The D2D3 form of uPAR acts as an immunotoxin and may cause diabetes and kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic ß cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored ß cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired ß cell proliferation and inhibited the bioenergetics of ß cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.

Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation.

Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.

Anks1a regulates COPII-mediated anterograde transport of receptor tyrosine kinases critical for tumorigenesis.

ErbB2 signalling, which is amplified by EphA2 binding, is an important therapeutic target for breast cancer. Despite the importance of the EphA2/ErbB2 complex in promoting breast tumorigenesis, the mechanism by which these receptor tyrosine kinases (RTKs) are exported from the endoplasmic reticulum (ER) remains poorly understood. Here we report that the PTB adaptor Anks1a is specifically localized to the ER on its own serine phosphorylation. Once there, Anks1a acts as an important regulator of COPII-mediated EphA2 ER export. The Anks1a ankyrin repeat domain binds EphA2 and causes it to accumulate at sites of ER exit. Simultaneously, the Anks1a PTB domain binds Sec23. This induces internalization of EphA2 via COPII vesicles, while Anks1a remains behind on the ER membrane. EphA2 also binds ErbB2 in the ER and seems to load ErbB2 into growing COPII carriers. Together, our study reveals a novel mechanism that regulates the loading of RTKs into COPII vesicles.

Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.

Transient receptor potential channel 6 (TRPC6) protects podocytes during complement-mediated glomerular disease.

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.

Dynamin rings: not just for fission.

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin-mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin-coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin-binding and -regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell.

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival.

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-ß1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-ß1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-ß response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

Direct dynamin-actin interactions regulate the actin cytoskeleton.

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

Transient activation of the MAP kinase signaling pathway by the forward signaling of EphA4 in PC12 cells.

In the present study, we demonstrate that ephrin-A5 is able to induce a transient increase of MAP kinase activity in PC12 cells. However, the effects of ephrin-A5 on the MAP kinase signaling pathway are about three-fold less than that of EGF. In addition, we demonstrate that EphA4 is the only Eph member expressed in PC12 cells, and that tyrosine phosphorylation induced by ephrin-A5 treatment is consistent with the magnitude and longevity of MAP kinase activation. Experiments using the Ras dominant negative mutant N17Ras reveal that Ras plays a pivotal role in ephrin-A5-induced MAP kinase activation in PC12 cells. Importantly, we found that the EphA4 receptor is rapidly internalized by endocytosis upon engagement of ephrin-A5, leading to a subsequent reduction in the MAP kinase activation. Together, these data suggest a novel regulatory mechanism of differential Ras-MAP kinase signaling kinetics exhibited by the forward signaling of EphA4 in PC12 cells.

Identification of phosphotyrosine binding domain-containing proteins as novel downstream targets of the EphA8 signaling function.

Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway.

The EphA8 receptor induces sustained MAP kinase activation to promote neurite outgrowth in neuronal cells.

Recent studies in our laboratory demonstrate that ligand-mediated activation of the EphA8 receptor critically regulates cell adhesion and migration. In this report, we show that the EphA8 receptor induces neurite outgrowth in NG108-15 cells in the absence of ligand stimulation. Using various deletion mutants lacking specific intracytoplasmic regions, we confirm that the tyrosine kinase domain of EphA8 is important for inducing neurite outgrowth. However, the tyrosine kinase activity of EphA8 is not crucial for neurite outgrowth induction. Treatment with various inhibitors further reveals that the mitogen-activated protein kinase (MAPK) signaling pathway is critical for neurite outgrowth induced by EphA8. Consistent with these results, EphA8 expression induced a sustained increase in the activity of MAPK, whereas ligand-mediated EphA8 activation had no further modulatory effects on MAP kinase activity. Additionally, activated MAPK relocalized from the cytoplasm to the nucleus in response to EphA8 transfection. These results collectively suggest that the EphA8 receptor is capable of inducing a sustained increase in MAPK activity, thereby promoting neurite outgrowth in neuronal cells.

The p110 gamma PI-3 kinase is required for EphA8-stimulated cell migration.

This study provides evidence that treatment with preclustered ephrin A5-Fc results in a substantial increase in the stability of the p110 gamma PI-3 kinase associated with EphA8, thereby enhancing PI-3 kinase activity and cell migration on a fibronectin substrate. In contrast, co-expression of a lipid kinase-inactive p110 gamma mutant together with EphA8 inhibits ligand-stimulated PI-3 kinase activity and cell migration on a fibronectin substrate, suggesting that the mutant has a dominant negative effect against the endogenous p110 gamma PI-3 kinase. Significantly, the tyrosine kinase activity of EphA8 is not important for either of these processes. Taken together, our results demonstrate that the stimulation of cell migration on a fibronectin substrate by the EphA8 receptor depends on the p110 gamma PI-3 kinase but is independent of a tyrosine kinase activity.

Identification of an enhancer region in the mouse ephA8 locus directing expression to the anterior region of the dorsal mesencephalon.

Eph receptors and ephrins are dynamically expressed in a wide range of regions of the vertebrate during embryogenesis. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. It is of considerable interest to elucidate how these expression domains are established in the development of the mesencephalon. In this study, we used a transgenic approach to define the cis-acting DNA regulatory elements involved in the anterior mesencephalon-specific expression of the mouse ephA8 gene. Our analyses of the temporal and spatial expression patterns of various ephA8/lacZ gene fusions in transgenic mice revealed that the 10-kb genomic DNA 5' immediately upstream of the ephA8 coding sequence is capable of directing lacZ expression in an ephA8-specific manner. Further deletion analyses of the ephA8 genomic region led to the identification of a 1-kb enhancer region, which directs expression in the embryo to the anterior region of the developing midbrain. This ephA8-specific regulatory DNA sequences can now be used in biochemical analyses to identify proteins modulating the anterior differentiation of the optic tectum, and in functional analyses to direct the expression of other developmentally important genes to this region.