagen mRNA with no change in the rate of transcription of the alpha1(I) collagen gene as assessed by Northern blotting with actinomycin D-treated fibroblasts and nuclear run-on assays. Expression of a truncated alpha1(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 alpha1(I) collagen mRNA. In vivo, the PI3K activity in fibroblasts may regulate basal levels of alpha1(I) collagen mRNA expression.

Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA.

Expression of the kinin B1 receptor is up-regulated in chronic inflammatory and fibrotic disorders; however, little is known about its role in fibrogenesis. We examined human embryonic lung fibroblasts that constitutively express the B1 receptor and report that engagement of the B1 receptor by des-Arg(10)-kallidin stabilized connective tissue growth factor (CTGF) mRNA, stimulated an increase in alpha1(I) collagen mRNA, and stimulated type I collagen production. These events were not observed in B2 receptor-activated fibroblasts. In addition, B1 receptor activation by des-Arg(10)-kallidin induced a rise in cytosolic Ca(2+) that is consistent with B1 receptor pharmacology. Our results show that the des-Arg(10)-kallidin-stimulated increase in alpha1(I) collagen mRNA was time- and dose-dependent, with a peak response observed at 20 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was also time- and dose-dependent, with a peak response observed at 4 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was blocked by the B1 receptor antagonist des-Arg(10),Leu(9)-kallidin. Inhibition of protein synthesis by cycloheximide did not block the des-Arg(10)-kallidin-induced increase in CTGF mRNA. These results suggest that engagement of the kinin B1 receptor contributes to fibrogenesis through increased expression of CTGF.

Regulation of connective tissue growth factor expression by prostaglandin E(2).

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.

Endothelial cell nitric oxide production in acute chest syndrome.

Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS.

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, alpha1(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 alpha1(I) procollagen protein levels than in alpha1(I) procollagen mRNA levels, suggesting that post-transcriptional events were responsible for the further decrease inalpha1(I) procollagen protein levels. The addition of transforming growth factor-beta to amino acid deprived fibroblasts increased alpha1(I) procollagen mRNA levels without affecting alpha1(I) procollagen protein levels, confirming a post-transcriptional site for regulatory control by amino acid deprivation. In the absence of ascorbic acid, alpha1(I) procollagen protein levels increased in amino acid deprived fibroblasts, but alpha1(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 alpha1(I) procollagen were intact. The addition of chloroquine, an inhibitor of lysosomal degradation of proteins, increased alpha1(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.

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.

Regulation of type I collagen mRNA in lung fibroblasts by cystine availability.

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.