Transforming growth factor-␤1 (TGF-␤1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-␤1 effects on mesenchymal cells, we hypothesized that TGF-␤1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-␤1 inhibition of branching morphogenesis. We show that addition of TGF-␤1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time-and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-␤1-induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-␤1 induces CTGF, we used SB431542, a specific inhibitor for TGF-␤ type I receptor (T␤RI)/ALK-5 to block TGF-␤1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-␤1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-␤1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-␤1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-␤1-induced CTGF expression are mediated by the T␤RI/ALK-5-dependent Smad2 signaling pathway. Keywords: connective tissue growth factor; TGF-␤1; ALK-5; Smad2; lung Normal lung development is highly coordinated by autocrine/ paracrine signaling, and by cell-extracellular matrix (ECM) and cell-cell interactions between the epithelium and mesenchyme (1-3). Peptide growth factors and transcriptional factors play an important role during the processes of lung branching morphogenesis (4). Small changes in the temporal or spatial expression of growth factors or transcriptional factors can lead to significant alterations in the final architecture of the lung and severe defects in the pulmonary function.Multiple lines of evidence have indicated that TGF-␤s are key negative regulators for lung branching morphogenesis. TGF-␤s belong to a family of closely related peptides, including TGF-␤1,

High tidal volume (V T ) ventilation plays a key role in ventilator induced lung injury and bronchopulmonary dysplasia. However, little is known about the effect of high V T on expression of growth factors that are critical to lung development. In a previous study, we demonstrated that connective tissue growth factor (CTGF) inhibits branching morphogenesis. In this study, we investigated the effect of high V T on CTGF expression in newborn rat lungs. Newborn rats were ventilated with normal V T (10 mL/kg) or high V T (25 mL/kg) for 6 h. Nonventilated animals served as controls. We found that high V T upregulated CTGF expression. To identify the potential signaling pathways mediating high V T induction of CTGF, newborn rats were ventilated with high V T for 1 or 3 h. Temporal expression of TGF-␤s, p-Smad2, Smad7, and CTGF was analyzed. High V T ventilation did not change gene expression of TGF-␤s and Smad7 but induced rapid and sustained expression of p-Smad2 that precedes increased CTGF expression. CTGF and p-Smad2 were localized in bronchiolar epithelial cells, alveolar walls and septa. These data suggest that high V T ventilation activates the Smad2 pathway, which may be responsible for downstream induction of CTGF expression in newborn rat lungs. M echanical ventilation is essential for managing prematurely born infants with respiratory failure. However, ventilation with high tidal volume (V T ) can lead to ventilator induced lung injury (VILI) and bronchopulmonary dysplasia (BPD) (1-3). The lung pathology of BPD is characterized by fewer and larger alveoli, dysmorphic and decreased capillary network, and variable interstitial fibrosis, suggesting abnormal lung development and injury repair processes (4,5). Most of the studies in VILI and BPD have been focused on high V T induced lung proinflammatory response (6 -9). Little is known about the effect of high V T on expression of growth factors that are key to lung development and injury repair in neonatal lungs.Connective tissue growth factor (CTGF) belongs to the CCN family of early gene products with a high degree of amino acid sequence homology and 38 conserved cysteine residues (10,11). CTGF promotes fibroblast proliferation, extracellular matrix (ECM) production, myofibroblast differentiation, and cell adhesion and migration (12-16). CTGF is a potent profibrotic cytokine and its mRNA and protein levels have been correlated with the degree of lung fibrosis in bleomycin-treated mice and in human fibrotic lung disorders (17)(18)(19). Previous studies have demonstrated that mechanical stress drastically induces CTGF expression in cultured fibroblasts (20,21). CTGF is closely linked to TGF-␤. Studies have demonstrated that TGF-␤ is a major inducer of CTGF expression in a variety of tissues and organs including the lung (11,(22)(23)(24). Activation of the Smad pathway plays a key role in TGF-␤ induction of CTGF expression (25,26). TGF-␤ stimulation of fibroblast proliferation, collagen synthesis, and myofibroblast differentiation is mediated via a CTG...

The Smad2/3 pathway plays a key role in mediating TGF-␤1 inhibition of branching morphogenesis and induction of connective tissue growth factor (CTGF) expression in embryonic lungs. Because a number of cell-specific interactions have been described between TGF-␤1-driven Smad signaling and the c-Jun N-terminal kinase (JNK) pathway, we have investigated the effects of JNK inhibition on TGF-␤1 activation of Smad2, inhibition of branching, induction of CTGF expression, and apoptosis in mouse embryonic lung explants. Mouse embryonic day 12.5 (E12.5) lung explants were treated with TGF-␤1 in the presence or absence of a specific pharmacologic JNK inhibitor (SP600125) and a specific JNK peptide inhibitor (JNKI). We found that TGF-␤1 activated the JNK pathway by stimulating c-Jun phosphorylation, which was blocked by JNK inhibitors. Treatment with SP600125 stimulated Smad2 phosphorylation and enhanced TGF-␤1-induced Smad2 phosphorylation. Treatment with JNK inhibitors also decreased normal branching morphogenesis and induced CTGF expression as well as augmented TGF-␤1 inhibition of branching and induction of CTGF expression. Furthermore, JNK inhibition-induced apoptosis. Our results demonstrate that inhibition of the JNK pathway promotes TGF-␤1-driven Smad2 responses in lung branching morphogenesis. These data suggest that the JNK pathway may antagonize TGF-␤1 dependent Smad2 signaling during mouse embryonic lung development.