Rafia S. Al-Lamki

Thioredoxin-2 Inhibits Mitochondria-Located ASK1-Mediated Apoptosis in a JNK-Independent Manner

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)–induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.

Evidence of Dysfunction of Endothelial Progenitors in Pulmonary Arterial Hypertension

RATIONALE Severe pulmonary arterial hypertension (PAH) is characterized by the formation of plexiform lesions and concentric intimal fibrosis in small pulmonary arteries. The origin of cells contributing to these vascular lesions is uncertain. Endogenous endothelial progenitor cells are potential contributors to this process. OBJECTIVES To determine whether progenitors are involved in the pathobiology of PAH. METHODS We performed immunohistochemistry to determine the expression of progenitor cell markers (CD133 and c-Kit) and the major homing signal pathway stromal cell-derived factor-1 and its chemokine receptor (CXCR4) in lung tissue from patients with idiopathic PAH, familial PAH, and PAH associated with congenital heart disease. Two separate flow cytometric methods were employed to determine peripheral blood circulating numbers of angiogenic progenitors. Late-outgrowth progenitor cells were expanded ex vivo from the peripheral blood of patients with mutations in the gene encoding bone morphogenetic protein receptor type II (BMPRII), and functional assays of migration, proliferation, and angiogenesis were undertaken. measurements and main results: There was a striking up-regulation of progenitor cell markers in remodeled arteries from all patients with PAH, specifically in plexiform lesions. These lesions also displayed increased stromal cell-derived factor-1 expression. Circulating angiogenic progenitor numbers in patients with PAH were increased compared with control subjects and functional studies of late-outgrowth progenitor cells from patients with PAH with BMPRII mutations revealed a hyperproliferative phenotype with impaired ability to form vascular networks. CONCLUSIONS These findings provide evidence of the involvement of progenitor cells in the vascular remodeling associated with PAH. Dysfunction of circulating progenitors in PAH may contribute to this process.

TNFR1- and TNFR2-mediated signaling pathways in human kidney are cell type-specific and differentially contribute to renal injury

In normal kidney, TNFR1 is expressed in glomerular and peritubular capillary EC, and some tubular cells, and colocalizes with inactive apoptosis signal‐regulating kinase‐1 (ASK1) phosphorylated at serine 967. Biopsies of rejecting or ischemic renal allografts, which show both tubular cell injury and proliferation, display down‐regulation of TNFR1 and activation of ASK1 as well as up‐regulation of TNFR2 on tubular cells, where it colocalizes with phosphorylated endothelial/epithelial tyrosine kinase (Etk). We have exploited receptor‐selective muteins and evaluated phosphorylation of receptor‐specific kinases to study TNF responses in situ. In organ culture, a TNFR1‐specific mutein changes phosphorylation of ASK1 to threonine 845, indicative of kinase activation. A TNFR2‐specific mutein down‐regulates TNFR1 in glomerular EC, up‐regulates TNFR2 and Etk in tubular cells, and induces phosphorylation of Etk. Wild‐type TNF induces TNFR2 and Etk and activates both ASK1 and Etk but does not down‐regulate TNFR1. Wild‐type TNF and TNFR1‐specific mutein trigger tubular cell apoptosis whereas wild‐type TNF and TNFR2‐specific mutein induce tubular cells to express proliferating cell nuclear antigen. Differential activation of ASK1 and Etk by regulated TNFRs in patient‐derived materials provides an explanation for diverse and opposing responses to TNF at distinct sites, and an in situ bioassay of TNFR signaling Al‐Lamki, R. S., Wang, J., Vandenabeele, P., Bradley, J. A., Thiru, S., Luo, D., Min, W., Pober, J. S., Bradley, J. R. TNFR1‐ and TNFR2‐mediated signaling pathways in human kidney are cell type‐specific and differentially contribute to renal injury. FASEB J. 19, 1637–1645 (2005)

Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.

Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with either of two cell surface receptors, TNFR-1 and TNFR-2. The levels and regulation of expression of these two receptors has been extensively analyzed in cultured cells, but little is known of TNFR expression in situ. We analyzed the expression of TNFR-1 and -2 in normal human renal kidney and in renal transplants undergoing acute cellular rejection. Immunohistochemistry and immunogold electron microscopy indicated a strong expression of TNFR-1 on the endothelium of glomeruli of normal kidney. Immunogold colocalization for TNFR-1 and a marker of the trans-Golgi network (TGN-46) demonstrated TNFR-1 within the Golgi complex in endothelial cells in normal kidney, confirming our previous studies with cultured cells. TNFR-1 expression was lost in glomeruli from acutely rejecting kidney, but TNFR-1 was detected in abundance on infiltrating leukocytes in the interstitium of allografts with acute rejection. In contrast, TNFR-2 was demonstrated predominantly in epithelial cells of distal convoluted tubule (DCT) in acute rejection kidney near TNF-expressing leukocytes. TNF was absent in normal kidney, but present in rejecting allograft. TNF was found in infiltrating leukocytes and in adjacent tubular epithelial cells. In situ hybridization showed TNFR-1 mRNA within the endothelium of the glomeruli and of a few arterioles in normal kidney, whereas TNFR-2 mRNA was seen in tubular epithelial cells of the DCT in acute transplant rejection. These data reveal that there is both differential expression and regulation of the two TNF receptors in human kidney.

Caveolae Participate in Tumor Necrosis Factor Receptor 1 Signaling and Internalization in a Human Endothelial Cell Line

Caveolae are abundant in endothelial cells (ECs) in situ but markedly diminished in cultured cells, making it difficult to assess their role in cytokine signaling. We report here that the human EC line EA.hy926 retains an abundant caveolar system in culture. Tumor necrosis factor (TNF) receptor 1 (TNFR1/CD120a) was enriched in caveolae and co-immunoprecipitated with caveolin-1 from caveolae isolated from these cells. To further investigate the role(s) of caveolae in TNF signaling in ECs, cells were treated with methyl-beta-cyclodextrin to disrupt caveolae. Methyl-beta-cyclodextrin did not alter total cell surface expression of TNFR1 or TNF-induced degradation of IkappaBalpha, a measure of nuclear factor-kappaB activation, but it did inhibit TNF-induced phosphorylation of Akt, a measure of phosphatidylinositol-3 kinase activation. Serum-induced phosphorylation of AKT was unaffected. Treatment with TNF induced disappearance of TNFR1 from caveolae and dissociation from caveolin-1 within 5 minutes. In contrast to transferrin receptor, internalized TNFR1 did not co-localize with clathrin, except possibly in the Golgi, at any time point examined. By 60 minutes of treatment with TNF, TNFR1 appeared in endosomes. We conclude that caveolae function in ECs to allow TNFR1 to activate phosphatidylinositol-3 kinase and Akt, perhaps through receptor cross talk, and that ligand-induced internalization and trafficking of TNFR1 to endosomes may originate directly from this compartment.

ENDOTHELIAL CELLS IN ALLOGRAFT REJECTION

In organ transplantation, blood borne cells and macromolecules (e.g., antibodies) of the host immune system are brought into direct contact with the endothelial cell lining of graft vessels. In this location, graft endothelial cells play several roles in allograft rejection, including the initiation of rejection responses by presentation of alloantigen to circulating T cells; the development of inflammation and thrombosis; and as targets of injury and agents of repair.

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pumps constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henles loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Smad-Dependent and Smad-Independent Induction of Id1 by Prostacyclin Analogues Inhibits Proliferation of Pulmonary Artery Smooth Muscle Cells In Vitro and In Vivo

Rationale: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking. Objective: Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling. Methods and Results: Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression. Conclusions: Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.

TL1A Both Promotes and Protects from Renal Inflammation and Injury

Death receptor 3 (DR3), a member of the TNF receptor (TNFR) superfamily, is induced in human renal tubular epithelial cells (TEC) in response to injury. This study examined the expression and actions of TL1A, the principal ligand for DR3. In histologically normal tissue from biopsy or nephrectomy specimens of renal allografts, TL1A mRNA and protein were expressed in vascular endothelial cells but not in TEC. In specimens of acute or antibody-mediated allograft rejection, vascular endothelial cells and infiltrating leukocytes expressed increased TL1A mRNA and protein, but TEC expressed TL1A protein without mRNA, consistent with uptake of exogenous ligand. Addition of TL1A to organ cultures of human or mouse kidney caused activation of NF-kappaB, expression of TNFR2, activation of caspase-3, and apoptosis in TEC. Inhibition of NF-kappaB activation increased TL1A-mediated caspase-3 activation and apoptosis of TEC, but it did not reduce the induction of TNFR2. In organ culture of DR3-deficient mouse kidneys, addition of TL1A induced TNFR2 but did not activate NF-kappaB and did not increase apoptosis of TEC. These data suggest that TL1A may contribute to renal inflammation and injury through DR3-mediated activation of NF-kappaB and caspase-3, respectively, but that an unidentified receptor may mediate the NF-kappaB-independent induction of TNFR2 in TEC.

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.