Decay-accelerating Factor mRNA Research Articles

Decay-accelerating factor promotes endometrial cells proliferation and motility under ovarian hormone stimulation

The intent of the study was to explore the elevating expression of decay-accelerating factor(DAF) exerts influence on biological behaviors of endometrial stromal cells except in classical immunology on the basis of bioinformatic statistics and clinical miscarriages findings suggesting its potential role in the establishment of endometrial receptivity. We confirmed that DAF locates on the cellular surface of endometrial epithelium and stroma. By using plasmid transfection to down-regulate DAF expression in primary endometrial stromal cells(ESCs), we discovered that DAF expression in ESCs increases in response to estradiol and progesterone stimulation in dose- and time-dependent manners; moreover, tamoxifen and RU486 stimulations to block estrogen receptors(ERs) and progesterone receptors(PRs) respectively result in reduced DAF mRNA and protein, and it is more obvious to block PRs. Meanwhile, knocked-down DAF in ESCs weakens the proliferation, migration and invasion of endometrial cells. Cell cycle analysis showed knocked-down DAF accumulates cells in S phase and diminishes cells in G0/G1 phase, which substantiates DAF mediates endometrial cells proliferation. In conclusion, DAF is a potential molecule involving in endometrial cellular proliferation and motility to verify up-expressed DAF during the WOI may facilitate endometrial physiobiological behavior changes, which shed light on DAF function and potential role in the endometrial receptivity establishment.

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF.

Binding of human antigen R (HuR) to an AU-rich element (ARE) in the 3′untranslated region (3′UTR) reduces the expression of decay accelerating factor (DAF)

We have investigated the role of the 3′untranslated region (3′UTR) in the expression of decay accelerating factor (DAF), one of the major membrane regulators of Complement activation. We show here that the 3′UTR of DAF contains an adenylate uridine rich element (ARE) AUUUAUUUAUAUUUAUUUA, which belongs to Class II Cluster 4 of the AU-rich element-containing mRNA (ARED) database. Enhanced Green Fluorescent Protein (EGFP) Reporter constructs containing the DAF 3′UTR showed reduced levels of expression when transfected into a variety of cell lines compared to 3′UTR reporter constructs without the ARE sequence. Furthermore, the inhibitor of mRNA transcription Actinomycin D had a much stronger effect on mRNA half-life of the ARE-containing 3′UTR demonstrating that this ARE destabilises the mRNA. Electrophoretic Mobility Shift Assays (EMSA) using biotinylated RNA probes, demonstrated that cytoplasmic Human antigen R (HuR) bound to the DAF ARE. Transfection experiments using HuR specific siRNA increased DAF expression whilst plasmids containing the coding sequence of HuR had the opposite effect, demonstrating that HuR reduced the stability of DAF mRNA and suggesting that it is of importance in regulating the expression of DAF. These data suggest that modulators of HuR could potentially be used to alter DAF expression and therefore increase the susceptibility of malignant cells to immunotherapy.

A Novel Class of Vascular Endothelial Growth Factor-responsive Genes That Require Forkhead Activity for Expression

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Decay-accelerating factor in the periovulatory rat ovary

One of the most prominent inflammatory reactions is the activation of the complement system. The activated complement system does not distinguish between pathogens and the host cell. In order to prevent autologous complement-mediated attack, host cells express a variety of both membrane-bound and fluid-phase complement regulatory proteins which control activity of the complement cascade by acting on convertase enzymes or the membrane-attack complex. Although the process of ovulation is facilitated by the inflammatory reaction, this reaction has the potential to cause serious damage to growing follicles, ovulated follicles, and other important ovarian tissues. This study was undertaken to characterize the expression and regulation of decay-accelerating factor (DAF), a complement regulator, as a potential mediator of ovarian tissue protection from ovulatory inflammation. DNA microarray and Northern blot analyses showed that an ovulatory gonadotropin stimulus dramatically yet transiently induced DAF mRNA expression in the immature rat ovary. Northern blot and PCR analyses revealed that of the three known DAF isoforms, glycosylphosphatidylinositol (GPI)-, soluble-, and transmembrane-(TM) DAF, GPI-DAF was the predominant form. In situ hybridization localized GPI-DAF mRNA expression in the theca-interstitial cells of the periovulatory ovary. Neither the anti-progestin RU486 nor the cyclooxygenase inhibitor indomethacin significantly inhibited human chorionic gonadotropin (hCG)-induced GPI-DAF mRNA expression in vivo. In vitro theca cell culture studies indicated that hCG induces GPI-DAF mRNA expression through the protein kinase A pathway. This study suggests that gonadotropin-induced GPI-DAF may be involved in the protection of ovarian tissues from the potential attack by the complement system activated by the inflammatory response associated with ovulation.

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employed to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.

Release of decay-accelerating factor into stools of patients with colorectal cancer by means of cleavage at the site of glycosylphosphatidylinositol anchor

The expression of decay-accelerating factor (DAF), a cell-membrane–complement regulator, is enhanced in colorectal cancer, and DAF is detected in the stools of patients with colorectal cancer. In this study, to elucidate mechanisms whereby DAF is released into the colonic lumen, we analyzed and compared the properties of DAF in stools and colorectal-cancer tissues. Stool specimens taken before surgery and tissue samples from surgically resected colorectal cancers were obtained from 21 patients. We analyzed DAF in stool and tissue specimens using immunoblotting, ultracentrifugation, and phase separation with Triton X-114. We analyzed the expression profile of DAF mRNA in cancer tissues using reverse transcription–polymerase chain reaction to determine whether DAF transcripts for a secretory form of DAF were present. With the use of immunoblotting, stool DAF was detected as a broad band with a molecular weight of around 70,000 kDa that migrated slightly more slowly than cancer-tissue DAF. About 90% of stool DAF was present as a soluble form that remained in the 100,000 g supernatant after ultracentrifugation. On phase separation with Triton X-114, the soluble stool DAF was partitioned mainly into the aqueous phase, indicating its hydrophilic nature and lack of the fatty-acid glycosylphosphatidylinositol anchor component. In colorectal cancer tissues, reverse transcription–polymerase chain reaction experiments revealed a nonspliced DAF messenger RNA that encodes a secretory form of DAF in just 2 of the 21 specimens examined. These data suggest that DAF is released from colorectal cancer cells by way of cleavage of membrane-bound DAF at the site of the glycosylphosphatidylinositol anchor.

Decay-accelerating factor induction by tumour necrosis factor-alpha, through a phosphatidylinositol-3 kinase and protein kinase C-dependent pathway, protects murine vascular endothelial cells against complement deposition.

We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription-polymerase chain reaction (RT-PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-alpha (TNF-alpha) increased EC DAF expression, detectable at 6 hr and maximal at 24-48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-alpha. DAF upregulation was mediated via a protein kinase C (PKC)alpha, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury.

In Vivo and in Vitro Evidence Suggest That HB-EGF Regulates Endometrial Expression of Human Decay-Accelerating Factor

Human endometrium expresses the critical complement component C3 in a cyclic fashion, with the highest expression in the secretory phase. As activated complement can kill cells, self or foreign, the secretory endometrial epithelium protects itself by concomitant expression of complement-protective proteins. The objectives of our present study were to describe the spatial and temporal regulation of the complement-protective protein decay-accelerating factor (DAF) in human endometrium and to identify local regulators of its expression. To describe the cyclic regulation of DAF, immunohistochemistry was performed using the IH4 monoclonal antibody on secretory phase endometrial biopsies taken from normal fertile volunteers in LH-timed cycles (n = 114). DAF expression in human endometrium was predominantly localized to the apical membrane of glandular and luminal epithelium. DAF expression, as assessed by histological scoring analysis, was minimal in the proliferative and early secretory phases and increased markedly on approximately day LH +7 (lumen) and LH +8 (glands). Maximal expression was seen in both glands and lumen by LH +8, and this persisted into menses. Using the RL95-2 endometrial epithelial cancer cell line as a model system, we next examined the cellular regulation of DAF. Treatment with E2 and progesterone, alone or in combination, had little effect on DAF expression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment increased cell surface and total DAF protein, increasing the signal by 260% on flow cytometry and by 200% on Western blot analysis. Stimulation of DAF protein expression was dose dependent, with maximal expression seen at 1 ng/ml. The stimulatory effects of HB-EGF were also observed at the mRNA level. EGF had effects similar to those of HB-EGF on DAF mRNA and protein expression, suggesting that the HB-EGF effect was mediated at least in part by the Her1 EGF receptor subunit. These studies suggest that DAF expression in the midsecretory phase is stimulated by HB-EGF or other members of the EGF family and may function to protect the epithelial integrity of human endometrium in the face of increased complement expression.

In vivo and in vitro evidence suggest that HB-EGF regulates endometrial expression of human decay-accelerating factor.

Human endometrium expresses the critical complement component C3 in a cyclic fashion, with the highest expression in the secretory phase. As activated complement can kill cells, self or foreign, the secretory endometrial epithelium protects itself by concomitant expression of complement-protective proteins. The objectives of our present study were to describe the spatial and temporal regulation of the complement-protective protein decay-accelerating factor (DAF) in human endometrium and to identify local regulators of its expression. To describe the cyclic regulation of DAF, immunohistochemistry was performed using the IH4 monoclonal antibody on secretory phase endometrial biopsies taken from normal fertile volunteers in LH-timed cycles (n = 114). DAF expression in human endometrium was predominantly localized to the apical membrane of glandular and luminal epithelium. DAF expression, as assessed by histological scoring analysis, was minimal in the proliferative and early secretory phases and increased markedly on approximately day LH +7 (lumen) and LH +8 (glands). Maximal expression was seen in both glands and lumen by LH +8, and this persisted into menses. Using the RL95-2 endometrial epithelial cancer cell line as a model system, we next examined the cellular regulation of DAF. Treatment with E2 and progesterone, alone or in combination, had little effect on DAF expression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment increased cell surface and total DAF protein, increasing the signal by 260% on flow cytometry and by 200% on Western blot analysis. Stimulation of DAF protein expression was dose dependent, with maximal expression seen at 1 ng/ml. The stimulatory effects of HB-EGF were also observed at the mRNA level. EGF had effects similar to those of HB-EGF on DAF mRNA and protein expression, suggesting that the HB-EGF effect was mediated at least in part by the Her1 EGF receptor subunit. These studies suggest that DAF expression in the midsecretory phase is stimulated by HB-EGF or other members of the EGF family and may function to protect the epithelial integrity of human endometrium in the face of increased complement expression.

Sodium butyrate enhances complement-mediated cell injury via down-regulation of decay-accelerating factor expression in colonic cancer cells.

Decay-accelerating factor (DAF) expressed on the surface of colonic cancer cells presents a barrier to complement-mediated clearance by contributing to the ineffectiveness of the humoral immune response. In this study, to investigate the mechanisms responsible for the anti-tumor effects of butyrate, we evaluated how butyrate modulates DAF expression in colonic cancer cells. Three colonic cancer cell lines (HT-29, Caco-2, and T84 cells) were studied. DAF protein expression was assessed by western blot, and DAF mRNA expression was evaluated by northern blot. Complement C3 deposition on the surface of colonic cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). The promoter activity of the DAF gene was assessed by a reporter gene-luciferase assay. Butyrate reduced the basal and interleukin-4 (IL-4)- and tumor necrosis factor-alpha (TNF-alpha)-induced expression of DAF protein and mRNA in HT-29 cells. It increased the susceptibility to complement attack and enhanced C3 deposition on HT-29 cells. The inhibitory effect of butyrate on DAF mRNA expression was also observed in T84 and Caco-2 cells. Butyrate decreased basal DAF expression at both transcriptional and post-transcriptional levels. The inhibitory effect of butyrate on IL-4-induced DAF expression was closely associated with a blockade of IL-4-induced DAF mRNA stability. TNF-alpha-induced transcriptional activation and the increased stability of the DAF gene were also blocked by butyrate. Similar but weak effects were induced by trichostatin A, a potent histone deacetylase inhibitor, suggesting that histone acetylation might participate in butyrate activity. These observations indicate that both a down-regulation of DAF expression and the induction of susceptibility to complement attack contribute to the anti-tumor effects of butyrate in colonic cancer.

Intestinal trefoil factor induces decay-accelerating factor expression and enhances the protective activities against complement activation in intestinal epithelial cells.

Mucosal damage induces a massive influx of serum complement components into the lumen. The epithelium produces a number of factors that can potentially ameliorate injury including intestinal trefoil factor (ITF), a small protease-resistant peptide produced and secreted onto the mucosal surface by goblet cells, and decay-accelerating factor (DAF), a protein produced by columnar epithelium which protects the host tissue from autologous complement injury. However, coordination of these intrinsic defensive products has not been delineated. DAF protein and mRNA expression were evaluated by immunoblotting and Northern blotting, respectively. NF-kappaB-DNA binding activity and DAF promoter activity were assessed by an electrophoretic gel mobility shift assay and a reporter gene luciferase assay, respectively. ITF induced a dose- and time-dependent increase in DAF protein and mRNA expression in human (HT-29 and T84) and rat (IEC-6) intestinal epithelial cells. In differentiated T84 cells grown on cell culture inserts, basolateral stimulation with ITF strongly enhanced DAF expression, but apical stimulation had no effects. The C3 deposition induced by complement activation was significantly blocked by the treatment with ITF. In HT-29 cells, ITF increased the stability of DAF mRNA. ITF also enhanced the promoter activity of the DAF gene via NF-kappaB motif and induced activation of NF-kappaB-DNA binding activity. ITF promotes protection of epithelial cells from complement activation via up-regulation of DAF expression, contributing to a robust mucosal defense.

Epidermal growth factor induces expression of decay-accelerating factor in human colonic cancer cells via the mitogen-activated protein kinase pathway

The expression of decay-accelerating factor (DAF), a complement regulatory protein, is enhanced in colorectal cancer. In this study, to elucidate mechanisms for enhanced DAF expression, we studied the effects of growth factors on DAF expression in HT-29 human colonic cancer cells. Cells were treated with epidermal growth factor (EGF), insulin-like growth factor-I, platelet-derived growth factor, and transforming growth factor-β. DAF protein expression and mRNA expression were determined with enzyme immunoassay and Northern blot analysis. The signaling pathways that target DAF expression in response to growth factor stimulation were characterized by using various inhibitors of the signal transduction pathway. EGF induced significant increases in DAF protein and mRNA expression in HT-29 cells; the other growth factors had a weak effect or no effect. The EGF-induced DAF expression was inhibited by mitogen-activated protein (MAP) kinase kinase inhibitor PD 98059 but not by phosphatidylinositol-3 kinase inhibitor, phospholipase Cγ inhibitor, or protein kinase C inhibitor. When we analyzed the phosphorylation state of the MAP kinase by immunoblot analysis, phosphorylated p44/p42 MAP kinase was detected in EGFstimulated HT-29 cells, and the addition of PD 98059 abrogated the phosphorylation. These results indicate that EGF regulates DAF expression in HT-29 cells via the signaling pathway that depends on the activation of MAP kinase. (J Lab Clin Med 2001;138:186-92)

Polymorphic expression of decay-accelerating factor in human colorectal cancer.

We have previously shown that expression of decay-accelerating factor (DAF), a complement regulatory protein, is enhanced immunohistochemically on the luminal surface of cancer glands in human colorectal cancer and is detected in stool specimens of patients with colorectal cancer. The amount of DAF present in the stools might be influenced by the stability of DAF on the cell surface which is regulated by biochemical properties such as glycosylation of the protein. In the present study, to help elucidate the mechanism for the release of DAF from human colorectal cancers, we biochemically analyzed DAF expression by western and northern blotting by using surgically resected specimens of colorectal cancers. Surgically resected colorectal cancer tissues were obtained from 10 patients. Expression of DAF was determined by western and northern blotting, and glycosylation of DAF protein was analyzed with glycosidase digestion. Northern blot analysis demonstrated that the expression of DAF mRNA in colorectal cancer was enhanced two- to threefold compared with normal tissues. In western blotting, expression of DAF protein in the cancer tissue was increased, and heterogeneity in the apparent molecular weight of DAF was observed among patients. When o-linked sugars were removed, this heterogeneity of DAF size diminished. The polymorphic expression of DAF in colorectal cancer is likely to reflect variability in the o-glycosylation of the protein. We speculate that this variability could affect the stability of DAF on the surfaces of cancer cells and, in turn, the amount of DAF shed into the stools of colorectal cancer patients.

Alternative exon usage in the 3′region of a single gene generates glycosylphosphatidylinositol-anchored and transmembrane forms of rat decay-accelerating factor

Several cDNA clones encoding genetic homologues of human decay-accelerating factor (DAF) were isolated from rat testis cDNA libraries and reverse transcription-polymerase chain reaction products. Sequence analysis revealed that rat DAF exists as two membrane forms, a glycosylphosphatidylinositol (GPI)-anchored and a transmembrane (TM) form. Southern blotting analysis showed that GPI- and TM-form mRNAs of rat DAF are derived from a single gene, as is the case for guinea pig, though both forms of mouse DAF mRNA are derived from two genes. Gene analysis of the C-terminal region of rat DAF indicated that both forms of mRNA are presumably generated by alternative splicing of exons which encode a GPI/3′-untranslated (3′UT) region and a TM/3′UT region in the 3′ end of the Ser/Thr-rich region. Northern blot analysis indicated that rat GPI-DAF mRNA was present in all tissues examined except for liver, while rat TM-DAF mRNA was preferentially expressed in testis. Infant rat testis barely expressed TM-DAF and membrane cofactor protein (MCP) but highly expressed 5I2 antigen (5I2Ag, rat Crry). However, adult rat testis showed increased expression of the 1.6-kb transcript of rat GPI-DAF, TM-DAF, MCP, and the 0.7-kb transcript of CD59, with a decreased level of 5I2Ag expression. These results suggest that rat 5I2Ag/Crry may play an important role in regulating complement activation during the early stages of testis development, and that DAF, MCP, and CD59 expressed in testis may enable sperm to survive complement attack in the mucus of the female genital organ, and/or to interact with an ovum.

Alternative exon usage in the 3' region of a single gene generates glycosylphosphatidylinositol-anchored and transmembrane forms of rat decay-accelerating factor.

Several cDNA clones encoding genetic homologues of human decay-accelerating factor (DAF) were isolated from rat testis cDNA libraries and reverse transcription-polymerase chain reaction products. Sequence analysis revealed that rat DAF exists as two membrane forms, a glycosylphosphatidylinositol (GPI)-anchored and a transmembrane (TM) form. Southern blotting analysis showed that GPI- and TM-form mRNAs of rat DAF are derived from a single gene, as is the case for guinea pig, though both forms of mouse DAF mRNA are derived from two genes. Gene analysis of the C-terminal region of rat DAF indicated that both forms of mRNA are presumably generated by alternative splicing of exons which encode a GPI/3'-untranslated (3'UT) region and a TM/3'UT region in the 3' end of the Ser/Thr-rich region. Northern blot analysis indicated that rat GPI-DAF mRNA was present in all tissues examined except for liver, while rat TM-DAF mRNA was preferentially expressed in testis. Infant rat testis barely expressed TM-DAF and membrane cofactor protein (MCP) but highly expressed 5I2 antigen (5I2Ag, rat Crry). However, adult rat testis showed increased expression of the 1.6-kb transcript of rat GPI-DAF, TM-DAF, MCP, and the 0.7-kb transcript of CD59, with a decreased level of 5I2Ag expression. These results suggest that rat 5I2Ag/Crry may play an important role in regulating complement activation during the early stages of testis development, and that DAF, MCP, and CD59 expressed in testis may enable sperm to survive complement attack in the mucus of the female genital organ, and/or to interact with an ovum.

Induction of Decay-Accelerating Factor by Cytokines or the Membrane-Attack Complex Protects Vascular Endothelial Cells Against Complement Deposition

AbstractVascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

Induction of Decay-Accelerating Factor by Cytokines or the Membrane-Attack Complex Protects Vascular Endothelial Cells Against Complement Deposition

Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNF) and interferon gamma (IFNγ) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

Expression of decay accelerating factor mRNA and complement C3 mRNA in human diseased kidney

Decay accelerating factor (DAF), a product of mesangial cells in vitro, is expressed on the surface of cells and is a candidate for the focal suppression of complement activation. It is not clear at present whether the levels of expression of DAF and intrarenal C3 synthesis correlate with the level of tissue injury. Immunohistochemistry for DAF and C3 and nonradioactive in situ hybridization with digoxigenin-labeled oligonucleotide probe for DAF and C3 mRNA were performed in 22 tissue samples of kidneys from patients with IgA nephropathy (IgAN), 6 with membranous nephropathy (MN), 6 with lupus nephritis (LN), and five normal kidneys. In the normal kidney, DAF was confined to the juxtaglomerular apparatus and little or no C3 was detected; however, a few glomerular cells were positive for DAF mRNA but no C3 mRNA positive cells were detected. In diseased kidneys, DAF and C3 as well as their mRNAs were detected in mesangial cells, tubular cells and infiltrating cells. Glomerular epithelial cells and Bowman's capsule cells contained little or no DAF and C3 but were positive for their mRNAs. The mean percentages of mesangial cells positive for DAF and C3 mRNAs were 49.3 +/- 11.5% and 50.7 +/- 10.3% in IgAN, and 17.0 +/- 6.3% and 19.4 +/- 9.0% in MN, respectively. The percentage of mesangial cells positive for DAF and C3 mRNAs among intraglomerular cells correlated positively with the degree of mesangial proliferation and glomerular sclerosis in IgAN. In contrast, in LN the percentage of glomerular cells positive for DAF mRNA correlated negatively with the degree of glomerular injury, while the percentage of cells positive for C3 mRNA did not change with the progression of the disease. The ratio of C3 mRNA/DAF mRNA of glomerular cells correlated with the degree of glomerular injury in both IgAN and LN. In the tubulointerstitium, the percentage of cells expressing mRNA, and C3 mRNA/DAF mRNA radio correlated with the degree of tubular atrophy and interstitial broadening in both IgAN and LN. We conclude that DAF and C3 mRNAs are synthesized in human diseased kidneys, and that a balance between locally synthesized DAF and C3 may be important in the progression of glomerulonephritis.

Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa.