The recent identification of the SMAD family of signal transducer proteins has unravelled the mechanisms by which transforming growth factor-beta (TGF-beta) signals from the cell membrane to the nucleus. Pathway-restricted SMADs are phosphorylated by specific cell-surface receptors that have serine/threonine kinase activity, then they oligomerize with the common mediator Smad4 and translocate to the nucleus where they direct transcription to effect the cell's response to TGF-beta. Inhibitory SMADs have been identified that block the activation of these pathway-restricted SMADs.

Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis.

Smad proteins play a key role in the intracellular signalling of transforming growth factor β (TGFβ), which elicits a large variety of cellular responses. Upon TGFβ receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGFβ. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGFβ and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGFβ responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGFβ. In addition, TGFβ induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGFβ-responsive regions of several other genes suggests that this may be a widely used motif in TGFβ-regulated transcription.

The generation of mice lacking speci®c components of the transforming growth factor-b (TGF-b) signal tranduction pathway shows that TGF-b is a key player in the development and physiology of the cardiovascular system. Both pro-and anti-angiogenic properties have been ascribed to TGF-b, for which the molecular mechanisms are unclear. Here we report that TGF-b can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF-b induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF-b/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF-b/ ALK1 pathway induces endothelial cell migration and proliferation. We identi®ed genes that are induced speci®cally by TGF-b-mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF-b/ALK1-induced (and Smad-dependent) migration, while induction of plasminogen activator inhibitor-1 by activated ALK5 may contribute to the TGF-b-induced maturation of blood vessels. Our results suggest that TGF-b regulates the activation state of the endothelium via a ®ne balance between ALK5 and ALK1 signalling.

TGF-beta signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-beta receptor induces phosphorylation of two such proteins, Smad2 and Smad3, which form hetero-oligomeric complex(es) with Smad4/DPC4 that translocate to the nucleus, where they then regulate transcriptional responses. However, the mechanisms by which the intracellular signals of TGF-beta are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signalling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses.