Cell-cell signaling roles for reactive oxygen species (ROS) generated in response to growth factors/cytokines in nonphagocytic cells are not well defined. In this study, we show that fibroblasts isolated from lungs of patients with idiopathic pulmonary fibrosis (IPF) generate extracellular hydrogen peroxide (H2O2) in response to the multifunctional cytokine, transforming growth factor-beta1 (TGF-beta1). In contrast, TGF-beta1 stimulation of small airway epithelial cells (SAECs) does not result in detectable levels of extracellular H2O2. IPF fibroblasts independently stimulated with TGF-beta1 induce loss of viability and death of overlying SAECs when cocultured in a compartmentalized Transwell system. These effects on SAECs are inhibited by the addition of catalase to the coculture system or by the selective enzymatic blockade of H2O2 production by IPF fibroblasts. IPF fibroblasts heterogeneously express alpha-smooth muscle actin stress fibers, a marker of myofibroblast differentiation. Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast phenotype. Thus, myofibroblast secretion of H2O2 functions as a diffusible death signal for lung epithelial cells. This novel mechanism for intercellular ROS signaling may be important in physiological/pathophysiological processes characterized by regenerating epithelial cells and activated myofibroblasts.

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-β1 (TGF-β1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-β1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-β1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-β1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-β1 stimulation. TGF-β1-induced EMP is FAK- dependent, whereas TGF-β1-induced apoptosis is favored when FAK signaling is inhibited.

Carnitine palmitoyltransferase I (CPT I) catalyzes the formation of acylcarnitine, the first step in the oxidation of longchain fatty acids in mitochondria. The enzyme exists as liver (L-CPT I) and muscle (M-CPT I) isoforms that are encoded by separate genes. Genetic deficiency of L-CPT I, which has been reported in 16 patients from 13 families, is characterized by episodes of hypoketotic hypoglycemia beginning in early childhood and is usually associated with fasting or illness. To date, only two mutations associated with L-CPT I deficiency have been reported. In the present study we have identified and characterized the mutations underlying L-CPT I deficiency in six patients: five with classic symptoms of L-CPT I deficiency and one with symptoms that have not previously been associated with this disorder (muscle cramps and pain). Transfection of the mutant L-CPT I cDNAs in COS cells resulted in L-CPT I mRNA levels that were comparable to those expressed from the wild-type construct. Western blotting revealed lower levels of each of the mutant proteins, indicating that the low enzyme activity associated with these mutations was due, at least in part, to protein instability. The patient with atypical symptoms had ϳ 20% of normal L-CPT I activity and was homozygous for a mutation (c.1436C → T) that substituted leucine for proline at codon 479. Assays performed with his cultured skin fibroblasts indicated that this mutation confers partial resistance to the inhibitory effects of malonyl-CoA. The demonstration of L-CPT I deficiency in this patient suggests that the spectrum of clinical sequelae associated with loss or alteration of L-CPT I function may be broader than was previously recognized .-Brown, N.

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