Introduction The transforming growth factor β (TGFβ) family of secreted factors regulates various biological processes, including cell proliferation, differentiation and apoptosis (Massague, 1998). TGFβs signal through cell-surface serine-threonine kinase type II and type I receptors. TGFβ binding to TGFβ type II (TβRII) receptor triggers its associaion with the TGFβ type I (TβRI) receptor (Massague, 1998). TβRII phosphorylates and activates TβRI, which, in turn, phosphorylates receptor- associated (RA) Smads (Smad2 and Smad3). RA-Smads bind Smad4 and translocate to the nucleus where they regulate transcription of target genes (Massague, 1998). In addition to Smads, TGFβ can activate Jun N-terminal kinase (JNK) (Atfi et al., 1997; Engel et al., 1999; Frey and Mulder, 1997), extracellular signal-regulated kinase (ERK) (Hartsough and Mulder, 1995), p38 mitogen-activated protein kinase (p38MAPK) (Hanafusa et al., 1999), and Akt (Bakin et al., 2000). Smad-dependent signaling has been shown to be required for the antiproliferative activity of TGFβ, and components of this pathway are frequently mutated or silenced in several human cancers (de Caestecker et al., 2000). Tumors, however, frequently express high levels of TGFβ and inhibition of TGFβ signaling has been shown to reduce tumor invasiveness and metastasis (Akhurst and Balmain, 1999; Barrack, 1997; Cui et al., 1996; Hojo et al., 1999). A number of studies provide evidence that TGFβ contributes to tumor cell invasion and metastasis by inducing mesenchymal transdifferentiation in epithelial cells (EMT) and stimulating cell migration (Akhurst and Balmain, 1999; Barrack, 1997; Oft et al., 1998). This TGFβ-mediated fibroblastic transdifferentiation is a complex process associated with alterations in epithelial cell junctions, changes in cell morphology, reorganization of the cell cytoskeleton, expression of fibroblastic markers (fibronectin, vimentin), and enhancement of cell migration (Bakin et al., 2000; Miettinen et al., 1994; Piek et al., 1999b). The molecular mechanisms of TGFβ-mediated EMT and cell migration are not entirely understood. Studies with TGFβ receptors have shown that a truncated TGFβ/bone morphogenic protein (BMP) type I receptor, Alk2, blocks EMT in mouse NMuMG cells (Miettinen et al., 1994). Adenoviral expression of constitutively active human TβRI/Alk5 together with Smad2/3 can induce EMT in these cells (Piek et al., 1999b). Expression of a dominant-negative truncated form of TβRII decreases the formation of invasive spindle tumours (Portella et al., 1998). Adenoviral expression of Smad2/3 induced EMT only in the context of expression of constitutively active Alk5 (Piek et al., 1999b). Overexpression of Smad7, an inhibitor of Smad-dependent signaling, or dominant-negative Smad3 did not affect the transdifferentiation, arguing against 3193 Transforming growth factor β (TGFβ) contributes to tumor progression by inducing an epithelial to mesenchymal transdifferentiation (EMT) and cell migration. We found that TGFβ-induced EMT was blocked by inhibiting activation of p38 mitogen-activated protein kinase (MAPK) with H-7, a protein kinase C inhibitor, and with SB202190, a direct inhibitor of p38MAPK. Inhibition of the p38MAPK pathway affected TGFβ-mediated phosphorylation of ATF2, but did not inhibit phosphorylation of Smad2. SB202190 impaired TGFβ- mediated changes in cell shape and reorganization of the actin cytoskeleton. Forced expression of dominant-negative (DN) MAPK kinase 3 (MKK3) inhibited TGFβ-mediated activation of p38MAPK and EMT. Expression of DN-p38α impaired TGFβ-induced EMT. Inhibition of p38MAPK blocked TGFβ-induced migration of non-tumor and tumor mammary epithelial cells. TGFβ induced activation of the p38MAPK pathway within 15 minutes. Expression of TGFβ type II (TβRII) and type I (TβRI/Alk5) kinase- inactive receptors blocked EMT and activation of p38MAPK, whereas expression of constitutively active Alk5-T204D resulted in EMT and phosphorylation of MKK3/6 and p38MAPK. Finally, dominant-negative Rac1N17 blocked TGFβ-induced activation of the p38MAPK pathway and EMT, suggesting that Rac1 mediates activation of the p38MAPK pathway. These studies suggest that the p38MAPK pathway is required for TGFβ-mediated EMT and cell migration. Key words: p38MAPK, TGFβ, Epithelial-mesenchymal transition, Cell migration, Rac1 Summary p38 mitogen-activated protein kinase is required for TGFβ-mediated fibroblastic transdifferentiation and cell migration Andrei V. Bakin 1 , Cammie Rinehart 1 , Anne K. Tomlinson 1 and Carlos L. Arteaga 1,2,3, * 1 Departments of Medicine and 2 Cancer Biology, and 3 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, 777 Preston Research Building, Nashville, TN 37232, USA *Author for correspondence (e-mail: carlos.arteaga@mcmail.vanderbilt.edu) Accepted 23 April 2002 Journal of Cell Science 115, 3193-3206 (2002) © The Company of Biologists Ltd Research Article