Bernhard Schmierer

TGFβ–SMAD signal transduction: molecular specificity and functional flexibility

Ligands of the transforming growth factor-β (TGFβ) superfamily of growth factors initiate signal transduction through a bewildering complexity of ligand–receptor interactions. Signalling then converges to nuclear accumulation of transcriptionally active SMAD complexes and gives rise to a plethora of specific functional responses in both embryos and adult organisms. Current research is focused on the mechanisms that regulate SMAD activity to evoke cell-type-specific and context-dependent transcriptional programmes. An equally important challenge is understanding the functional role of signal strength and duration. How are these quantitative aspects of the extracellular signal regulated? How are they then sensed and interpreted, and how do they affect responses?

Transcription Factor Binding in Human Cells Occurs in Dense Clusters Formed around Cohesin Anchor Sites

During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.

Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor β-Dependent Nuclear Accumulation of Smads

ABSTRACT Upon transforming growth factor β (TGF-β) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-β-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-β-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-β-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

Analysis of Smad nucleocytoplasmic shuttling in living cells.

Transforming growth factor β (TGF-β) signalling leads to phosphorylation and activation of receptor-regulated Smad2 and Smad3, which form complexes with Smad4 and accumulate in the nucleus. The Smads, however, do not seem to reside statically in the cytoplasm in the absence of signalling or in the nucleus upon TGF-β stimulation, but have been suggested to shuttle continuously between these cellular compartments in both the absence and presence of TGF-β. Here we investigate this nucleocytoplasmic shuttling in detail in living cells using fusions of Smad2 and Smad4 with enhanced GFP. We first establish that the GFPSmad fusions behave like wild-type Smads in a variety of cellular assays. We go on to demonstrate directly, using photobleaching experiments, that Smad2 and Smad4 shuttle between the cytoplasm and nucleus in both TGF-β-induced cells and in uninduced cells. In uninduced cells, GFPSmad2 is less mobile in the cytoplasm than is GFPSmad4, suggesting that it may be tethered there. In addition, we show that both GFPSmad2 and GFPSmad4 undergo a substantial decrease in mobility in the nucleus upon TGF-β stimulation, suggesting that active complexes of Smads are tethered in the nucleus, whereas unactivated Smads are more freely diffusible. We propose that regulated cytoplasmic and nuclear retention may play a role in determining the distribution of Smads between the cytoplasm and the nucleus in both uninduced cells and upon TGF-β induction.

Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells

The catecholamine‐mediated modulation of the cytokine network has primarily been demonstrated for leukocytes. Whereas catecholamines decrease the LPS‐induced production of IL‐6 by leukocytes, serum levels of IL‐6 are dramatically increased by the catecholamine epinephrine in animal endotoxemia models. We now demonstrate that epi‐nephrine as well as norepinephrine can induce IL‐6 in an endothelial cell line (HMEC‐1). Furthermore, these catecholamines could even potentiate the LPS‐induced IL‐6 protein production. The synergistic effect of cat‐echolamines and LPS could be reproduced in primary human skin microvascular endothelial cells. The cate‐cholamine‐induced IL‐6 stimulation is based on increased IL‐6 mRNA levels. RNA stability assays revealed that this regulation is not a result of enhanced RNA stability and therefore is most likely due to an increased transcription. Treatment with cycloheximide indicated that new protein synthesis is not necessary for this transcriptional up‐regulation of IL‐6 mRNA. Prein‐cubation with α and β receptor antagonists showed that the effect is mediated by β1‐ and β2‐adrenergic receptors. Thus, endothelial cells might be a possible source of increased IL‐6 production observed in situations such as stress or septic shock, in which catecholamines are elevated due to endogenous production or exogenous application.—Gornikiewicz, A., Sautner, T., Brostjan, C., Schmierer, B., Függer, R., Roth, E., Muhlbacher, F., Bergmann, M. Catecholamines up‐regu‐late lipopolysaccharide‐induced IL‐6 production in human microvascular endothelial cells. FASEB J. 14, 1093–1100 (2000)

Two highly related regulatory subunits of PP2A exert opposite effects on TGF-β/Activin/Nodal signalling

We identify Bα (PPP2R2A) and Bδ (PPP2R2D), two highly related members of the B family of regulatory subunits of the protein phosphatase PP2A, as important modulators of TGF-β/Activin/Nodal signalling that affect the pathway in opposite ways. Knockdown of Bα in Xenopus embryos or mammalian tissue culture cells suppresses TGF-β/Activin/Nodal-dependent responses, whereas knockdown of Bδ enhances these responses. Moreover, in Drosophila, overexpression of Smad2 rescues a severe wing phenotype caused by overexpression of the single Drosophila PP2A B subunit Twins. We show that, in vertebrates, Bα enhances TGF-β/Activin/Nodal signalling by stabilising the basal levels of type I receptor, whereas Bδ negatively modulates these pathways by restricting receptor activity. Thus, these highly related members of the same subfamily of PP2A regulatory subunits differentially regulate TGF-β/Activin/Nodal signalling to elicit opposing biological outcomes.

Asparagine and aspartate hydroxylation of the cytoskeletal ankyrin family is catalyzed by factor-inhibiting hypoxia-inducible factor.

Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13–24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.

CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response

Here, we report that genome editing by CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in immortalized human retinal pigment epithelial cells.

Controlling long-term signaling: receptor dynamics determine attenuation and refractory behavior of the TGF-β pathway.

Ligand binding, but not receptor activity, results in desensitization and long-term dampening of cellular responsiveness to TGF-β. Turning Off TGF-β Responsiveness Transforming growth factor–β (TGF-β) signaling can have both tumor-suppressing and tumor-promoting activity. Vizán et al., through a combination of assays in cultured cells and mathematical modeling, provide an explanation for context-dependent responsiveness. In response to ligand binding, the type I and type II receptors, which comprise the ligand-activated TGF-β receptors, were rapidly depleted from the cell surface, leaving the cells unable to respond to subsequent exposures of TGF-β until ligand was depleted from the medium and receptors had reaccumulated at the cell surface. Mathematical modeling suggested that when autocrine TGF-β production exceeded a certain threshold, cellular responsiveness to TGF-β was limited. Indeed, cancer cells that produced the greatest amount of TGF-β exhibited the least responsiveness to an acute TGF-β stimulation. Understanding the complex dynamics of growth factor signaling requires both mechanistic and kinetic information. Although signaling dynamics have been studied for pathways downstream of receptor tyrosine kinases and G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors, they have not been investigated for the transforming growth factor–β (TGF-β) superfamily pathways. Using an integrative experimental and mathematical modeling approach, we dissected the dynamic behavior of the TGF-β to Smad pathway, which is mediated by type I and type II receptor serine/threonine kinases, in response to acute, chronic, and repeated ligand stimulations. TGF-β exposure produced a transient response that attenuated over time, resulting in desensitized cells that were refractory to further acute stimulation. This loss of signaling competence depended on ligand binding, but not on receptor activity, and was restored only after the ligand had been depleted. Furthermore, TGF-β binding triggered the rapid depletion of signaling-competent receptors from the cell surface, with the type I and type II receptors exhibiting different degradation and trafficking kinetics. A computational model of TGF-β signal transduction from the membrane to the nucleus that incorporates our experimental findings predicts that autocrine signaling, such as that associated with tumorigenesis, severely compromises the TGF-β response, which we confirmed experimentally. Thus, we have shown that the long-term signaling behavior of the TGF-β pathway is determined by receptor dynamics, does not require TGF-β–induced gene expression, and influences context-dependent responses in vivo.

Hypoxia-dependent sequestration of an oxygen sensor by a widespread structural motif can shape the hypoxic response - a predictive kinetic model

BackgroundThe activity of the heterodimeric transcription factor hypoxia inducible factor (HIF) is regulated by the post-translational, oxygen-dependent hydroxylation of its α-subunit by members of the prolyl hydroxylase domain (PHD or EGLN)-family and by factor inhibiting HIF (FIH). PHD-dependent hydroxylation targets HIFα for rapid proteasomal degradation; FIH-catalysed asparaginyl-hydroxylation of the C-terminal transactivation domain (CAD) of HIFα suppresses the CAD-dependent subset of the extensive transcriptional responses induced by HIF. FIH can also hydroxylate ankyrin-repeat domain (ARD) proteins, a large group of proteins which are functionally unrelated but share common structural features. Competition by ARD proteins for FIH is hypothesised to affect FIH activity towards HIFα; however the extent of this competition and its effect on the HIF-dependent hypoxic response are unknown.ResultsTo analyse if and in which way the FIH/ARD protein interaction affects HIF-activity, we created a rate equation model. Our model predicts that an oxygen-regulated sequestration of FIH by ARD proteins significantly shapes the input/output characteristics of the HIF system. The FIH/ARD protein interaction is predicted to create an oxygen threshold for HIFα CAD-hydroxylation and to significantly sharpen the signal/response curves, which not only focuses HIFα CAD-hydroxylation into a defined range of oxygen tensions, but also makes the response ultrasensitive to varying oxygen tensions. Our model further suggests that the hydroxylation status of the ARD protein pool can encode the strength and the duration of a hypoxic episode, which may allow cells to memorise these features for a certain time period after reoxygenation.ConclusionsThe FIH/ARD protein interaction has the potential to contribute to oxygen-range finding, can sensitise the response to changes in oxygen levels, and can provide a memory of the strength and the duration of a hypoxic episode. These emergent properties are predicted to significantly shape the characteristics of HIF activity in animal cells. We argue that the FIH/ARD interaction should be taken into account in studies of the effect of pharmacological inhibition of the HIF-hydroxylases and propose that the interaction of a signalling sensor with a large group of proteins might be a general mechanism for the regulation of signalling pathways.