Browsing by Subject "Rho-GTPase"

Activation of Wnt signaling pathways is critical to a variety of developmental events across all animal taxa. These highly evolutionarily conserved pathways are also important in the adult organism for maintaining homeostasis of self-renewing tissues. Because of its role in such important physiological processes, deregulation of Wnt signaling can have severe consequences; indeed, inappropriate activation of this pathway has been implicated in multiple human diseases, including cancer.

Upon binding their cellular receptors, canonical Wnt ligands, like Wnt 3A, stimulate the stabilization, accumulation, and nuclear translocation of a multifunctional cellular protein βcatenin, the consequence of which is induction of βcatenin-dependent transcription. This work describes the identification and characterization of two Wnt3A-stimulated intracellular signaling pathways activated in parallel to βcatenin stabilization: the RhoA pathway and the ERK pathway. These two auxiliary pathways do not affect βcatenin stability, accumulation, or subcellular localization; rather, they modulate βcatenin -dependent transcriptional activity through other mechanisms. As a result of their influence on βcatenin-dependent transcription, these pathways instruct cell fate decisions in C3H10T1/2 mesenchymal stem cells, in particular inhibition of adipogenesis and promotion of osteoblastogenesis.

Expression microarray analysis and biochemical and pharmacological techniques were used to further characterize the two Wnt3A-stimulated auxiliary pathways in C3H10T1/2 cells. Remarkably, each pathway influences βcatenin function via a novel mechanism. In the Wnt3A/RhoA pathway, Wnt3A-stimulated trimeric G proteins activate a RhoA-ROCK-SRF cascade. Activated SRF can cooperate with βcatenin to enhance the induction of Wnt3A target genes, like Ctgf, that also contain SRF binding sites within regulatory elements. In the Wnt3A/ERK pathway, Wnt3A transactivates the EGFR in a concentration-dependent manner, leading ultimately to ERK activation, which interacts with and promotes βcatenin/Tcf4 interaction and enhances induction of βcatenin/Tcf4 target genes.

These data emphasize the complexity of Wnt signaling and have intriguing implications regarding cross-regulation of the pathway, especially in stem cells. Also, since not all cells are capable of responding to Wnt3A by activation of these auxiliary pathways, this work identifies novel mechanisms that could underlie cell type-specific responses to Wnts and provides mechanistic insight into cellular responses to Wnt concentration gradients. Moreover, this work identifies novel transcriptional mechanisms important for promoting osteogenic cell fate specification, which could ultimately provide new therapeutic targets in disease states with bone loss or ineffective bone formation.

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for unipolar or multipolar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by two-component reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We investigate the behavior of a simple class of models and show that while the timescale of competition varies enormously depending on model parameters, a single factor explains a large majority of this variation. The dominant factor concerns the degree to which the maximal active GTPase concentration in a cluster approaches a “saturation point” determined by model parameters. We further show that the Rho-GTPase polarity machinery in the budding yeast S. cerevisiae, which normally generates only one bud through competition, can be manipulated to generate multiple buds

in ways consistent with this theoretical framework. We suggest that both saturation and the effect of saturation on competition reflect fundamental properties of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism, which predict whether the system will generate unipolar or multipolar outcomes.