The MEK Pathway Is Required for Stimulation of p21WAF1/CIP1 by Transforming Growth Factor-β

Abstract

Transforming growth factor-␤ (TGF-␤)can induce the cyclin-dependent kinase inhibitors p21 and p15 in a variety of cell types. We have shown previously that Smad3 is required for the growth inhibitory activity of TGF-␤, whereas overexpression of Smads is not sufficient to activate the expression of p21 in HaCaT cells. These data suggest that an additional signaling pathway may be involved in stimulating p21 in HaCaT cells. Given the recent finding that the mitogen-activated protein kinase (MAPK) pathway can cause p21 induction and arrest cells, we examined the involvement of this pathway for p21 and p15 induction by TGF-␤. We found that TGF-␤ can regulate the MAPK pathway, leading to the increased transactivation ability of transcription factor Elk. Constitutively active components in the MAPK pathway activate p21 expression, and inhibitors or dominant negative constructs for the MAPK pathway significantly decrease p21 induction by TGF-␤. Both constitutively active MEK and inhibitors for MEK have no effect on Smad activity, including DNA binding, localization, and interaction with coactivator p300/CBP. These findings suggest that the MAPK pathway may be an independent pathway that is involved in p21 and p15 induction by TGF-␤.

Blockade of Smad4 in transformed keratinocytes containing a Ras oncogene leads to hyperactivation of the Ras-dependent Erk signalling pathway associated with progression to undifferentiated carcinomas

“…This response was blocked in 3DdnS transfectants (Figure 3a), while no di erences for TGF-b 1 up-regulation of p21 Cip1 were observed between control PC and dominant-negative PdnS transfectants (Figure 3b,c). Since the Erk MAP kinase signalling pathway has also been implicated in the regulation of p21 Cip1 by TGF-b in epithelial cells (Yue et al, 1998;Hu et al, 1999), we studied the e ects of PD098059, a speci®c inhibitor of MAP kinase kinase (MEK) (Simon et al, 1996), on p21 Cip1 protein induction by TGF-b 1 . PD098059, at a concentration of 50 mM (that is shown to inhibit e ciently Erk signalling activity in PDV cells, see Smad4 and Ras co-operate for tumour progression M lglesias et al below), consistently reduced (by 35 ± 50%) TGF-b 1 -stimulated p21 Cip1 expression in PC and PdnS clones, while it did not a ect the enhancement of p21 Cip1 by the growth factor in 3DC cells ( Figure 3c).…”

“…The signalling pathways involved in these TGF-binduced gene responses remain an open question (see Massague , 1998). Thus, p21 Cip1 appears to be a downstream target gene regulated by Smad4 in carcinoma cells (Grau et al, 1997;Hunt et al, 1998), while it has been reported that Ras and the MAP kinase pathway are required for up-regulation of p21 Cip1 by TGF-b in intestinal epithelial cells and human keratinocytes, respectively (Yue et al, 1998;Hu et al, 1999). The same appears to occur for TGF-bmediated induction of other genes, such as the 3TP-lux reporter construct, which contains a known TGF-binducible plasminogen activator inhibitor-1 (PAI-1) promoter (Grau et al, 1997;Mucsi et al, 1996;de Winter et al, 1997).…”

“…Recently, Kretzschmar et al (1999) found that oncogenic Ras, acting via MAP kinases, inhibits TGF-b-induced nuclear accumulation of Smad2 and Smad3. However, another report has shown that constitutively active MEK, or inhibitors for MEK, have no e ect on Smad activity in human keratinocytes (Hu et al, 1999).…”

A Novel Transforming Growth Factor-β Receptor-interacting Protein That Is Also a Light Chain of the Motor Protein Dynein

“…Despite advances in our understanding of the mechanisms by which the Smad and Ras/Mapk cascades mediate some TGF␤ effects, these pathways seem to regulate primarily transcriptional events (Hocevar et al, 1999;Hu et al, 1999;Sporn and Vilcek, 2000; Mulder, 2000a, 2001). However, TGF␤ is multifunctional and its biological responses are diverse.…”

“…In addition to the Ras/Mapk and Smad pathways, several proteins have been identified based upon their interaction with the TGF␤ receptors (Yue and Mulder, 2001). Furthermore, various Smad-interacting proteins have also been identified, including SARA and Dab2, which interact with both Smads and the TGF␤ receptors (Tsukazaki et al, 1998;Hocevar et al, 2001;Yue and Mulder, 2001).Despite advances in our understanding of the mechanisms by which the Smad and Ras/Mapk cascades mediate some TGF␤ effects, these pathways seem to regulate primarily transcriptional events (Hocevar et al, 1999;Hu et al, 1999;Sporn and Vilcek, 2000; Mulder, 2000a, 2001). However, TGF␤ is multifunctional and its biological responses are diverse.…”