Inhibition of JNK Enhances TGF-β1-Activated Smad2 Signaling in Mouse Embryonic Lung

Abstract

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.

A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung

“…Second, the use of different strains of mice, at variable gestational ages, and different doses of JNK inhibitors can lead to significantly different results. To illustrate, in contrast to our findings, the JNK inhibitor SP600125 at a dose of 10 μM, used in lung explants obtained from CD1 mice at embryonic day 12.5, induced endogenous CTGF expression, TGFβ1-induced CTGF expression, increased DNA fragmentation and cleaved caspase 3 [19]. Adult JNK1 null mutant mice have been reported to have increased susceptibility to hyperoxia [6].…”

“…Firstly, the JNK pathway has been implicated in TGF-β1 signaling in lung cells [19,24,26-29], specifically CTGF [19,26,28] and cell death [19,27] as well as myofibroblast transformation [24]. Secondly, hyperoxia has been shown to upregulate TGF-β1 in premature rat lungs [30].…”

“…Another molecule that has been implicated as a downstream mediator of TGF-β1 signaling in the newborn lung is connective tissue growth factor (CTGF) [18]. Recently, the JNK pathway has been implicated for TGF-β1-mediated effects in the developing lung [19]. …”

FGF2-mediated attenuation of myofibroblast activation is modulated by distinct MAPK signaling pathways in human dermal fibroblasts

“…ERK has also been demonstrated to have the ability to phosphorylate serines and threonines in the linker region of SMAD2/3, and mutation of specific ERK-target serine and threonine residues of SMAD3 results in potentiated SMAD3 signaling, suggesting that ERK can inhibit SMAD3 signaling via this mechanism [38]. Previous reports also demonstrate the ability of JNK activation to antagonize TGFβ signaling and TGFβ-mediated SMAD activation [39–41], in support of our findings that pharmacological inhibition of JNK signaling results in increased fibroblast activation.…”

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

“…The cell-specific differences in crosstalk between MAPK and Smad pathways are also evident with the JNK pathway. Where active JNK enhances or maintains TGFβ-mediated expression in fibroblasts(41) and mesangial cells(42) through phosphorylation of C-terminal Smad2, inhibiting JNK activity in embryonic lung explants increases both endogenous and TGFβ-induced Smad phosphorylation (45) suggesting that active JNK antagonizes TGFβ-induced pSmad2. In yet another study, TGFβ-activated JNK was shown to only phosphorylate Smad3 outside the C-terminal SXS motif in Mv1Lu cells (46).…”