EndoPDI, a Novel Protein-disulfide Isomerase-like Protein That Is Preferentially Expressed in Endothelial Cells Acts as a Stress Survival Factor
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m308124200
ISSN1083-351X
AutoresD C Sullivan, Łukasz Huminiecki, John W. Moore, Joseph J. Boyle, Richard Poulsom, D. Creamer, Juliet N. Barker, Roy Bicknell,
Tópico(s)Cardiovascular Effects of Exercise
ResumoWe have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium. We have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium. Protein-disulfide isomerase (PDI) 1The abbreviations used are: PDIprotein-disulfide isomeraseERendoplasmic reticulumEndoPDIendothelial protein-disulfide isomeraseHUVEChuman umbilical vein endothelial cellsHDMEChuman dermal microvascular endothelial cellsFACSfluorescence-activated cell sortingsiRNAshort interfering RNAvWFvon Willebrand factorFITCfluorescein isothiocyanateTrxthioredoxin. is a ubiquitously expressed multifunctional protein found in the endoplasmic reticulum (ER). It constitutes around 0.8% of total cellular protein and can reach near millimolar concentrations in the ER lumen of some tissues. PDI plays a role in protein folding because of its ability to catalyze the formation of native disulfide bonds and disulfide bond rearrangement (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar). Proteins targeted for secretion by the cell are inserted into and translocated across the ER membrane and enter the ER lumen in an unfolded state. PDI, together with a variety of other folding factors and molecular chaperones resident in the ER correctly fold the proteins ready for secretion (2Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar). The accumulation of misfolded proteins in the ER, known as the Unfolded Protein Response, results in increased transcription of chaperones and folding catalysts. Proteins that fail to fold correctly are relocated to the cytosol for proteasomal degradation. protein-disulfide isomerase endoplasmic reticulum endothelial protein-disulfide isomerase human umbilical vein endothelial cells human dermal microvascular endothelial cells fluorescence-activated cell sorting short interfering RNA von Willebrand factor fluorescein isothiocyanate thioredoxin. PDI is a modular protein consisting of a, b, b′, a′, and c domains (3Edman J.C. Ellis L. Blacher R.W. Roth R.A. Rutter W.J. Nature. 1985; 317: 267-270Crossref PubMed Scopus (477) Google Scholar). The a and a′ domains show sequence and structural homology to thioredoxin (Trx) and both contain the active site WCGHCK motif, constituting two independent catalytic sites for thiol-disulfide bond exchange reactions (4Hawkins H.C. Freedman R.B. Biochem. J. 1991; 275: 335-339Crossref PubMed Scopus (124) Google Scholar, 5LaMantia M.L. Lennarz W.J. Cell. 1993; 74: 899-908Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 6Lyles M.M. Gilbert H.F. J. Biol. Chem. 1994; 269: 30946-30952Abstract Full Text PDF PubMed Google Scholar, 7Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 11725-11735Crossref PubMed Scopus (143) Google Scholar). A rate-limiting step in the folding of many newly synthesized proteins is the formation of disulfide bridges (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar) and the presence of WCGHCK in PDI is essential for this process, as confirmed by the loss of PDI activity following mutation of the cysteine residues within these motifs (5LaMantia M.L. Lennarz W.J. Cell. 1993; 74: 899-908Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 8Yao Y. Zhou Y. Wang C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar). The b and b′ domains also have the thioredoxin structural fold but lack the active site motif. Thus, PDI contains both redox active and inactive thioredoxin modules. The C-terminal c domain, a putative Ca2+ binding region, is rich in acidic amino acids and contains the -KDEL motif, which is necessary and sufficient for the retention of a polypeptide within the lumen of the ER. The C-terminal domain is, however, not necessary for the enzymatic, chaperone (see below) or disulfide isomerase activities of PDI (9Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakovski T. Notbohm H. Kivirikko K.I. EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (54) Google Scholar). In fact, the smallest PDI fragment showing an efficient catalysis of disulfide bond rearrangement has been shown to be a construct containing the b′-a′-c modules (7Darby N.J. Creighton T.E. Biochemistry. 1995; 34: 11725-11735Crossref PubMed Scopus (143) Google Scholar). In addition to its disulfide isomerase activity, PDI also shows chaperone activity, for example it can function as the β-subunit of prolyl-4 hydroxylase, preventing the misfolding and aggregation of the α-subunit (10John D.C. Grant M.E. Bulleid N.J. EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (95) Google Scholar). This function is similar to that of some molecular chaperones such as Hsp90 in other proteins (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar). Furthermore, PDI is able to interact with and correctly fold type X collagen polypeptides that contain no cysteine residues (11McLaughlin S.H. Bulleid N.J. Biochem. J. 1998; 331: 793-800Crossref PubMed Scopus (45) Google Scholar). There is now an increasing family of protein-disulfide isomerases, each having two or more thioredoxin or catalytically inactive b domains (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar, 12Clissold P.M. Bicknell R. Bioessays. 2003; 25: 603-611Crossref PubMed Scopus (35) Google Scholar). Sequence homology between members is poor, their relatedness lying in the structural similarity of the thioredoxin-like fold (12Clissold P.M. Bicknell R. Bioessays. 2003; 25: 603-611Crossref PubMed Scopus (35) Google Scholar). It has been proposed that different PDIs may show different substrate specificities (1Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar) and support for this has been provided in studies showing that ERp57 is specific for the folding of N-glycosylated proteins (13Elliott J.G. Oliver J.D. High S. J. Biol. Chem. 1997; 272: 13849-13855Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 14Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Science. 1997; 275: 86-88Crossref PubMed Scopus (347) Google Scholar). We now report the identification of a novel PDI-like protein, which we have called EndoPDI because of its high expression in endothelial cells. This tissue-specific expression is unusual among members of the PDI family (12Clissold P.M. Bicknell R. Bioessays. 2003; 25: 603-611Crossref PubMed Scopus (35) Google Scholar). Previous work has shown that PDI, the archetypal member of the PDI family, can protect neurons and endothelial cells from hypoxic cell death. Thus, we have investigated whether EndoPDI can similarly protect endothelial cells from stress-induced apoptosis and compared its activity to that of PDI. In contrast to PDI, which is essential for the survival of endothelial cells in the resting as well as the stressed state, EndoPDI protects endothelial cells only under conditions of stress. We have sought a possible protective role of EndoPDI under hypoxia and found that loss of EndoPDI results in reduced secretion of adrenomedullin and endothelin-1 together with a reduction in membrane-bound CD105. While EndoPDI is essential for folding of CD105, PDI is not, illustrating differences at the molecular level in the mechanisms of protection afforded by two molecules. Bioinformatic Methods—EndoPDI was initially found as a gene preferentially expressed in vascular endothelial cells by analysis of expression data deposited in SAGEmap (15Lash A.E. Tolstoshev C.M. Wagner L. Schuler G.D. Strausberg R.L. Riggins G.J. Altschul S.F. Genome. Res. 2000; 10: 1051-1060Crossref PubMed Scopus (354) Google Scholar). Briefly, the SAGEmap data set was downloaded from the project website (www.ncbi.nlm.nih.gov/SAGE/) in February 2001 and deposited in a MySQL data base. Only normal tissue libraries (total = 37) were used in the analysis. There were two libraries representing vascular endothelium; SAGE Duke HMVEC and SAGE Duke HMVEC+VEGF. The Preferential Expression Measure (PEM) was used to identify genes preferentially expressed in vascular endothelium. PEM = log(o/e), where o is the observed SAGE tag count in vascular endothelium, and e is the expected tag count if the distribution was uniform across the libraries. e = (G * N/T), where G is the total number of SAGE tags for a given gene, N is the total number of tags for vascular endothelium (110, 460), and T is the total number of tags in all normal libraries (1, 077, 231). The vascular endothelial PEM score for EndoPDI was 1.941. The highest vascular endothelial score yet seen is attributed to EGF-containing fibulin-like extracellular matrix protein-1 at 2.081. Von Willebrand factor (vWF) is a well characterized endothelial-specific gene that had a PEM score of 1.847. Culture of Endothelial Cells—Human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics BioWhittaker (Wokingham, Berkshire, UK) and were cultured in MCDB131 medium (Invitrogen) containing 20% fetal calf serum (Sigma-Aldrich), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, 5 IU/ml heparin, and 50 μg/ml endothelial cell growth supplement (Sigma, Dorset, UK). Cells were routinely split 1 in 3 and were used up to the 8th passage. Isolation of Full-length cDNA for EndoPDI—Total RNA was extracted from HDMEC and 1 μg was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). The first 270 nucleotides at the 5′-end were then amplified by PCR using the upstream primer 5′-CCGGTACCCCCGCGCGCCCAGGACGCCTCCTCC-3′, designed to include a KpnI restriction endonuclease site, and the downstream primer 5′-GCAGCATCCAGTTTTCCAGT-3′ and the PCR product cloned into the topo II PCR vector (Invitrogen) and sequenced. An IMAGE clone (3356029) containing partial EndoPDI cDNA sequence was identified from the Unigene data base. The clone is complete at the 3′-end but is truncated by 255 nucleotides at the 5′-end. The unique BstUI site at position 267 was used to enable the ligation of the IMAGE clone EndoPDI insert with the cloned 270 nucleotides of the 5′-end to give a full-length cDNA of EndoPDI. Production of Polyclonal Antibodies to EndoPDI and Western Blotting—The peptide of sequence ADGEDGQDPHSKC was synthesized by the Protein and Peptide Chemistry Department of Cancer Research UK, using standard techniques. This sequence corresponds to amino acids 52–63 of the human EndoPDI protein sequence with an additional cysteine residue added to the C terminus to enable coupling to a carrier protein. The peptide was coupled to Imject® Maleimide-activated mcKLH (Pierce) following the manufacturer's instructions and diluted with Freund's adjuvant before injection into rabbits. A standard immunization protocol was followed with 200 μg of immunogen used for the first injection and 100 μg of immunogen for subsequent boosts. The test bleeds were screened against pre-bleeds by ELISA to identify the presence of antibodies to EndoPDI in the rabbit serum. Serum that was found to contain EndoPDI antibodies by ELISA was used at 1:100 dilution in Western blotting experiments for the detection of EndoPDI protein by standard Western blotting techniques. Antibodies to PDI were purchased from Bioquote Ltd. (North Yorkshire, England) for use in Western blotting. Preparation of a Recombinant EndoPDI Construct for Riboprobe Synthesis—A 300-bp region of the 3′-UTR of EndoPDI was amplified by PCR from plasmid DNA containing the EndoPDI clone described above using 5′-TGTGGCTCCTGAGTTGAGTG-3′ as the upstream forward primer and 5′-ACTCAGGCACGGTCAGAAGT-3′ as the reverse downstream primer. The Basic Local Alignment Search Tool (BLAST) (16Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) was used to ensure that the chosen region of EndoPDI did not have homology to other sequences. The PCR product was cloned into pCRII-TOPO (Invitrogen) following the manufacturer's instructions and sequenced to confirm identity and orientation. Riboprobes were transcribed (MAXIscript in vitro transcription kit, Ambion AMS Ltd, Witney, Oxon, UK) from linearized plasmids in the presence of [32P]UTP (Amersham Biosciences) to give radioactively labeled probe. RNase Protection Assay—Total RNA was extracted from cells in culture using TRI reagent (Sigma). RNase protection assays were performed in duplicate on 10–30 μg of total RNA as described (16Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). To attenuate the signal strength of the highly abundant loading control, U6 small nuclear RNA (accession no. X01366), a riboprobe of significantly lower specific activity was prepared by addition of unlabeled CTP to the labeling reaction. The protected fragment size for EndoPDI was 300 nucleotides. In each assay, a positive and negative (tRNA only) control and undigested riboprobes were analyzed. Intensity of signal, quantified on a PhosphorImager (Molecular Dynamics, Chesham, Buckinghamshire, UK) was calculated as the ratio of the signal of interest to U6 mRNA to correct for variations in loading. In Situ Hybridization—In situ hybridization analysis was performed with radioactively labeled probes as described in Poulsom et al., 1998 (17Poulsom R. Longcroft J.M. Jeffery R.E. Rogers L.A. Steel J.H. Eur. J. Histochem. 1998; 42: 121-132PubMed Google Scholar). The EndoPDI-specific probe used for in situ hybridization was the same as that used for the RNase protection assay described above. Human tissue was collected with full ethical approval during routine pathology, fixed in formalin, and embedded in paraffin. Multiple Tissue Array Analysis—Human multiple tissue expression arrays (Clontech, Oxford, UK) with poly(A)+ RNA from different tissues were used for analysis of the distribution of EndoPDI mRNA in human tissues. DNA from the same region of EndoPDI used for riboprobe production was used for preparation of a cDNA probe. The cDNA was labeled with [32P]dCTP (Rediprime random primer labeling kit, Amersham Biosciences) and hybridized at 65 °C overnight in ExpressHyb (Clontech) solution following the manufacturer's instructions. Transfection of Microvascular Endothelial Cells with siRNA—EndoPDI specific (5′-GAAGCTGTGAAGTACCAGGTT-3′) and PDI-specific (5′-GACCTCCCCTTCAAAGTTGTT-3′) siRNA oligos were synthesized using the Silencer™ siRNA Construction kit (Ambion, Huntingdon, UK) following the manufacturer's instructions. HDMEC (5 × 105 cells) were plated onto 0.2% gelatin coated 10-cm Petri dishes and incubated for 24 h. Cells were then transfected with 10 nm EndoPDI and/or PDI siRNA using oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. The cells were then incubated for 24 h prior to hypoxia for 16 h (0.1% O2,5%CO2, and 94.9% N2) or continuation of normoxic exposure for 16 h before performing FACS analysis as described. FACS Analysis—To distinguish apoptotic from necrotic cells, double staining for exposed phosphatidylserine and propidium iodide (PI) exclusion was performed as follows: Cells were harvested, washed twice with PBS, and resuspended in binding buffer (10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2). 5 μl of annexin V-FITC antibody (BD Pharmingen, San Diego) and 10 μl of PI (50 μg/ml) were added to the cells. After incubation for 15 min in the dark at room temperature, the cells were analyzed by a FACScan. Controls of unstained cells, cells stained with annexin V-FITC only, and cells stained with PI only were used to set up compensation and quadrants. The cell surface expression of CD105 was quantified by incubating 105 cells per tube with 50 μl (10 μl/ml in phosphate-buffered saline) of monoclonal antibody to CD105 on ice for 1 h and washed twice with cold phosphate-buffered saline. After incubation with FITC-labeled rabbit anti-mouse F(ab)2 (1/40 DAKO) for 30 min on ice, the cells were washed and resuspended in 0.3 ml of 2% buffered formalin and analyzed on a FACScan. Measurement of Endothelin-1 Secretion by ELISA—The secretion of endothelin-1 by endothelial cells was measured using a human endothelin-1 ELISA (R&D Systems, Abindgdon, UK) following the manufacturer's instructions. Briefly, 50,000 cells were treated with RNAi oligos as before, the medium collected and diluted 1/25 for use in the ELISA. Measurement of Adrenomedullin by Radioimmunoassay—The secretion of adrenomedullin by endothelial cells was measured using an adrenomedullin radioimmunoassay (Peninsula Laboratories Europe, Ltd, Merseyside, England) following the manufacturer's instructions. Briefly, 50,000 cells were treated with RNAi oligos as before, the medium collected and used in the radioimmunoassay. Identification of EndoPDI using Bioinformatic and cDNA Sequence Analysis—We utilized serial analysis of gene expression (SAGE) libraries (www.ncbi.nlm.nih.gov/SAGE) to find genes that are highly expressed in endothelial cells. Using this approach we identified a novel gene, which we subsequently called EndoPDI, that is highly expressed in both VEGF-stimulated and quiescent microvascular endothelial cells (HMVEC) with counts of 1224 and 741 tags per million, respectively (Table I). The library with the next highest tag count for EndoPDI was the foreskin fibroblast library with 204 tags per million, i.e. less than a third of that for HMVEC. Homologues of EndoPDI have been identified in the mouse, rat, Xenopus, Drosophila, and mosquito (Fig. 1A). While PDI has only two APWCGHC thioredoxin motifs, EndoPDI has three (Fig. 1B); however, both have in common a C-terminal KDEL sequence, which is characteristic of proteins that are retained within the endoplasmic reticulum. We used the Neural Network program, SignalIP (18Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4942) Google Scholar) to analyze the protein sequence of EndoPDI and found that it contains a signal peptide, MPARPGRLLPLLARPAALTALLLLLLGHGGGGRW at the N terminus with the most likely cleavage site being located between positions 32 and 33 (GGG-RW). All other members of the PDI family contain a domain that has the thioredoxin structural fold, also called the b domain, but contain no thioredoxin active site. Using the structure prediction program VAST (19Gibrat J.F. Madej T. Bryant S.H. Curr. Opin. Struct. Biol. 1996; 6: 377-385Crossref PubMed Scopus (890) Google Scholar) we found that EndoPDI has the structure ao, a, a′, c, with no b domain (Fig. 1B).Table IExpression of EndoPDI in SAGE libraries of normal tissues The NCBI database for serial analysis of gene expression (SAGE) was used to examine the relative expression of EndoPDI. EndoPDI expression was found in a total of 211 libraries, and of these, 26 were derived from normal tissues. The tags per million counts for EndoPDI in these 26 libraries is shown in the table.Cell/Tissue typeTags per millionSAGE Duke HMVEC + VEGF (vascular normal endothelium cell line)1224SAGE Duke HMVEC (vascular normal endothelium cell line)741Foreskin fibroblasts204SAGE TSU (prostate normal cell line)175SAGE PR317 (prostate normal SAGE microdissected)117SAGE H126 (pancreas epithelium ductal normal cell line)91SAGE 293 CTRL (kidney normal cell line)90SAGE IOSE29 11 (ovary epithelium normal cell line)82SAGE 293 IND (kidney normal cell line)63SAGE HOSE 4 (ovary epithelium normal)61SAGE Chen Normal Pr (prostate normal bulk)60SAGE mammary epithelium (mammary gland epithelium ductal normal)60SAGE NHA (5th) brain normal SAGE astrocyte57SAGE breast myoepithelial (mammary gland normal myoepithelial)51SAGE NC2 (epithelium normal colon)40SAGE NC1 (epithelium normal colon)39SAGE normal gastric body epithelium (stomach normal)39SAGE PERITO 13 (normal peritoneum)36SAGE normal pediatric cortex H1571 (normal cortex juvenile)25SAGE normal lung22SAGE Duke thalamus (normal bulk thalamus)20SAGE normal spinal cord18SAGE normal pool (6th) (brain normal)15SAGE normal liver14SAGE normal heart11SAGE BB542 whitematter (brain normal)10 Open table in a new tab Genomic Organization, Chromosomal Localization, and Tissue Distribution of EndoPDI—The putative full-length gene encoding EndoPDI has been found in genome databases (accession no. BD127641) and mapped to human chromosome 6 at position 6p25.2. This region of chromosome 6 also encodes another molecule containing thioredoxin-like domains called PICOT (20Witte S. Villalba M. Bi K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). We performed RNase protection analysis to examine the expression of EndoPDI in vitro in ten different cell types (Fig. 2). As expected, EndoPDI expression was highest in endothelial cells. The other cell lines tested were MRC-5 (fibroblast), SY-SH-SY (neuroblastoma), SK23 (skin fibroblast), MDA468 (breast carcinoma), NCIH520 (squamous lung carcinoma), ZR75 (estrogen-dependent breast carcinoma), and HL60 (promyelocytic leukemia). We found that EndoPDI expression was greatest in large vessel endothelial cells (HUVEC) compared with microvascular endothelial cells (HDMEC), with the immortalized cell line HMME displaying the lowest expression among the endothelial cells. The promyelocytic leukemia cell line, HL60 displayed a 2.5-fold higher expression of EndoPDI than MDA468, the next highest expressing cell line. Expression of EndoPDI in HL60 may reflect the common cell lineage of hematopoetic and endothelial cells in that both originate from hemangioblasts in the embryonic blood islands. The expression of EndoPDI in the remaining 6 cell lines was at least 4-fold lower than in endothelial cells. Tissue Expression Array Studies—Human multiple tissue expression arrays were used to determine the relative expression of EndoPDI mRNA in human tissues (Fig. 3A). The blots contained poly(A)+ RNA from 72 different human tissues. EndoPDI was detected in 20 of 72 tissue spots. The highest levels were found in lymph node, followed by stomach then heart. The high expression in the stomach was unexpected since the stomach is not a particularly well vascularized organ, and in light of the hypoxic induction of EndoPDI described later, not known to be hypoxic. Arrays containing tissue from matched tumor and normal tissue samples were used to determine whether EndoPDI is up-regulated in human tumors (Fig. 3B). Up-regulation of endoPDI was found in tumors of the cervix, uterus, stomach, and lung. In Situ Hybridization Studies—In situ hybridization studies were performed in order to define expression of EndoPDI in human tissues in vivo (Fig. 4). Expression was found to be rare and seen only in the vasculature of human melanoma (A–D), the syncytiotrophoblasts of placenta (E and F), in macrophages and the microvasculature of the atherosclerotic plaque (G and H) and in the keratinocytes of a hair follicle (I and J). EndoPDI Is Up-regulated by Hypoxia in HDMEC—We next investigated whether EndoPDI is regulated by hypoxia. Using RNase protection analysis we found that EndoPDI is 2-fold up-regulated after 1 h hypoxia in HDMEC with a maximal 2.5-fold induction after 16 h hypoxia (Fig. 5A). Western analysis confirmed the protein to be 3-fold up-regulated by hypoxia (Fig. 5B). Loss of EndoPDI Causes Increased Apoptotic Cell Death in Microvascular Endothelial Cells in Hypoxia but Not Normoxia—Since the up-regulation of PDI has been previously shown to have a protective effect against apoptotic cell death induced by hypoxia in neuronal cells (21Tanaka S. Uehara T. Nomura Y. J. Biol. Chem. 2000; 275: 10388-10393Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), we investigated whether EndoPDI has a role in protecting endothelial cells from apoptosis under hypoxia. The approach we used was to down-regulate EndoPDI expression using specific siRNA oligos. The siRNA efficiently down-regulated EndoPDI mRNA (Fig. 6A) and protein (Fig. 6B). Cells were treated with either transfection reagents alone, EndoPDI-specific siRNA, or scrambled siRNA. Scrambled siRNA is siRNA that contains the same overall nucleotide composition as the gene-specific siRNA but has no homology to any known genes according to BLAST search results. We found that under normoxia, HDMEC that had been treated with transfection reagents alone (control) or with scrambled oligos expressed the same level of EndoPDI mRNA and protein, and this expression was completely blocked after transfection with EndoPDI-specific siRNA (Fig. 6). Similar results were observed when the cells were subjected to 16 h hypoxia (0.1% O2) in that the expression level of EndoPDI mRNA and protein was unaffected by treatment with scrambled siRNA (Fig. 6). Again under hypoxia, there was loss of EndoPDI expression after transfection with siRNA specific to EndoPDI. We used the same approach to down-regulate the expression of PDI using siRNA oligos specific to PDI. As with EndoPDI, we found that HDMEC treated with siRNA to PDI had lost PDI protein expression under both normoxia and hypoxia (Fig. 6C). We used siRNA to examine the effect of down-regulation of EndoPDI on HDMEC survival under hypoxia. We found that down-regulation of EndoPDI under normoxia had no effect on HDMEC survival (Fig. 7A). However, when HDMEC were treated with EndoPDI siRNA under hypoxia there was a significant increase in the apoptotic and necrotic cell populations (Fig. 7B). Loss of PDI Causes Increases Apoptotic Cell Death in Microvascular Endothelial Cells in Both Normoxia and Hypoxia— The effect of EndoPDI on endothelial cell behavior was compared with that of PDI. Using PDI-specific siRNA to down-regulate PDI, we performed FACS analysis to determine the extent of apoptosis resulting from the loss of PDI (Fig. 8). In contrast to EndoPDI, we found that loss of PDI caused a high level of apoptosis in normoxia as well as under hypoxia. In fact, under the same conditions, PDI down-regulation resulted in 55 and 48% of the cell population being apoptotic in normoxia and hypoxia, respectively, whereas 12% of the cell population was apoptotic under hypoxia after down-regulation of EndoPDI and only 4.5% apoptotic under normoxia. The Effect of Lack of EndoPDI and PDI Expression on the Secretion or Cell Surface Expression of Hypoxically Induced Endothelial Survival Factors—Under hypoxia, endothelial cells produce a number of molecules that act as hypoxia survival factors. Examples of such molecules are endothelin-1 (22Shichiri M. Kato H. Marumo F. Hirata Y. Hypertension. 1997; 30: 1198-1203Crossref PubMed Scopus (159) Google Scholar) adrenomedullin (23Oehler M.K. Norbury C. Hague S. Rees M.C. Bicknell R. Oncogene. 2001; 20: 2937-2945Crossref PubMed Scopus (103) Google Scholar) and CD105 (24Li C. Issa R. Kumar P. Hampson I.N. Lopez-Novoa J.M. Bernabeu C. Kumar S. J. Cell Sci. 2003; 116: 2677-2685Cross
Referência(s)