Earley S, Pauyo T, Drapp R, Tavares MJ, Liedtke W, Brayden JE. TRPV4-dependent dilation of peripheral resistance arteries influences arterial pressure. Am J Physiol Heart Circ Physiol 297: H1096 -H1102, 2009. First published July 17, 2009 doi:10.1152/ajpheart.00241.2009.-Transient receptor potential vanilloid 4 (TRPV4) channels have been implicated as mediators of calcium influx in both endothelial and vascular smooth muscle cells and are potentially important modulators of vascular tone. However, very little is known about the functional roles of TRPV4 in the resistance vasculature or how these channels influence hemodynamic properties. In the present study, we examined arterial vasomotor activity in vitro and recorded blood pressure dynamics in vivo using TRPV4 knockout (KO) mice. Acetylcholine-induced hyperpolarization and vasodilation were reduced by ϳ75% in mesenteric resistance arteries from TRPV4 KO versus wild-type (WT) mice. Furthermore, 11,12-epoxyeicosatrienoic acid (EET), a putative endothelium-derived hyperpolarizing factor, activated a TRPV4-like cation current and hyperpolarized the membrane of vascular smooth muscle cells, resulting in the dilation of mesenteric arteries from WT mice. In contrast, 11,12-EET had no effect on membrane potential, diameter, or ionic currents in the mesenteric arteries from TRPV4 KO mice. A disruption of the endothelium reduced 11,12-EET-induced hyperpolarization and vasodilatation by ϳ50%. A similar inhibition of these responses was observed following the block of endothelial (small and intermediate conductance) or smooth muscle (large conductance) K ϩ channels, suggesting a link between 11,12-EET activity, TRPV4, and K ϩ channels in endothelial and smooth muscle cells. Finally, we found that hypertension induced by the inhibition of nitric oxide synthase was greater in TRPV4 KO compared with WT mice. These results support the conclusion that both endothelial and smooth muscle TRPV4 channels are critically involved in the vasodilation of mesenteric arteries in response to endothelial-derived factors and suggest that in vivo this mechanism opposes the effects of hypertensive stimuli.calcium-activated potassium channels; endothelium; vascular smooth muscle; blood pressure; epoxyeicosatrienoic acid; transient receptor potential vanilloid 4 THE VASCULAR ENDOTHELIUM produces a variety of potent vasoactive factors that are critically important for blood flow and blood pressure regulation. Epoxyeicosatrienoic acids (EETs) are produced by cytochrome P-450 epoxygenase enzymes from arachidonic acid and act as an endothelium-derived hyperpolarizing factor (EDHF) in some vascular beds (3, 24). Vasodilation and smooth muscle cell hyperpolarization in response to EETs are associated with the increased activity of large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels. The vanilloid transient receptor potential channel TRPV4 can be activated by EETs, suggesting that the channel can also serve as a cell-surface receptor for these compounds. We recently demonstrated that in cerebr...

Craniofacial anomalies (CFA) are the most frequent human congenital disease and a major cause of infant mortality and childhood morbidity. Although CFA appear to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathomechanisms for the majority of CFA are currently unknown. Here we reveal an unknown role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient for Wwp2 develop malformations of the craniofacial region. Wwp2 is present in cartilage where its expression is controlled by Sox9. Our studies demonstrate that Wwp2 influences craniofacial patterning through its interactions with Goosecoid (Gsc), a paired-like homeobox transcription factor that plays an important role in craniofacial development. We show that Wwp2 associated Gsc is a transcriptional activator of the key cartilage regulatory protein Sox6. Wwp2 interacts with Gsc to facilitate its mono-ubiquitination, a post-translational modification required for optimal transcriptional activation of Gsc. Our results identify the first physiological pathway regulated by Wwp2 in vivo as well as identify a unique non-proteolytic mechanism through which the Wwp2 controls craniofacial development.

Summary The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an auto-inhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.

Mutations in the coactivator CREB-binding protein (CBP) are a major cause of the human skeletal dysplasia Rubinstein-Taybi syndrome (RTS); however, the mechanism by which these mutations affect skeletal mineralization and patterning is unknown. Here, we report the identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as a key regulator of CBP activity and demonstrate that its functions map to both osteoprogenitor cells and mature osteoblasts. In osteoblasts, PDK1 activated the CREB/CBP complex, which in turn controlled runt-related transcription factor 2 (RUNX2) activation and expression of bone morphogenetic protein 2 (BMP2). These pathways also operated in vivo, as evidenced by recapitulation of RTS spectrum phenotypes with osteoblast-specific Pdk1 deletion in mice (Pdk1 osx mice) and by the genetic interactions observed in mice heterozygous for both osteoblast-specific Pdk1 deletion and either Runx2 or Creb deletion. Finally, treatment of Pdk1 osx and Cbp +/-embryos with BMPs in utero partially reversed their skeletal anomalies at birth. These findings illustrate the in vivo function of the PDK1-AKT-CREB/CBP pathway in bone formation and provide proof of principle for in utero growth factor supplementation as a potential therapy for skeletal dysplasias. IntroductionRubinstein-Taybi syndrome (RTS) was first described in 1963 as a condition characterized by impaired intellectual function, broad thumbs and halluces, craniofacial dysmorphism, and frequent fractures (1, 2). Characteristic craniofacial defects include a short philtrum, micrognathia, a high arched palate, and dental defects such as talon cusps, enamel hypoplasia, and abnormalities in tooth number (3). Genetically, RTS is associated with microdeletions, inversions, and translocations involving chromosome 16p13.3 (4). Mapping of the 16p13.3 region in patients with RTS has identified heterozygous mutations in CREB-binding protein (CBP) that account for approximately 40% of RTS cases (5). Cbp +/-mice show a similar range of defects as those in RTS, including neurodevelopmental and behavioral defects; oligodactyly; extra, split, or asymmetric vertebrae; calvarial hypomineralization; and delayed ossification at several sites (6-9). However, despite these advances in understanding the genetic basis of RTS, the mechanism by which these defects arise is unclear, and we have little insight into which pathways might function upstream and downstream of CBP during development.