Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve.

Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20-/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.

Identification of a peripheral blood gene signature predicting aortic valve calcification.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.

Disruption of CXCR4 signaling in pharyngeal neural crest cells causes DiGeorge syndrome-like malformations.

DiGeorge syndrome (DGS) is a congenital disease causing cardiac outflow tract anomalies, craniofacial dysmorphogenesis, thymus hypoplasia, and mental disorders. It results from defective development of neural crest cells (NCs) that colonize the pharyngeal arches and contribute to lower jaw, neck and heart tissues. Although TBX1 has been identified as the main gene accounting for the defects observed in human patients and mouse models, the molecular mechanisms underlying DGS etiology are poorly identified. The recent demonstrations that the SDF1/CXCR4 axis is implicated in NC chemotactic guidance and impaired in cortical interneurons of mouse DGS models prompted us to search for genetic interactions between Tbx1, Sdf1 (Cxcl12) and Cxcr4 in pharyngeal NCs and to investigate the effect of altering CXCR4 signaling on the ontogeny of their derivatives, which are affected in DGS. Here, we provide evidence that Cxcr4 and Sdf1 are genetically downstream of Tbx1 during pharyngeal NC development and that reduction of CXCR4 signaling causes misrouting of pharyngeal NCs in chick and dramatic morphological alterations in the mandibular skeleton, thymus and cranial sensory ganglia. Our results therefore support the possibility of a pivotal role for the SDF1/CXCR4 axis in DGS etiology.

The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/ß-catenin transcription pathway.

The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the ß-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.

FOXC1 haploinsufficiency due to 6p25 deletion in a patient with rapidly progressing aortic valve disease.

6p25 deletion is a rare but well-known entity. The main clinical features include an abnormal facial appearance, developmental delay, and ocular anomalies. Cardiac anomalies are frequently seen but remain poorly delineated. We describe a 4-year-old girl with 6p25.3 deletion, which includes the FOXC1 gene, typical dysmorphic features associated with developmental delay and oculo-motor anomalies. Aortic valve dysplasia was diagnosed early in life. The cardiac lesion progressed very rapidly between the age of 3 and 4 years requiring aortic valve replacement. Genomic analysis of blood and excised valve tissue showed down-regulation of FOXC1 but also FOXC2 expression in the diseased aortic valve. This allows us to speculate on the potential role of FOXC1 in aortic valve anomalies.

Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages.

BACKGROUND: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. RESULTS: By embryonic day 13.5, all rescued Raldh2(-/-) hearts exhibit severe, reproducible OFT septation defects, although wild-type and Raldh2(+/-) littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2(-/-) mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2(-/-) mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. CONCLUSIONS: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus.

P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

α6ß4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6ß4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6ß4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6ß4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.

Variations in the poly-histidine repeat motif of HOXA1 contribute to bicuspid aortic valve in mouse and zebrafish.

Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5-1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and the identification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1-1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1-/- mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.

Antibacterial polyelectrolyte micelles for coating stainless steel.

In this study, we report on the original synthesis and characterization of novel antimicrobial coatings for stainless steel by alternating the deposition of aqueous solutions of positively charged polyelectrolyte micelles doped with silver-based nanoparticles with a polyanion. The micelles are formed by electrostatic interaction between two oppositely charged polymers: a polycation bearing 3,4-dihydroxyphenylalanine units (DOPA, a major component of natural adhesives) and a polyanion (poly(styrene sulfonate), PSS) without using any block copolymer. DOPA units are exploited for their well-known ability to anchor to stainless steel and to form and stabilize biocidal silver nanoparticles (Ag(0)). The chlorine counteranion of the polycation forms and stabilizes biocidal silver chloride nanoparticles (AgCl). We demonstrate that two layers of micelles (alternated by PSS) doped with silver particles are enough to impart to the surface strong antibacterial activity against gram-negative E. coli. Moreover, micelles that are reservoirs of biocidal Ag(+) can be easily reactivated after depletion. This novel water-based approach is convenient, simple, and attractive for industrial applications.

Clay and DOPA containing polyelectrolyte multilayer film for imparting anticorrosion properties to galvanized steel.

A facile and green approach is developed to impart remarkable protection against corrosion to galvanized steel. A protecting multilayer film is formed by alternating the deposition of a polycation bearing catechol groups, used as corrosion inhibitors, with clay that induces barrier properties. This coating does not affect the esthetical aspect of the surface and does not release any toxic molecules in the environment.

Contribution of anatomical criteria to surgical approach for treatment of bilateral coronoid hyperplasia.

INTRODUCTION: Bilateral coronoid hyperplasia is a rare condition characterized by a progressive and painless limitation of mouth opening. The treatment consists of coronoidectomy by intraoral or coronal approach. There is no recommendation in the literature on the choice of the surgical approach according to the importance of the hypertrophy. The objective of our study is to search for predictive anatomical criteria of each approach. MATERIAL AND METHODS: These anatomical criteria were evaluated from 3D CT facial reconstructions of 4 male patients aged 4 to 30 years retrospectively after their surgery. A single intraoral approach was used for cases 1 and 2 (group A), a double approach for cases 3 and 4 (group B). Same measurements were performed on 10 male case controls (group C). We performed a descriptive analysis of our results due to an insufficient number of patients. RESULTS: The average width of the coronal processes was much greater in group B, when a double approach was necessary, than in groups A and C. In groups A and B, the average height of the coronoid notch is low compared to group C when there is a need for a coronal approach. The width/TZS ratio is increased in group B compared to group C. DISCUSSION: In our study, 3 criteria could be informative for the choice of the surgical approach: the width at the superior end of the coronoid process, the width/TZS ratio and the height of the coronoid notch but additional data are needed to confirm our therapeutic options.

Side-dependent effect in the response of valve endothelial cells to bidirectional shear stress.

Endothelial cells covering the aortic and ventricular sides of the aortic valve leaflets are exposed to different stresses, in particular wall shear stress (WSS). Biomechanical stimuli actively regulate valve tissue structure and induce remodeling events leading to valve dysfunction. Endothelial to mesenchymal transformation (EndMT), for example, has been associated with aortic valve disease. The biomechanical response of cells at different sides of the leaflets has not been clearly characterized. To analyze the mechanical response of valve endothelial cells (VECs) we developed a unique fluid activation device that applies physiologically relevant pulsatile WSS. We characterized the morphology and function of adult porcine aortic VECs derived from the opposite sides of aortic valve leaflets following exposure to different pulsatile WSS. We found that elongation and orientation of cells in response to pulsatile WSS depends on their side of origin. Quantification of gene expression confirms phenotypic differences between aortic and ventricular VECs. Aortic VECs exposed to pulsatile WSS similar to that in vivo at the tip of aortic side of the valve leaflet upregulated pro-EndMT (ACTA2, Snail, TGFß1) and inflammation (ICAM-1, VCAM-1) genes, whereas expression of endothelial markers like PECAM-1 was decreased. Conversely, ventricular-VECs showed strong increase of PECAM-1 expression and no activation of pro-EndMT marker. Finally, we found that stress-induced genes are upregulated in both cell types, at higher levels in ventricular compared to aortic VECs. Application of physiological shear stress levels using a fluid activation device therefore reveals functional differences in VECs derived from opposite sides of the aortic valve leaflets.

Mandibular osteonecrosis following herpes zoster infection: Report of a rare case with a literature review.

Any patient with a herpes zoster infection of the mandibular branch of the trigeminal nerve should benefit from early oral monitoring, especially in elderly population where traumatic dental prostheses are common, because osteonecrosis can occur.

Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease.

Among the aortic valve diseases, the bicuspid aortic valve (BAV) occurs when the aortic valve has two leaflets (cusps), rather than three, and represents the most common form of congenital cardiac malformation, affecting 1-2% of the population. Despite recent advances, the etiology of BAV is poorly understood. We have recently shown that Krox20 is expressed in endothelial and cardiac neural crest derivatives that normally contribute to aortic valve development and that lack of Krox20 in these cells leads to aortic valve defects including partially penetrant BAV formation. Dysregulated expression of endothelial nitric oxide synthase (Nos3) is associated with BAV. To investigate the relationship between Krox20 and Nos3 during aortic valve development, we performed inter-genetic cross. While single heterozygous mice had normal valve formation, the compound Krox20+/-;Nos3+/- mice had BAV malformations displaying an in vivo genetic interaction between these genes for normal valve morphogenesis. Moreover, in vivo and in vitro experiments demonstrate that Krox20 directly binds to Nos3 proximal promoter to activate its expression. Our data suggests that Krox20 is a regulator of nitric oxide in endothelial-derived cells in the development of the aortic valve and concludes on the interaction of Krox20 and Nos3 in BAV formation.

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.

Reduced aggrecan expression affects cardiac outflow tract development in zebrafish and is associated with bicuspid aortic valve disease in humans.

Hemodynamic forces have been known for a long time to regulate cardiogenic processes such as cardiac valve development. During embryonic development in vertebrates, the outflow tract (OFT) adjacent to the ventricle comes under increasing hemodynamic load as cardiogenesis proceeds. Consequently, extracellular matrix components are produced in this region as the cardiac cushions form which will eventually give rise to the aortic valves. The proteoglycan AGGRECAN is a key component of the aortic valves and is frequently found to be deregulated in a variety of aortic valve diseases. Here we demonstrate that aggrecan expression in the OFT of developing zebrafish embryos is hemodynamically dependent, a process presumably mediated by mechanosensitive channels. Furthermore, knockdown or knockout of aggrecan leads to failure of the OFT to develop resulting in stenosis. Based on these findings we analysed the expression of AGGRECAN in human bicuspid aortic valves (BAV). We found that in type 0 BAV there was a significant reduction in the expression of AGGRECAN. Our data indicate that aggrecan is required for OFT development and when its expression is reduced this is associated with BAV in humans.

Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes.

BACKGROUND: The mechanism involved in the onset of aortic valve (AoV) disease remains unclear despite its poor prognosis and frequency. Recently, we reported that Krox20 (EGR2 in humans) is involved in AoV development and dysfunction. AIM: Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases. METHODS: Transcriptional levels of Col1a2/COL1A2 and Krox20/EGR2 in AoVs from Krox20(+/-) mice and human patients operated on for severe aortic regurgitation were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Human control valves were obtained from three transplanted patients without AoV disease. Twenty-one heterozygous Krox20(+/-) mice were compared with 35 controls at different ages. Three independent measurements of valve thickness were performed on magnified tissue sections using Image J software. In vivo valve structure and function were evaluated using the high-frequency Vevo(®) 2100 echocardiogram. RESULTS: qRT-PCR analysis using AoVs from patients with severe aortic regurgitation showed a decrease in EGR2 expression associated with significant downregulation of COL1A2 expression (P<0.05). Similar results were observed in the AoVs of Krox20(+/-) mice. Anatomical examination revealed that incomplete invalidation of Krox20 caused significant thickening of the aortic leaflet compared with controls (145±22 vs. 75±24µm; P=0.01). Within the mutant group, this thickening worsened significantly over time (Krox20(+/-) mice aged>7 vs.<7months: 136±48 vs. 102±41µm; P<0.001). Moreover, the aortic leaflets of embryonic day 18.5 Krox20(+/-) embryos were significantly more thickened than those from controls, suggesting that this disease begins during embryonic development. Echo-Doppler analysis showed a significant increase in AoV dysfunction in heterozygous versus control mice (53% vs. 17%; P<0.001), suggesting a tight relationship between valve architecture and function. Morphometric analysis revealed that the most severe AoV dysfunction was always associated with the most thickened valves. Classic histological analysis revealed that mutant AoVs had extracellular matrix disorganization, with features of human myxomatous degeneration, including excess of proteoglycan deposition in spongiosa and reduction of collagen fibre in fibrosa, but no calcification. CONCLUSION: Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.

Loss of Krox20 results in aortic valve regurgitation and impaired transcriptional activation of fibrillar collagen genes.

AIMS: Heart valve maturation is achieved by the organization of extracellular matrix (ECM) and the distribution of valvular interstitial cells. However, the factors that regulate matrix components required for valvular structure and function are unknown. Based on the discovery of its specific expression in cardiac valves, we aimed to uncover the role of Krox20 (Egr-2) during valve development and disease. METHODS AND RESULTS: Using series of mouse genetic tools, we demonstrated that loss of function of Krox20 caused significant hyperplasia of the semilunar valves, while atrioventricular valves appeared normal. This defect was associated with an increase in valvular interstitial cell number and ECM volume. Echo Doppler analysis revealed that adult mutant mice had aortic insufficiency. Defective aortic valves (AoVs) in Krox20(-/-) mice had features of human AoV disease, including excess of proteoglycan deposition and reduction of collagen fibres. Furthermore, examination of diseased human AoVs revealed decreased expression of KROX20. To identify downstream targets of Krox20, we examined expression of fibrillar collagens in the AoV leaflets at different stages in the mouse. We found significant down-regulation of Col1a1, Col1a2, and Col3a1 in the semilunar valves of Krox20 mutant mice. Utilizing in vitro and in vivo experiments, we demonstrated that Col1a1 and Col3a1 are direct targets of Krox20 activation in interstitial cells of the AoV. CONCLUSION: This study identifies a previously unknown function of Krox20 during heart valve development. These results indicate that Krox20-mediated activation of fibrillar Col1a1 and Col3a1 genes is crucial to avoid postnatal degeneration of the AoV leaflets.

Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.