EndoPDI, a Novel Protein-disulfide Isomerase-like Protein That Is Preferentially Expressed in Endothelial Cells Acts as a Stress Survival Factor

Abstract

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. 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 at Willebrand factor is a well gene that a PEM score of of dermal microvascular endothelial cells and human umbilical vein endothelial cells were from and were in containing and endothelial cell were in and were used to the Isolation of cDNA for RNA was from and was using The at the were by using the to a and the and the into the and containing EndoPDI cDNA sequence was identified from the data base. The is at the but is by at the The site at was used to the of the EndoPDI with the of the to a full-length cDNA of of to EndoPDI and Western of sequence was synthesized by the Protein and of using This sequence to amino acids of the human EndoPDI protein sequence with an cysteine to the to to a The was to following the and with into A was with of used for the and of for The were by to identify the presence of to EndoPDI in the that was found to contain EndoPDI by was used at in Western for the of EndoPDI protein by Western to PDI were from for in Western of a EndoPDI for of the of EndoPDI was by from containing the EndoPDI using as the and as the The S.F. J. Res. 1997; 25: PubMed Scopus Google Scholar) was used to that the of EndoPDI not have homology to other The was into following the and to and were in transcription from in the presence of to RNA was from cells in using protection were in on of total RNA as S.F. J. Res. 1997; 25: PubMed Scopus Google Scholar). the of the RNA a of specific activity was by addition of to the The fragment for EndoPDI was In each a and and were of on a was as the of the of to mRNA to for in In situ hybridization analysis was with as in R. J. 1998; Google Scholar). The used for in situ hybridization was the as that used for the protection tissue was with in and in tissue expression with RNA from different were used for analysis of the distribution of EndoPDI mRNA in human tissues. from the of EndoPDI used for was used for of a cDNA The cDNA was with and at in following the of with specific and were synthesized using the following the were and for were with EndoPDI PDI using to the The cells were for to hypoxia for and or of for analysis as from for exposed and was as were with and in binding of and of were to the cells. for in the at the cells were by a of cells with and cells with only were used to set and The cell expression of was by cells with in of to on for and with with for on the cells were and in of and on a of by secretion of endothelin-1 by endothelial cells was using a human endothelin-1 following the Briefly, cells were with as the and for in the of by secretion of adrenomedullin by endothelial cells was using an adrenomedullin following the Briefly, cells were with as the and used in the of EndoPDI using Bioinformatic and cDNA Sequence analysis of gene expression libraries to genes that are expressed in endothelial cells. this we identified a novel gene, which we called EndoPDI, that is expressed in both and microvascular endothelial cells with of and tags The with the highest tag count for EndoPDI was the with tags a of that for of EndoPDI have been identified in the and While PDI has only two APWCGHC thioredoxin EndoPDI has three however, both have in a C-terminal which is of proteins that are within the endoplasmic We used the H. J. S. von Protein 1997; 10: PubMed Scopus Google Scholar) to the protein sequence of EndoPDI and found that it contains a at the N with the site between and other members of the PDI family contain a domain that has the thioredoxin structural also called the b domain, but contain no thioredoxin active the T. S.H. Biol. PubMed Scopus Google Scholar) we found that EndoPDI has the a, a′, with no b domain of EndoPDI in SAGE libraries of normal The for analysis of gene expression was used to the expression of EndoPDI expression was found in a total of and of were from normal tissues. The tags for EndoPDI in these libraries is shown in the Duke HMVEC normal endothelium cell Duke HMVEC normal endothelium cell normal cell normal SAGE normal cell normal cell normal cell normal cell normal normal SAGE normal normal normal normal normal normal Duke normal normal normal normal in a and of putative full-length gene EndoPDI has been found in and to human at This of also molecule containing thioredoxin-like domains called S. Y. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We protection analysis to the expression of EndoPDI in in different cell EndoPDI expression was highest in endothelial cells. The other cell were and We found that EndoPDI expression was in endothelial cells compared with microvascular endothelial cells with the cell the expression among the endothelial cells. The cell a expression of EndoPDI the highest cell Expression of EndoPDI in may the cell of and endothelial cells in that both from in the The expression of EndoPDI in the cell was at in endothelial cells. Expression tissue expression were used to the expression of EndoPDI mRNA in human The RNA from different human tissues. EndoPDI was in of tissue The highest were found in by The high expression in the was the is not a well and in of the hypoxic of EndoPDI not known to be containing tissue from tumor and normal tissue were used to whether EndoPDI is in human tumors of was found in tumors of the and In situ hybridization studies were in to expression of EndoPDI in human in Expression was found to be rare and seen only in the of human the syncytiotrophoblasts of and in and the of the atherosclerotic (G and and in the keratinocytes of a hair and EndoPDI by in investigated whether EndoPDI is by hypoxia. protection analysis we found that EndoPDI is hypoxia in with a hypoxia Western analysis confirmed the protein to be by hypoxia of EndoPDI in in but the of PDI has been shown to have a protective effect cell by hypoxia in cells S. T. Y. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google we investigated whether EndoPDI has a role in endothelial cells from apoptosis under hypoxia. The we used was to EndoPDI expression using specific The EndoPDI mRNA and protein were with or siRNA. is that contains the as the but has no homology to known genes to We found that under that been with or with expressed the level of EndoPDI mRNA and protein, and this expression was with results were observed the cells were to hypoxia in that the expression level of EndoPDI mRNA and protein was by with under was loss of EndoPDI expression with specific to We used the to the expression of PDI using specific to PDI. with EndoPDI, we found that with to PDI PDI protein expression under both normoxia and hypoxia We used to the effect of of EndoPDI on survival under hypoxia. We found that of EndoPDI under normoxia no effect on survival were with EndoPDI under hypoxia was a significant in the and cell of PDI in in and The effect of EndoPDI on endothelial cell was compared with that of PDI. to PDI, we analysis to the of apoptosis from the loss of PDI 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 PDI in and of the cell in normoxia and of the cell was under hypoxia of EndoPDI and only under The of of EndoPDI and PDI Expression on the or Expression of endothelial cells a number of molecules that as hypoxia survival of such molecules are endothelin-1 H. Y. 1997; PubMed Scopus Google Scholar) adrenomedullin C. S. Bicknell R. PubMed Scopus Google Scholar) and C. R. P. C. S. J. Sci. 2003; PubMed Scopus Google Scholar). We compared the expression of these molecules by endothelial cells under hypoxia of EndoPDI, PDI, or both EndoPDI and PDI There was a significant decrease in endothelin-1 expression both PDI and EndoPDI The reduction in endothelin-1 secretion with EndoPDI or PDI was the as secretion under with EndoPDI and PDI caused a but not significant reduction in the secretion of endothelin-1 compared with that of EndoPDI or PDI results that both PDI and EndoPDI have a role in the folding and secretion of endothelin-1 under hypoxia with protein having for endothelin-1 the In contrast, loss of EndoPDI expression reduced adrenomedullin secretion under loss of PDI expression effect Furthermore, was no significant in adrenomedullin secretion both PDI and EndoPDI compared with EndoPDI results that PDI has or no role in the folding and secretion of adrenomedullin under but that EndoPDI has for this is adrenomedullin the of both PDI and EndoPDI that are other chaperones that function in the folding and secretion of this called is an endothelial specific gene expression is by hypoxia and has been shown to protect endothelial cells from apoptosis under hypoxia C. R. P. C. S. J. Sci. 2003; PubMed Scopus Google Scholar). shows that hypoxia caused of expression on to a in cell expression as using the While to PDI effect on that to EndoPDI caused a reduction in expression of of the and that the two together expression. The effect of EndoPDI specific on expression is similar to that for with where a similar cell expression results C. R. P. C. S. J. Sci. 2003; PubMed Scopus Google Scholar). We that EndoPDI may folding in the of PDI, the is not the for a role for EndoPDI in folding this molecule under hypoxia. We a novel human protein-disulfide isomerase that we have called EndoPDI to its high and expression in endothelial cells. has shown that EndoPDI expression is by is only expressed in in hypoxic and protects endothelial cells from under hypoxia. We also show that the protective effect of EndoPDI under hypoxia be caused by a folding and chaperone activity on molecules. archetypal PDI, EndoPDI is a rare example of a PDI that has three domains containing the APWCGHC thioredoxin domain but no b The of a sequence and a well sequence support to this a full-length cDNA for The sequence and the C-terminal that PDI, EndoPDI be to the ER. EndoPDI to have an unusual of tissue expression. expected we expression in well such as and but we found expression in some of vascular such as the the of tissue expression for EndoPDI from the tissue expression is with the SAGE expression for example the SAGE data that the expression of EndoPDI in the is around in the Furthermore, the SAGE data that the expression of EndoPDI in the and is a that is confirmed by the tissue expression The of the other PDI family members between different tissue between different cell within the tissue and with the of the cell R.B. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). The only member of the PDI family to shown to have tissue expression is the specific PDI Biol. 1997; 16: PubMed Scopus Google Scholar). may be for the folding of specific proteins such as J. S. P. P. R. 1997; PubMed Scopus Google Scholar). We may similarly that EndoPDI may be for the folding of endothelial cell specific Expression of EndoPDI in endothelial cells is at in the or other tumor cell with the of A analysis identified EndoPDI as in that as the gene in H. J. PubMed Scopus Google Scholar). In of and the that cells are from the as endothelial expression in cells was not In situ analysis showed EndoPDI to be expressed in of known or putative tumors P. P. Biol. PubMed Scopus Google Scholar) atherosclerotic T. Biol. 1999; 19: PubMed Scopus Google Scholar) and the bulb of a hair J. PubMed Scopus Google Scholar). Thus, in tumors EndoPDI was present in the vascular endothelium in atherosclerotic in and microvascular endothelium of the G and and in keratinocytes of the hair bulb and expression was also seen in the syncytiotrophoblasts of the normal these it to that expression of EndoPDI in is a of cells are the active cell type yet identified R.A. J.D. Nature. 1995; Google Scholar) and many proteins that are specific to or expressed in for example and L. Bicknell R. Res. 2000; 10: PubMed Scopus Google Scholar, A. R. Y. S. Wang J. A. L. T. 2003; PubMed Scopus Google Scholar). Furthermore, unlike other cell endothelial cells are able to such as and R. J. Google Scholar). This is of in the survival and to of tumor and an to some that the There are a set of proteins found to be expressed in endothelial cells hypoxia J. 1998; Full Text PDF PubMed Scopus Google Scholar) and it is that EndoPDI be essential for the folding and of these proteins for survival under hypoxia. PDI is also hypoxia in endothelial cells C. S. J. PubMed Scopus Google Scholar) as well as in cells S. T. Y. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The of PDI in cells was shown to have a protective effect hypoxic cell in these cells S. T. Y. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). results show that PDI is an of endothelial cell survival under both normoxia and but that EndoPDI is expressed in and protects endothelial cells under hypoxia. We that EndoPDI in with PDI endothelial cells to show a for survival under hypoxia. this we the secretion of endothelin-1 and adrenomedullin together with the cell expression of CD105, molecules with known protective apoptosis in endothelial cells. three are known to be genes and to protect endothelial cells from under hypoxia H. Y. 1997; PubMed Scopus Google Scholar, C. R. P. C. S. J. Sci. 2003; PubMed Scopus Google Scholar, M.J. A. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We found that PDI and EndoPDI to be in the secretion of endothelin-1, EndoPDI may be more PDI in and adrenomedullin secretion from endothelial cells. In fact, of PDI effect on expression of EndoPDI and PDI together expression. In we have identified EndoPDI a novel protein-disulfide protein that is expressed in endothelial is by hypoxia and is expressed in the endothelium of tumors and atherosclerotic plaques. EndoPDI to be a for endothelial cell survival under hypoxic conditions to its secretion of endothelial cell survival work is to the role of PDI and EndoPDI in the hypoxic endothelial We for with the in situ analysis and for identification of We also for