Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the expression of the human COL1A2 gene by TNF-alpha

Authors:
Verrecchia F, Wagner EF and Mauviel A
In:
Source: EMBO Rep
Publication Date: (2002)
Issue: 3(11): 1069-1074
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic fibroblast, mouse (MEF) immort
Species: mouse
Tissue Origin: embryo
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment

Jun-N-terminal kinases (JNKs) are activated upon exposure of cells to cytokines, growth factors and environmental stresses. In the human a(2) type I collagen gene COL1A2 promoter a Smad3/4 gene target downstream of TGF-beta has been shown to participate in the regulatory programs activated by TGF-beta and TNF-alpha to control type I collagen expression. The authors aimed to reveal the role of JNK and NF-kappaB pathways downstream of TNF-alpha in the context of COL1A2 expression. Immortalized fibroblasts cell lines were derived from mouse embryos in which targeted disruption of the jnk1 gene had been performed. These JNK-/- fibroblast were nucleofected with either empty vector or a HA-tagged jnk1 expression vector. 24 hour post transfection cells were treated with TNF-alpha and cell lysates were analyzed by Western blotting. jnk1 expression restored the phosphorylation of c-Jun in response to TNF-alpha indicating its role in COL1A2 transactivation.

Abstract

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-kappaB pathways downstream of TNF-alpha in the context of alpha(2) type I collagen gene (COL1A2) expression. In JNK1-/--JNK2-/- (JNK-/-) fibroblasts, TNF-alpha inhibited basal COL1A2 expression but had no effect on TGF-beta-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-kappaB essential modulator-/- (NEMO-/-) fibroblasts, lack of NF-kappaB activation did not influence the antagonism exerted by TNF-alpha against TGF-beta but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-alpha, critical kinases upstream of the JNK and NF-kappaB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-alpha to inhibit the expression of a given gene, COL1A2, depending on its activation state.