Mechanism of AP-1-mediated gene expression by select organochlorines through the p38 MAPK pathway

Organochlorine compounds have been demonstrated tohave detrimental health effects in both wildlife andhumans, an effect largely attributed to their ability tomimic the hormone estrogen. Our laboratory has studiedcell signaling by environmental chemicals associated withthe estrogen receptor (ER) and more recently via ER-independent mechanisms. Here, we show that the organo-chlorine pesticide dichlorodiphenyltrichloroethane (DDT)and its metabolites induce a stress mitogen-activated pro-tein kinase (MAPK) that leads to AP-1 activation. Throughthe use of a dominant negative c-Fos mutant, we showthat DDT exposure induces the collagenase promoter in anAP-1-dependent manner. DDT stimulates an AP-1 complexshift at the DNA to one favoring c-Jun/c-Fos dimers thro-ugh both increasing c-Jun levels and by post-translationalactivation of c-Jun and c-Fos in HEK 293 and humanendometrial Ishikawa cells. DDT treatment induces phos-phorylation of ERK and p38, while JNK phosphorylationlevels are slightly decreased. Using pharmacological andmolecular inhibitors of the various MAPKs, we implicatethe p38 signaling cascade, and to a lesser extent ERK, asnecessary pathways for AP-1-mediated gene expressioninduction by organochlorines. Taken together, these resultsdemonstrate that organochlorines induce the collagenasepromoter via sequential activation of the p38 kinase cas-cade and AP-1.IntroductionActivator protein-1 (AP-1) is a generic term used to describetranscription factors that bind specifically to a DNA enhancersequence [TGA(G/C)TCA] called the 12-O-tetradecanylphor-bol 13-acetate (PMA)-responsive element, which is alsoreferred to as the AP-1 site. In addition, AP-1 componentscan bind and potentiate transcription from AP-1-related DNAelements (ex. cAMP response element) (1,2). Members of theJun and Fos families of proteins dimerize to preferentially bindAP-1 sites with high affinity and, hence, each dimer combina-tion makes up an AP-1 protein complex. Upon stimulation, Junand Fos proteins recruit p300/CBP co-activators that recruitother co-activators such as the p160s, which can directly bindnuclear hormone receptors like the estrogen receptor (ER) (3).AP-1 is a ubiquitous protein complex that can be induced bymultiple stimuli, leading to diverse cellular effects. For exam-ple, proliferation, differentiation, cellular stress and death haveall been associated with elevated AP-1 activity (2,4–10).Increased AP-1 activity leads to various pathological out-comes, such as carcinogenesis (11–14). Hence, AP-1 appearsto play diverse roles in regulation of the cell cycle. Determin-ing what exogenous compounds stimulate AP-1 and how theystimulate AP-1 is central to understanding the role of theenvironment in pathogenesis. Generally, the final outcome isdetermined by cell type, promoter context, associative proteinsand the type of stimuli.Environmental stimulation of AP-1 occurs through a combi-nation of signaling events, leading to an increased activity ofproteins that directly potentiate Jun and Fos family members oractivate transcription factors that regulate expression of junand fos. The mitogen-activated protein kinases (MAPKs) areserine/threonine protein kinases intimately involved in govern-ing cellular processes such as cell growth, proliferation, differ-entiation and apoptosis. AP-1 is a major target of the MAPKs.There are three main subfamilies of MAPKs: extracellularsignal-regulated kinase (ERK), c-Jun N-terminal kinase(JNK) and p38. Each MAPK subfamily is phosphorylated/activated by specific MAPK kinases, which are in turn phos-phorylated/activated by MAPK kinase kinases that tend torecognize multiple substrates. Currently it is believed thatgrowth and differentiation factors induce an ERK pathway,whereas the JNK and p38 pathways are more involved instress-mediated signaling (15). However, exceptions to thesegeneral concepts exist. For example, increased p38 activity hasbeen implicated in the initiation and progression of carcino-genesis (12,13). To complicate matters, new members of theMAPK family have recently been discovered, such as bigMAPK 1 (BMK1), also known as ERK5, that respond toboth stress signals and growth factors (16). Ultimately, thegiven effect a MAPK has on gene expression depends on thecellular and stimulatory context.Organochlorines represent a class of environmental com-pounds characterized by a chlorinated hydrocarbon backbone