Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge.

BACKGROUND: During infectious diseases, proinflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung, the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG (erythroblast transformation-specific-related gene) as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG were analyzed in cultured HUVECs (human umbilical vein ECs). Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5[PAC]-CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or lipopolysaccharide resulted in a rapid and substantial degradation of ERG within lung ECs but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek-a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. CONCLUSIONS: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge.

Background: During infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. Methods: Cytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNFα or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5(PAC)Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. Results: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek , a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. Conclusions: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

Formation of the glomerular microvasculature is regulated by VEGFR-3.

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.

Genetic Deletion of Emp2 Does Not Cause Proteinuric Kidney Disease in Mice.

Nephrotic syndrome is one of the most common glomerular diseases in children and can be classified on the basis of steroid responsiveness. While multiple genetic causes have been discovered for steroid resistant nephrotic syndrome, the genetics of steroid sensitive nephrotic syndrome remains elusive. Mutations in Epithelial Membrane Protein 2 (EMP2), a member of the GAS3/PMP22 tetraspan family of proteins, were recently implicated as putative monogenic cause of steroid sensitive nephrotic syndrome. We investigated this hypothesis by developing Emp2 reporter and knockout mouse models. In lacZ reporter mice (engineered to drive expression of the enzyme ß-galactosidase under the control of the endogenous murine Emp2 promoter), Emp2 promoter activity was not observed in podocytes but was particularly prominent in medium- and large-caliber arterial vessels in the kidney and other tissues where it localizes specifically in vascular smooth muscle cells (vSMCs) but not in the endothelium. Strong Emp2 expression was also found in non-vascular smooth muscle cells found in other organs like the stomach, bladder, and uterus. Global and podocyte-specific Emp2 knockout mice were viable and did not develop nephrotic syndrome showing no evidence of abnormal glomerular histology or ultrastructure. Altogether, our results do not support that loss of function of EMP2 represent a monogenic cause of proteinuric kidney disease. However, the expression pattern of Emp2 indicates that it may be relevant in smooth muscle function in various organs and tissues including the vasculature.

Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium.

To define the role of ERK1/2 signaling in the quiescent endothelium, we induced endothelial Erk2 knockout in adult Erk1-/- mice. This resulted in a rapid onset of hypertension, a decrease in eNOS expression, and an increase in endothelin-1 plasma levels, with all mice dying within 5 wk. Immunostaining and endothelial fate mapping showed a robust increase in TGFß signaling leading to widespread endothelial-to-mesenchymal transition (EndMT). Fibrosis affecting the cardiac conduction system was responsible for the universal lethality in these mice. Other findings included renal endotheliosis, loss of fenestrated endothelia in endocrine organs, and hemorrhages. An ensemble computational intelligence strategy, comprising deep learning and probabilistic programing of RNA-seq data, causally linked the loss of ERK1/2 in HUVECs in vitro to activation of TGFß signaling, EndMT, suppression of eNOS, and induction of endothelin-1 expression. All in silico predictions were verified in vitro and in vivo. In summary, these data establish the key role played by ERK1/2 signaling in the maintenance of vascular normalcy.

Glomerular endothelial cell maturation depends on ADAM10, a key regulator of Notch signaling.

The principal function of glomeruli is to filter blood through a highly specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial a disintegrin and metalloprotease 10 (ADAM10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.

Ascending Vasa Recta Are Angiopoietin/Tie2-Dependent Lymphatic-Like Vessels.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

Glomerular mesangial cell recruitment and function require the co-receptor neuropilin-1.

Proteinuria has been reported in cancer patients receiving agents that target the transmembrane receptor neuropilin-1 (Nrp1) suggesting potential adverse effects on glomerular function. Here we show that Nrp1 is highly expressed by mesangial cells and that genetic deletion of the Nrp1 gene from PDGF receptor-ß+ mesangial cells results in proteinuric disease and glomerulosclerosis, leading to renal failure and death within 6 wk of age in mice. The major defect is a failure of mesangial cell migration that is required to establish the mature glomerular tuft. In vitro data show that the potent chemotactic effect of PDGFB is lost in Nrp1-deficient mesangial cells. Biochemical analyses reveal that Nrp1 is required for PDGFB-dependent phosphorylation of p130 Crk-associated substrate (p130Cas), a large-scaffold molecule that is involved in motility of other cell types. In stark contrast, matrix adhesion and activation of ERK and Akt, which mediate proliferation of mesangial cells in response to PDGFB, are unaffected by the absence of Nrp1. Taken together, these results identify a critical cell-autonomous role for Nrp1 in the migratory behavior of mesangial cells and may help explain the renal effects that occur in patients receiving Nrp1-inhibitory drugs.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

Review series: The cell biology of renal filtration.

The function of the kidney, filtering blood and concentrating metabolic waste into urine, takes place in an intricate and functionally elegant structure called the renal glomerulus. Normal glomerular function retains circulating cells and valuable macromolecular components of plasma in blood, resulting in urine with just trace amounts of proteins. Endothelial cells of glomerular capillaries, the podocytes wrapped around them, and the fused extracellular matrix these cells form altogether comprise the glomerular filtration barrier, a dynamic and highly selective filter that sieves on the basis of molecular size and electrical charge. Current understanding of the structural organization and the cellular and molecular basis of renal filtration draws from studies of human glomerular diseases and animal models of glomerular dysfunction.

Loss of the podocyte-expressed transcription factor Tcf21/Pod1 results in podocyte differentiation defects and FSGS.

Podocytes are terminally differentiated cells with an elaborate cytoskeleton and are critical components of the glomerular barrier. We identified a bHLH transcription factor, Tcf21, that is highly expressed in developing and mature podocytes. Because conventional Tcf21 knockout mice die in the perinatal period with major cardiopulmonary defects, we generated a conditional Tcf21 knockout mouse to explore the role of this transcription factor in podocytes in vivo. Tcf21 was deleted from podocytes and podocyte progenitors using podocin-cre (podTcf21) and wnt4-cre (wnt4creTcf21) driver strains, respectively. Loss of Tcf21 from capillary-loop stage podocytes (podTcf21) results in simplified glomeruli with a decreased number of endothelial and mesangial cells. By 5 weeks of age, 40% of podTcf21 mice develop massive proteinuria and lesions similar to FSGS. Notably, the remaining 60% of mice do not develop proteinuria even when aged to 8 months. By contrast, earlier deletion of Tcf21 from podocyte precursors (wnt4creTcf21) results in a profound developmental arrest of podocyte differentiation and renal failure in 100% of mice during the perinatal period. Taken together, our results demonstrate a critical role for Tcf21 in the differentiation and maintenance of podocytes. Identification of direct targets of this transcription factor may provide new therapeutic avenues for proteinuric renal disease, including FSGS.

Yap- and Cdc42-dependent nephrogenesis and morphogenesis during mouse kidney development.

Yap is a transcriptional co-activator that regulates cell proliferation and apoptosis downstream of the Hippo kinase pathway. We investigated Yap function during mouse kidney development using a conditional knockout strategy that specifically inactivated Yap within the nephrogenic lineage. We found that Yap is essential for nephron induction and morphogenesis, surprisingly, in a manner independent of regulation of cell proliferation and apoptosis. We used microarray analysis to identify a suite of novel Yap-dependent genes that function during nephron formation and have been implicated in morphogenesis. Previous in vitro studies have indicated that Yap can respond to mechanical stresses in cultured cells downstream of the small GTPases RhoA. We find that tissue-specific inactivation of the Rho GTPase Cdc42 causes a severe defect in nephrogenesis that strikingly phenocopies loss of Yap. Ablation of Cdc42 decreases nuclear localization of Yap, leading to a reduction of Yap-dependent gene expression. We propose that Yap responds to Cdc42-dependent signals in nephron progenitor cells to activate a genetic program required to shape the functioning nephron.

Podocyte-specific loss of Cdc42 leads to congenital nephropathy.

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.

Cell migration by a FRS2-adaptor dependent membrane relocation of ret receptors.

During development neural progenitor cells migrate with extraordinary precision to inhabit tissues and organs far from their initial position. Little is known about the cellular basis for directional guidance by tyrosine kinase receptors (RTKs). RET is a RTK with important functions in guiding the migration of neuronal cells, and RET dysregulation leads to clinical disease such as agangliosis of the colon. We show here that RET migration in neuroepitheliomal and non-neuronal cells is elicited by the activation of specific signaling pathways initiated by the competitive recruitment of the FRS2 adaptor molecule to tyrosine 1062 (Y1062) in RET. FRS2 selectively recruited RET to focal complexes and led to activation of SRC family kinases and focal adhesion kinase (FAK). Activation of SRC depended on its direct interaction with RET at a different intracellular tyrosine (Y981) and activation of molecular signaling from these two separate sites in concert regulated migration. Our data suggest that an important function for FRS2 is to concentrate RET in membrane foci, leading to an engagement of specific signaling complexes localized in these membrane domains.

Structure and chemical inhibition of the RET tyrosine kinase domain.

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.

Engineering the recruitment of phosphotyrosine binding domain-containing adaptor proteins reveals distinct roles for RET receptor-mediated cell survival.

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.

Distinct turnover of alternatively spliced isoforms of the RET kinase receptor mediated by differential recruitment of the Cbl ubiquitin ligase.

Alternative splicing of transcripts encoding the RET kinase receptor leads to isoforms differing in their cytoplasmic tail. Although in vitro studies have demonstrated a higher transforming activity of the long RET isoform (RET51), only the short isoform (RET9) can rescue the effects of a RET null mutation in the enteric nervous system and kidney development. The molecular basis underlying the distinct functions of the two RET isoforms is not understood. Here we demonstrated that activated RET51 associated more strongly with the ubiquitin ligase Cbl than did RET9, leading to increased ubiquitylation and faster turnover of RET51. The association of Cbl with RET was indirect and was mediated through Grb2. A constitutive complex of Grb2 and Cbl could be recruited to both receptor isoforms via docking of Shc to phosphorylated Tyr-1062 in RET. A mutant Shc protein unable to recruit the Grb2.Cbl complex decreased the turnover and prolonged the half-life of RET9, thus ascribing a previously unknown negative role to the Shc adaptor molecule. In addition, phosphorylation of Tyr-1096, which is present in RET51 but absent in RET9, endowed the longer isoform with a second route to recruit the Grb2.Cbl complex. These findings establish a mechanism for the differential down-regulation of RET9 and RET51 signaling that could explain the apparently paradoxical activities of these two RET isoforms. More generally, these results illustrate how alternative splicing can regulate the half-life and function of a growth factor receptor.