Transforming growth factor-beta (TGF-beta) has been associated with the onset of cardiac cell hypertrophy, but the mechanisms underlying this dissociation are not completely understood. By a previous study, we investigated the involvement of a MAP3K, ZAK, which in cultured H9c2 cardiac cells is a positive mediator of cell hypertrophy. Our results showed that expression of a dominant-negative form of ZAK inhibited the characteristic TGF-beta-induced features of cardiac hypertrophy, including increased cell size, elevated expression of atrial natriuretic factor (ANF), and increased organization of actin fibers. Furthermore, dominant-negative MKK7 effectively blocked both TGF-beta-and ZAK-induced ANF expression. In contrast, a JNK/SAPK specific inhibitor, sp600125, had little effect on TGF-beta- or ZAK-induced ANF expression. Our findings suggest that a ZAK mediates TGF-beta-induced cardiac hypertrophic growth via a novel TGF-beta signaling pathway that can be summarized as TGF-beta > ZAK > MKK7 > ANF. (C) 2004 Elsevier Inc. All rights reserved.