VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis.

Ectodomain shedding and regulated intracellular proteolysis can determine the fate or function of cell surface proteins. Fms-related tyrosine kinase (FLT) or VEGF receptor 1 is a high-affinity cell surface VEGF-A receptor tyrosine kinase that is constitutively cleaved to release an NH-terminal VEGF-A binding ectodomain that, once shed, can antagonize the effects of VEGF-A in the extracellular milieu. We evaluated the effect of VEGF-A on FLT1 cleavage in native cells and in transient and stable expression systems.

EGF regulation of proximal tubule cell proliferation and VEGF-A secretion.

Proximal tubule cell (PTC) proliferation is critical for tubular regeneration and recovery from acute kidney injury. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF-A) are important for the maintenance of tubulointerstitial integrity and can stimulate PTC proliferation. We utilized HK-2 cells, an immortalized human PTC line, to characterize the EGF-dependent regulation of VEGF-A secretion and proliferation in PTCs.

Aldosterone regulates a 5' variant sgk1 transcript via a shared hormone response element in the sgk1 5' regulatory region.

We previously identified a 5' variant alternate transcript of Sgk1 (Sgk1_v3) encoding an NH-terminal variant Sgk1 isoform, Sgk1_i3 that, like Sgk1, is expressed in the distal convoluted tubule, connecting tubule and collecting duct and can stimulate epithelial Na transport (Am J Physiol Renal Physiol 303: F1527-F1533, 2012). We now demonstrate that, similar to Sgk1, aldosterone and glucocorticoids stimulate Sgk1_v3 expression in cell lines from the collecting duct and airway epithelia. In mice, short term aldosterone infusion and maneuvers that increase endogenous aldosterone secretion including dietary Na deprivation and K loading increases distal nephron Sgk1_v3 expression in vivo.

Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage.

FLT1 is a cell surface VEGF receptor which is cleaved to release an N-terminal ectodomain which binds VEGF and PlGF and can antagonize the effects of VEGF in the extracellular milieu. To further evaluate FLT1 processing we expressed tagged FLT1 constructs in HEK293 and COS7 cells where we demonstrate, by deletion mapping, that the cleavage site is immediately adjacent to the transmembrane domain (TMD) between residues 759 and 763. Cleavage reciprocally regulates free VEGF in conditioned media and we show that the cleavage site is also transferable to another transmembrane receptor.

N-terminal cleavage and release of the ectodomain of Flt1 is mediated via ADAM10 and ADAM 17 and regulated by VEGFR2 and the Flt1 intracellular domain.

Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1), can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage.

Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist.

FLT1 and its soluble form (sFLT1) arise as alternate transcripts from the same gene and sFLT1 can antagonize the effect of vascular endothelial growth factor (VEGF) on its cognate receptors. We investigated the effect of VEGF and protein kinase C (PKC) activation on sFLT1 abundance. We demonstrated that VEGF stimulates sFLT1 and FLT1 mRNA and protein levels in vascular endothelial cells via VEGFR2 and PKC.

A regulated NH2-terminal Sgk1 variant with enhanced function is expressed in the collecting duct.

Sgk1 is a relatively unstable kinase that regulates epithelial Na(+) transport in the distal nephron of the kidney. We identified a 5' variant alternate transcript of human Sgk1 (Sgk1_v3) that is expressed in the connecting tubule and collecting duct, is regulated by aldosterone and insulin, and is predicted to encode an NH(2)-terminal variant Sgk1 isoform, Sgk1_i3. Sgk1_i3 contains a polybasic motif, KKR, in its NH(2) terminus that regulates ubiquitination and stability of the expressed protein in HEK293 cells.

Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia.

Nedd4-2, a E3 ubiquitin ligase, regulates epithelial sodium channel-mediated transcellular Na(+) transport in the collecting duct. We investigated the effect of Nedd4-2 on the junctional complex and paracellular conductance in mpkCCD(c14) cells, a collecting duct cell line. We demonstrate that Nedd4-2 coimmunoprecipitated with and reduced the expression of transfected occludin in HEK293 cells.

Alternate processing of Flt1 transcripts is directed by conserved cis-elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation.

The vascular endothelial growth factor receptor, Flt1 is a transmembrane receptor co-expressed with an alternate transcript encoding a secreted form, sFlt1, that functions as a competitive inhibitor of Flt1. Despite shared transcription start sites and upstream regulatory elements, sFlt1 is in far greater excess of Flt1 in the human placenta. Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts.

Glycosylphosphatidylinositol-specific phospholipase D improves glucose tolerance.

Insulin regulation of energy metabolism is complex and involves numerous signaling cascades. Insulin has been suggested to stimulate a phospholipase that cleaves glycosylphosphatidylinositols resulting in the generation of an inositol glycan that serves as an insulin mediator. To determine if glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) may play a role in glucose metabolism, we examined the effect of overexpressing GPI-PLD using adenovirus-mediated gene transfer in C57BL/6 mice.

A recently evolved novel trophoblast-enriched secreted form of fms-like tyrosine kinase-1 variant is up-regulated in hypoxia and preeclampsia.

Context: Recent published studies indicate a possible role for sFlt1 in the development of preeclampsia.

Objective: The objective of the study was to investigate the expression and regulation of sFlt1-e15a, a recently described novel C-terminal variant isoform of sFlt1.

Design: The studies included a computational comparative analysis of the genomic locus of sFlt1 across vertebrate species; an assessment of sFlt1 variants in human and rhesus cells and tissues; an analysis of sFlt1 variants transiently expressed in HeLa and COS-7 cells; an evaluation of the effect of hypoxia on sFlt1 expression in trophoblasts; and a comparison of placental sFlt1 expression between pregnancies complicated by preeclampsia and control pregnancies.

An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function.

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2 terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5' variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2 terminus.

Nedd4-2 isoforms ubiquitinate individual epithelial sodium channel subunits and reduce surface expression and function of the epithelial sodium channel.

We previously reported the existence of multiple isoforms of human Nedd4-2 (Am J Physiol Renal Physiol 285: F916-F929, 2003). When overexpressed in M-1 collecting duct epithelia, full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH(2)-terminal C2 domain (Nedd4-2DeltaC2), and Nedd4-2 lacking WW domains 2 and 3 (Nedd4-2DeltaWW2,3) variably reduce benzamil-sensitive Na(+) transport. We investigated the effect of each of the Nedd4-2 isoforms on cell surface expression and ubiquitination of ENaC subunits.

Statin therapy reduces serum levels of glycosylphosphatidylinositol-specific phospholipase D.

Statin therapy is associated with changes in low-density, very low-density, and high- density lipoprotein metabolism. The effect of statin therapy on a minor high-density lipoprotein particle containing glycosylphosphatidylinositol-specific phospholipase D has not been examined. Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) has been implicated in triglyceride metabolism.

Glycosylphosphatidylinositol-specific phospholipase d in nonalcoholic Fatty liver disease: a preliminary study.

Context: Recent studies demonstrated that de novo lipogenesis is increased in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD also have plasma lipid abnormalities. These lipid abnormalities may in part be related to insulin resistance, which is common in patients with NAFLD.

Glycosylphosphatidylinositol-specific phospholipase D influences triglyceride-rich lipoprotein metabolism.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a minor HDL-associated protein. Because many minor HDL-associated proteins exchange between different lipoprotein classes during the postprandial state and are also involved in triglyceride (TG) metabolism, we hypothesized that GPI-PLD may play a role in the metabolism of TG-rich lipoproteins. To test this hypothesis, we examined the distribution of GPI-PLD among lipoprotein classes during a fat tolerance test in C57BL/6 and LDL receptor-deficient (LDLR(-/-)) mice fed either a chow or high-fructose diet.

Adenoviral vectors expressing human endostatin-angiostatin and soluble Tie2: enhanced suppression of tumor growth and antiangiogenic effects in a prostate tumor model.

Angiogenesis is essential for prostate cancer development and metastasis. Antiangiogenic therapy targeting tumor neovasculature, therefore, represents a promising approach for prostate cancer treatment. We hypothesized that adenoviral-mediated delivery of a combination of antiangiogenic factors might have an enhanced antitumor response.

Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity.

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal b-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified.

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism.

GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown.