RhoGEFs (guanine nucleotide exchange factors), positioned upstream of RhoA, mediate a calcium-independent mechanism of Ca 2+ sensitization involved in the regulation of vascular smooth muscle contraction. We have previously suggested that p63RhoGEF plays a crucial role in regulating vascular tone and, consequently, in controlling blood pressure. Our long-term aim is to identify inhibitors of p63RhoGEF that can suppress unwanted vascular constriction. Conventional preclinical drug testing has primarily employed mouse models, a process that is both costly and time-consuming. In comparison, zebrafish bioassays provide a more economical and readily accessible alternative. Given these advantages, we redirected our investigations to zebrafish for functional studies of p63RhoGEF. To validate the physiological relevance of this approach, we obtained a series of evidence that the zebrafish p63RhoGEF-like protein serves as a functional equivalent of its mammalian counterpart in vitro. For example, we demonstrated that full-length zebrafish p63RhoGEF triggers RhoA activation following agonist stimulation, in a manner comparable to mammalian p63RhoGEF. In this work, we utilized constitutively inactive recombinant RhoA, which irreversibly traps activated RhoGEFs, in a pull-down assay to investigate whether such stimulation activates zebrafish p63RhoGEF and propagates signaling to downstream effectors. Our current investigation aims to evaluate the functional capacity of the zebrafish p63RhoGEF-like protein, to develop a zebrafish model for testing its function and screening p63RhoGEF inhibitors. This endeavor will contribute to the understanding of potential therapies for pathological vascular constriction.
Sakai et al. (Sun,) studied this question.