Signal Transduction
Fig 20.12 Signal integration through different transcriptional partners.
Through interaction with different DNA-binding transcription factors, Smad proteins form nodes of integration that produce different signalling outputs. Image adapted from Massagué et al.3
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Smad3 act together to repress expression of ID1. In epithelial cells this is necessary for effective arrest of the cell cycle. Thus TGF and TNFact in concert to slow down the cell cycle, reducing DNA replication and therefore protecting the organism against DNA damaging agents. c-Jun, in complex with c-Fos (AP1 complex, see Figure 19.1, page 579) aids Smads 2 and 3 in the induction of extracellular matrix proteins and proteases in response to TGF .74
Smads also cooperate with the Wnt pathway, in both a TGF -independent and a dependent manner. We have already mentioned that in the absence of a TGF signal, Smad4 interacts with -catenin/Tcf and this complex activates expression of c-myc (Figure 20.11).53 Cooperation between Smads and Wnt in the presence of TGF occurs in expression of the Xenopus homeobox gene twin (Xtwn). Here Smads 3 and 4 team up with -catenin/Lcf in the transformation of dorsal mesoderm to form the Spemann organizer.75
Inflammatory mediators, such as TNF- , IFN- , and IL-1 , activate the transcription factors NF- B/RelA and IRF3, which combine with Smads 2 and 4, leading to the induction of the inhibitory Smad7. In this way, they abrogate the response to BMP or TGF (see below and Figure 20.16). Coactivators such as CBP or p300 also serve as platforms for pathway crosstalk. An example is the collaboration between BMP2 and LIF, leading (E3) to activation of Smad1/Smad4 and STAT3 respectively, each binding to different sites in p300 and cooperatively activating the glial fibrillary acidic protein promoter.76
The Smad linker region: hotspot for kinases and an E3-ligase
The linker region of the receptor-regulated Smads contains numerous phosphorylation sites which are targeted by various kinases, including
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Fig 20.13 Opposing effects of BMP and FGF signalling on the translocation of Smad complexes into the nucleus.
The linker region of Smad proteins contains several phosphorylation sites, targets of CDK2/4, ERKs1 and 2 and GSK3 . The PPxY motif binds the E3ubiquitin ligases Smurf. Phosphorylation by ERK facilitates binding of Smurf1 (an ubiquitin E3 ligase). In embryonic development, the BMP signal is counteracted by secretion of trapping proteins such as noggin and chordin and also by FGF (1). This, through the Ras–ERK pathway causes phosphorylation of serine residues in the linker region (2). ERK-mediated phosphorylation of Smad1 facilitates the binding of Smurf1 to PPxY (3). The Smurf/Smad complex cannot bind the nuclear pore protein Nup214 and remains in the cytoplasm. Moreover, Smurf1 causes destruction of Smad. In the nucleus, linker phosphorylation of Smads and Smurf-mediated destruction also occurs (4) but the kinase remains unknown.
CDK2 and CDK4, GSK3 , and MAPkinases (ERK, JNK, and p38) (Figure 20.13). Phosphorylation of Smad3 by CDK4, and of Smad1 by MAPkinases, causes cytosolic retention and subsequent degradation.77–79 The functional consequences of phosphorylation by GSK3 are not known. Phosphorylation of Smad1 by ERK2 has been studied in more detail and we treat this topic in the context of neural development in Xenopus laevis.80
In the early gastrula stage, the dorsal ectoderm transforms into neuroectoderm, which, at a later point provides the neural plate from which the notochord develops.81 The ectoderm develops into dermis under the influence of BMP, but at the dorsal site of the embryo the signal is opposed by FGF, migrating from the mesodermal marginal zone,82,83 and by chordin and noggin (and other factors) that trap the BMP ligand. These diffuse from the Spemann organizer at the onset of gastrulation (Figure 20.15). FGF opposes the action of BMP and it also presents a neuroinductive signal. The opposing action of FGF is mediated through its receptor tyrosine kinase (FGFR), activating the Ras–ERK pathway and resulting in phosphorylation of the linker region of Smad1. This favours the interaction of Smad1 with Smurf1, an E3-ubiquitin ligase, thereby blocking the nuclear import binding site located
ID1 is required for progression through G1, one of its functions being to disable the action
of Rb. ID proteins are helix–loop–helix proteins that lack a DNA binding domain. They bind to other helix–loop–helix proteins and, in so doing, prevent their access to DNA. In this way they prevent the action of,
for instance, MyoD. Their expression is blocked in senescent fibroblasts.
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in the MH2 domain. Smad1 fails to enter the nucleus and is degraded following ubiquitylation (figure 20.13).78
There are other instances in which the activities of the FGF and BMP pathways have opposing effects. These include the pairs FGF4/BMP2 in limb bud formation, FGF10/BMP4 in lung morphogenesis, FGF2/BMP4 in cranial suture fusion and FGF8/BMP4 in the initiation of tooth development.80
These processes may also be coordinated by signal integration at the level of Smad1.
Hans Spemann and the organizer
‘How does that harmonious interlocking of separate processes come about which makes up the complete process of development? Do they go on side by side independently of each other (by ‘self-differentiation’, Roux), but from the very beginning so in equilibrium that they form the highly complicated end product of the complete organism, or is their influence on each other one of mutual stimulation, advancement or limitation?’
The question was put by Hans Spemann, professor of zoology at the University of Freiburg im Breisgau.84 With his graduate student, Hilde Mangold, he examined the transplantation of structures from the giant embryos of amphibians. Portions of surface cells (presumptive ectoderm) were removed and then implanted into an intact embryo. In many instances the implanted cells adopted the fate of the cells surrounding the new insertion site, but cells from a limited area, namely the region of the upper and lateral blastopore
lip failed to do so. These gave rise to the formation of a nearly complete secondary dorsal axis, commencing with the formation of a second medullary plate (Medullarplatte) and giving rise to the formation of a secondary: neural tube (Neuralrohr), notochord (chorda), prevertebral discs (Urwirbel), kidneys (Niere), and even gut (Darmlumen). This area was named ‘organizer’ and we now know it as the ‘Spemann organizer’ (see Figures 20.14 and 20.15).
Of several hundred chimeric embryos, only five survived, and these formed the basis for the famous paper that was published in 1924.85 Spemann and Mangold were the first to prove the reality of ‘induction’, by which one group of cells influences the developmental fate of the other. The identity of the inducing signal long remained the holy grail of developmental
biology, but now, after about 80 years, we begin to realize that it constitutes a number of secreted factors, amongst which the antagonists of BMP play an important role.
In 1935 Spemann was awarded the Nobel Prize in Physiology or Medicine ‘for his discovery of the organizer effect in embryonic development’. He was relieved of his professorial position in 1937, his place being taken by Otto
Mangold, more acceptable to the regime and previously the husband of Hilde who had been killed in a kitchen explosion 13 years earlier.
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The linker region of Smad3 is also phosphorylated by the cell-division cycle kinases CDK2 and CDK4. As their phosphorylation sites overlap with those of ERK, the consequences are similar and Smad3 remains sequestered in the cytoplasm to face destruction by Smurf. Tumour cells that over-express these
kinases may therefore be less sensitive to the growth inhibitory effect of TGF .77
The transcriptional activity of the Smads is also inhibited by PKC-mediated phosphorylation of the DNA-binding MH1 domain and enhanced by binding of Ca2 –calmodulin.87 For example, Ca2 –calmodulin enhances the ventral mesoderm-forming activity of Smad1 (BMP) and blocks the activity
Fig 20.14 Secondary dorsal axis formation after implanting “the organizer”.
Fig 20.15 Neuroectoderm induction through FGF and the release of the BMP-traps noggin and chordin. Neuroectoderm formation in Xenopus laevis occurs through inhibition of the BMP signal by release of noggin and chordin from the Spemann organizer and through diffusion of FGF from the mesoderm marginal zone into dorsal ectoderm. Neuroectoderm develops into notochord and gives rise to the brain.
The BMP proteins are involved in the development of the craniofacial complex
(teeth, peridontium, and jaws). The discovery of stem cells in dental pulp now offers the possibility of regenerative therapy of the craniofacial complex. These rather fancy words disguise the idea that this approach may ultimately offer the possibility of regenerating whole teeth after extraction.86
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