Shannon J. Odelberg - US grants

DESCRIPTION (provided by applicant): Newts have a remarkable capacity to regenerate several anatomical structures and organs including their limbs, spinal cords, heart, tails, eye lenses, retinas, and upper and lower jaws. These regenerative processes are dependent upon the dedifferentiation of fully-differentiated cells in the vicinity of the amputation site. This degree of cellular plasticity is unique to organisms with marked regenerative capabilities and is not observed in mammals. However, our laboratory has recently demonstrated that terminally- differentiated mouse myotubes can be induced to dedifferentiate when stimulated with a protein extract from regenerating newt limbs. These results indicate that the signaling pathways for cellular dedifferentiation are intact in extracellular signals that initiate dedifferentiation. The goal of this proposal is to identify the newt genes that initiate dedifferentiation and control cellular plasticity in mammalian cells. Candidate cellular plasticity genes will be identified by performing differential display analysis and suppression subtractive cDNA hybridization between early limb regenerates and non regenerating limb tissues. Sequence analysis, degree of induction, and cellular expression patterns will be used as a basis for selecting candidate genes for further study. Genes that exhibit a significant induction and contain sequences suggesting they encode secreted proteins such as growth factors, cytokines or other ligands will be tested for their ability to initiate cellular dedifferentiation by treating cultured mouse myotubes with conditioned medium from cells expressing these candidate genes. Genes expressed in the underlying stump tissue that contain sequences suggesting they encode cellular protein such as receptors, kinases or transcription factors could be genes that respond to the ectopically expressing them in mouse myotubes. Using these methods, we expect to identify newt genes that function in regulating cellular plasticity in mammalian cells. Identifying these genes would have important implication for the future of regenerative medicine.

PROJECT SUMMARY/ABSTRACT Sepsis is a catastrophic systemic inflammatory host response to infection, which can lead to vascular leak, edema, organ failure, and death. Despite intense study, few therapeutic strategies other than nonspecific supportive care have been developed and death rates remain as high as 60-70% in cases of septic shock. More than 750,000 Americans contract sepsis each year and more of these patients die than those that succumb to breast cancer, prostate cancer, and AIDS combined. It is known that agonists found in septic patients, such as inflammatory cytokines, VEGF, thrombin, microparticles, bacterial toxins, and bacteria themselves induce the vascular instability and edema that help trigger septic pathophysiology. Our preliminary data suggest that the direct and immediate effects of these endothelial-disrupting agents may be mediated by diverse receptors that signal via a common convergence point, the intracellular GTPase ARF6. ARF6 appears to control trafficking of cell-cell junction proteins and is distinct from the canonical inflammatory pathways that regulate transcription (e.g., those activating NF-?B). In animal models of inflammatory disease, inhibiting the activation of ARF6 either through conditional genetic ablation or chemical inhibition stabilizes the vasculature, decreases inflammation, and increases survival rates. Therefore, we hypothesize that activation of ARF6 during sepsis induces pathologic vascular leak, which contributes to multi-organ failure and death and that pharmacologic inhibition or genetic ablation of ARF6 or its activating ARF-GEFs will stabilize human and mouse endothelium exposed to septic insults and increase survival rates in animal models of sepsis. We realize that the septic response in mice may not completely mimic the human response. Therefore, we will assess the similarities and differences between the species in regards to ARF-GEF?ARF6 pathway and its control of vascular integrity. In Aim 1, we will determine whether ARF6 represents a convergence point for regulating vascular permeability induced by agonists generated in the septic milieu. We will use defined agonists that are present in the plasma of sepsis patients to determine whether these agonists signal through ARF6 or other ARF family members to induce paracellular permeability of both human and mouse endothelium. We will identify the ARF-GEFs and adaptor proteins involved in these signaling processes. In Aim 2, we will individually ablate Arf6 and Arno (a known ARF6-GEF) in mice and use chemical inhibition of ARF6 in several animal models of sepsis to determine whether removal or inhibition of ARF6 activity stabilizes the vasculature and increases survival rates. To more closely mimic clinical situations, we will also use ARF6 inhibition as an adjuvant to antibiotics to assess whether combination therapy can reduce vascular leak and mortality rates in these animal models. In Aim 3, we examine the efficacy of ARF6 inhibition in in vitro human models of sepsis by assessing endothelial integrity following ARF6 blockade and exposure to plasma or microparticles from septic patients. In vitro whole blood models of sepsis will also be used to assess ARF6 function.

DESCRIPTION (provided by applicant): Inflammatory and autoimmune diseases have a major impact on health care in the United States. For example, rheumatoid arthritis (RA) affects approximately 1.3 million American adults. In its progressive form the disease has debilitating effects including painful inflammation and destruction of the joints of the fingers, wrists, knees, hips, and vertebral column and leads to reduced life expectancy. Vascular instability and angiogenesis are hallmarks of RA and other inflammatory diseases. Indeed, a breakdown of the vascular endothelium may be the critical first step in the pathogenesis of many autoimmue diseases including RA. Arthritis-inducing cytokines, such as TNF-? and IL-1ß, signal through a pathway that activates NF-?B and increases the transcription of target genes that fight infection. Many RA patients are treated with biologic agents that inhibit this pathway but by doing so, leave the patient immunocompromised. We have recently identified a molecular pathway that is activated by IL-1ß but diverges from the canonical NF-?B-pathway at the level of the IL-1R adapter protein MYD88. MYD88 binds to ARNO, an ARF-GEF that activates the small GTPase ARF6. Active ARF6 disrupts adherens junctions by reducing the levels of cell surface VE-cadherin, which leads to vascular destabilization and increased permeability. We have shown that blocking the activity of ARF6 by inhibiting its activation with the ARF6 small molecule inhibitor SecinH3 reduces both the progression of arthritis and acute inflammation in standard in vivo mouse models of human disease without inhibiting NF-?B activation and rendering the mouse immunocompromised. We hypothesize that the activation of ARF6 is common to all RA-inducing inflammatory pathways and that inhibiting ARF6 activation would reduce vascular permeability and its debilitating sequelae without affecting the beneficial immunomodulatory effects arising from NF-?B activation. If true, we would expect that mice harboring endothelial-specific deficiencies in Arf6 or other members of this divergent pathway would exhibit marked resiliency in mouse models of arthritis and acute inflammation but would not be immunosuppressed. We will test our hypothesis by pursuing the following three aims. In Aim 1, we will examine the roles of ARF6 and NF-?B in controlling endothelial barrier function following TNF-? receptor (TNFR) and toll-like receptor (TLR) stimulation. In Aim 2, we will examine whether TNFR- and TLR-dependent inflammatory pathways induce endothelial permeability by activating ARF-GEFs and disrupting adherens junctions. In Aim 3, we will determine whether endothelial expression of ARF6 is required for arthritic progression and acute inflammation in mouse models of these diseases. The successful completion of these aims will allow us to assess whether this divergent pathway controls cytokine-induced vascular permeability and arthritic progression in mouse disease models and would indicate whether the pursuit of therapeutic strategies to inhibit this pathway might be a useful approach for treating rheumatoid arthritis and other related inflammatory diseases.

? DESCRIPTION (provided by applicant): Activating mutations in two G??proteins of the q class (G?q), GNAQ and GNA11, are the drivers of oncogenesis in approximately 80% of uveal melanomas. Similar activating mutations of GNAQ are also found in patients with Sturge-Weber syndrome, which can manifest itself as glaucoma and vascular malformations of the conjunctiva, choroid, retina, and episclera. Uveal melanoma is the most common primary ocular tumor, and in approximately 50% of the cases, the tumor will metastasize to other organs, primarily the liver. Once metastasized, the disease is invariably fatal. Activating GNAQ and GNA11 mutations drive uveal melanoma oncogenesis via the control of several recently identified signaling pathways, including phospholipase C- ?/protein kinase C (PLC-?/PKC) and TRIO-RhoA/Rac1 pathways, which activate MAPK/ERK and YAP to induce AP1- and YAP-TEAD-mediated transcription. However, the mechanism(s) by which G?q proteins activate multiple downstream pathways has not been completely elucidated. Preliminary data suggest that the small GTPase ARF6 may act as an immediate downstream effector of activated GNAQ/GNA11 to control all of the currently known oncogenic G?q signaling pathways. Other preliminary data also suggest that G?q may signal through ARF6 to activate ?-catenin signaling by promoting the relocalization of ?-catenin from the cell membrane to the nucleus where it can mediate gene transcription. Preliminary data also support the in vivo role of ARF6 in uveal melanoma. When ARF6 is silenced in uveal melanoma by shRNA or is inhibited with a small molecule inhibitor, tumor establishment and growth is significantly inhibited in an orthotopic xenograft mouse model. Based on these preliminary data, the following aims will be pursued. In Aim 1, we will investigate whether activated G?q proteins induce ?-catenin signaling via activation of ARF6 in uveal melanoma. We will employ gene silencing via RNA interference and small molecule inhibition of selected targets to assess intracellular localization, transcriptional activity, and function of ?-catenin in uveal melanoma. In Aim 2, we will elucidate the role of ARF6 in orchestrating known downstream signaling pathways of oncogenic G?q. The same strategies used in Aim 1 will be employed to determine whether ARF6 acts as an immediate downstream effector of activating GNAQ/GNA11 mutations to control (PLC-?/PKC) and TRIORhoA/ Rac1 pathways and AP-1 and YAP-TEAD-mediated transcription. In Aim 3, we will assess the in vivo function of ARF6 in tumor establishment and growth by using orthotopic xenograft models of human uveal melanoma. The successful completion of these aims will allow us to determine whether ARF6 plays a critical role in G?q signaling, thus providing a promising therapeutic target for the development of drugs that could be used to treat uveal melanoma and possibly other G?q-related disorders such as Sturge-Weber syndrome.