JBC INTERFERin siRNA transfection reagent

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M201503200 on March 7, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16900-16905, May 10, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/19/16900    most recent
M201503200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, F.
Right arrow Articles by Wu, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, F.
Right arrow Articles by Wu, Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Processing of Pro-atrial Natriuretic Peptide by Corin in Cardiac Myocytes*

Faye Wu, Wei Yan, Junliang Pan, John Morser, and Qingyu WuDagger

From the Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94804

Received for publication, February 13, 2002, and in revised form, March 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Corin is a type II transmembrane serine protease abundantly expressed in the heart. In a previous study using transfected 293 cells, we showed that corin converted pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), suggesting that corin is likely the pro-ANP convertase. Because other serine proteases such as thrombin and kallikrein had previously also been shown to cleave pro-ANP in vitro, it remained to demonstrate that corin is indeed the endogenous pro-ANP convertase in cardiomyocytes. In this study, we examined pro-ANP processing in a murine cardiac muscle cell line, HL-5. Northern analysis showed that corin mRNA was present in HL-5 cells. In HL-5 cells transfected with a plasmid expressing pro-ANP, recombinant pro-ANP was converted to mature ANP as determined by Western analysis, indicating the presence of the endogenous pro-ANP convertase in these cells. The processed recombinant ANP was shown to be active in an enzyme-linked immunosorbent assay-based cGMP assay in baby hamster kidney cells. The processing of recombinant pro-ANP in HL-5 cells was highly sequence-specific, because mutation R98A, but not mutations R101A and R102A, in pro-ANP prevented the conversion of pro-ANP to ANP. Expression of recombinant wild-type corin enhanced the processing of pro-ANP in HL-5 cells. In contrast, overexpression of active site mutant corin S985A or transfection of oligonucleotide small interfering RNA duplexes directed against the mouse corin gene completely inhibited the processing of recombinant pro-ANP in HL-5 cells. These results indicate that corin is the physiological pro-ANP convertase in cardiac myocytes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Atrial natriuretic peptide (ANP)1 is a cardiac hormone stored in the dense granules of cardiac myocytes. In response to high blood pressure, ANP is secreted into the circulation. In target organs such as kidney and peripheral vessels, ANP binds to its receptor and stimulates the intrinsic guanylyl cyclase activity of the receptor, leading to production of intracellular cGMP. The biological effects of ANP are to promote salt excretion, reduce blood volume, and relax vessel tension, thereby reducing blood pressure (1, 2). The biological importance of the ANP-mediated pathway in maintaining normal blood pressure has been demonstrated in a number of studies. In knockout mice, for example, deficiency in either ANP or its receptor led to spontaneous hypertension (3, 4). High plasma concentrations of ANP and brain-type natriuretic peptide (BNP) are found in patients with congestive heart failure. The levels of these natriuretic peptides are often correlated with the extent of ventricular dysfunction and development of cardiac arrhythmias (5, 6). ANP and BNP have been used as therapeutic agents in patients with decompensated congestive heart failure and acute myocardial infarction to improve cardiac function and clinical status (7, 8). Administration of ANP has also been used as therapy in patients with renal failure (9).

In cardiac myocytes, ANP is synthesized as a 126-amino acid prepropeptide (10, 11). After the signal peptide is removed, pro-ANP is stored in the dense granules of the cell. Upon secretion from the dense granules, pro-ANP is activated on the surface of cardiac myocytes by proteolytic cleavage at residue arginine 98, generating an N-terminal propeptide and a mature 26-amino acid C-terminal peptide that is biologically active (12, 13). Several studies showed that a high-molecular-weight trypsin-like enzyme associated with the membrane of cardiac myocytes was responsible for the activation cleavage of pro-ANP (14-16). Despite tremendous efforts, the identity of the pro-ANP convertase remained unknown for many years.

Recently, we cloned a unique serine protease, corin, from the human heart (17). Sequence analysis indicates that corin is a mosaic protein composed of a distinctive assortment of domains. At its N terminus, corin has a cytoplasmic domain and an integral transmembrane domain. In the extracellular region of corin, there are two frizzled-like cysteine-rich motifs, eight low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus (17, 18). The overall topology of corin is similar to those of other type II transmembrane serine proteases of the trypsin superfamily (19), such as hepsin (20, 21), and enterokinase (22). In a functional study using transfected human embryonic kidney 293 cells, we showed that recombinant human corin converted pro-ANP to ANP, suggesting that corin is likely the long-sought pro-ANP convertase (23).

To further demonstrate that corin is indeed the endogenous enzyme responsible for the processing of pro-ANP in cardiac myocytes, we studied pro-ANP processing in a murine cardiac myocytic cell line, HL-5. Here we show that expression of recombinant human wild-type corin enhanced the processing of pro-ANP in HL-5 cells. In contrast, overexpression of active site mutant corin S985A or transfection of oligonucleotide siRNA duplexes directed against the mouse corin gene completely blocked the processing of pro-ANP. These results strongly support corin being the physiological pro-ANP convertase in the heart.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Penicillin, streptomycin, L-glutamine, fetal bovine serum (FBS), and cell culture medium were purchased from Invitrogen (Rockville, MD). The murine cardiac myocytic cell line HL-5 was kindly provided by Dr. William C. Claycomb (Louisiana State University Medical Center, New Orleans, LA). Human embryonic kidney 293 cells and baby hamster kidney (BHK) cells were obtained from the American Type Culture Collection (ATCC) and maintained at the Core Facility at Berlex Biosciences. Anti-V5 antibody was purchased from Invitrogen (Carlsbad, CA). All other chemical reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell Culture-- HL-5 cells were cultured in Ex-Cell 320 medium (JRH Biosciences, Lenexa, KS) containing 10% FBS, 15 µg/ml insulin, 50 µg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), 1 µM retinoic acid, 0.1 mM norepinephrine, 100 µg/ml penicillin/streptomycin, 292 µg/ml L-glutamine, and 0.1 mM MEM non-essential amino acids. Human 293 cells were cultured in alpha -MEM (Life Technologies Inc.) supplemented with 10% FBS and 1% L-glutamine. BHK cells were cultured in MEM (Invitrogen) supplemented with 10% FBS. All cells were cultured at 37 °C in humidified incubators with 5% CO2 and 95% air.

Reverse Transcriptase-PCR (RT-PCR)-- To examine cardiac-specific gene expression in HL-5 cells, mRNA was isolated from HL-5 cells using a commercial mRNA preparation kit (Micro-Fast Track 2.0 mRNA isolation kit, Invitrogen). RT-PCR reactions were carried out using the One-Step RT-PCR Advantage Kit (CLONTECH, Palo Alto, CA) with 30 cycles of amplification (30-s annealing at 65 °C, 1-min extension at 68 °C, and 30-s denaturation at 94 °C). The following oligonucleotide primers specific for mouse cardiac genes were used in this study: corin (sense 5'-CGCTGCATTGCGGCTGAGTGGGTG-3' and antisense 5'-CTGGCCGTCACATCTCCTGGA-3') (24); pro-ANP (sense 5'-CAGAGTGGGCAGAGACAGCA-3' and antisense 5'-TTGCTTTCAAGAGGGCAGATCTAT-3') (25); alpha -cardiac myosin heavy chain (alpha -MHC) (sense 5'-CTGCTGGAGAGGTTATTCCTCG-3' and antisense 5'-GGAAGAGTGAGCGGCGCATCAAGG-3') (26); connexin43 (sense 5'-GTTCAAGTACGGGATTGAAGAGCACGGCAA-3' and antisense 5'-TGGTTTTCTCCGTGGGACGTGAGAGGAAGC-3') (27); alpha -cardiac actin (sense 5'-TGTTACGTCGCCTTGGATTTGAG-3' and antisense 5'-AAGAGAGAGACATATCAGAAGC-3') (28); and alpha -skeletal actin (sense 5'-TATTCCTTCGTGACCACAGCTGAACGT-3' and antisense 5'-CGCGAACGCAGACGCGAGTGCGC-3') (28).

Northern Analysis-- Poly(A)+ RNA samples from mouse heart and lung were purchased from CLONTECH. mRNA from HL-5 cells was isolated using the Micro-Fast Track 2.0 mRNA isolation kit (Invitrogen). RNA samples (2.5 µg each) were fractionated on denaturing agarose gels and transferred onto nitrocellulose membranes. Northern hybridization was performed with a 32P-labeled mouse corin cDNA probe at 42 °C overnight in a solution containing 40% formamide, 5% Denhardt's solution, 6× SSC, 100 µg/ml salmon sperm DNA, and 0.1% SDS. As a control for mRNA sample loading, Northern blots were reprobed with a mouse beta -actin cDNA probe (CLONTECH).

Site-directed Mutagenesis-- Plasmid constructs expressing human wild-type corin (pcDNACorin), active site mutant corin S985A (pcDNACorinS985A), human wild-type pro-ANP (pcDNAproANP), and mutant pro-ANPs R101A (pcDNAproANPR101A) and R102A (pcDNAproANPR102A) were generated by site-directed mutagenesis and described previously (23). Plasmids expressing active site mutant prothrombin S205A (pPTS205A) and active site mutant hepsin S353A (pHepsinS353A) were generated by a PCR-based mutagenesis method. Plasmid pcDNAproANPR98A expressing mutant pro-ANP R98A, in which residue Arg-98 was replaced by Ala, was constructed by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA) with sense primer 5'-ATCTGCCCTCCTAAAAAGCAA-3' and antisense primer 5'-GATCTCCGCAGGCTCGGCAGGGGCAGTGAGCAG-3'. To facilitate detection of recombinant proteins, a tag sequence was included in the constructs that expresses a viral V5 and a His tag at the C terminus of recombinant pro-ANP. All plasmid constructs were confirmed by DNA sequencing.

Transfection and Western Analysis-- Transient transfection was performed in HL-5 or 293 cells using Lipofectin (Invitrogen) according to the manufacturer's instructions. Conditioned medium was collected 48 or 72 h after transfection. To analyze pro-ANP processing, recombinant pro-ANP and its derivatives in the conditioned medium were immunoprecipitated by an anti-V5 antibody (Invitrogen). Proteins were separated by SDS-PAGE and analyzed by Western blotting using a horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen).

cGMP Assay-- To examine the activity of recombinant ANP, a cGMP assay was performed using an enzyme immunoassay (EIA) kit (Biotrak, Amersham Biosciences, Inc.). In this assay, BHK cells were grown in 96-well plates in MEM medium supplemented with 10% FBS and 1% of L-glutamine. Confluent cells were washed once with serum-free medium. The conditioned medium (180 µl) containing recombinant pro-ANP and its derivatives from transfected HL-5 or 293 cells was added to each well and incubated at 37 °C for 10 min. The cells were lysed by addition of a lysis buffer (20 µl/well) containing 2% dodecyl trimethylammonium and 50 mM sodium acetate, pH 5.8. The intracellular cGMP concentration in ANP-stimulated BHK cells was determined with the Biotrak EIA kit. Each experimental condition was assayed in quadruplicate.

Effects of Protease Inhibitors-- Recombinant pro-ANP was expressed in 293 cells transfected with the pro-ANP-expressing plasmid. The conditioned medium containing recombinant pro-ANP was incubated with HL-5 cells at 37 °C for 4 h in the presence of one of the following: 2.5 mg/ml aprotinin, 20 mg/ml benzamidine, 25 mg/ml leupeptin, or 12.5 mg/ml soybean trypsin inhibitor. The conditioned medium was collected, and the processing of pro-ANP was analyzed by SDS-PAGE and Western blotting using an anti-V5 antibody.

Effects of RNA Interference-- Oligonucleotide siRNAs were designed based on sequences specific for mouse corin cDNA (5'-AACCACCGCCCUCCGUGCA-3' and 5'-AACACAAGCACCUGCAUGAAC-3') (24). Antisense and sense siRNA oligonucleotides with dTdT 3'-overhang were synthesized and annealed by Dharmacon Research, Inc. (Lafayette, CO). Transfection of HL-5 and 293 cells with the siRNA duplexes was performed according to the manufacturer's instruction. Briefly, HL-5 cells or 293 cells expressing corin were grown in 24-well cell culture plates. Increasing concentrations of siRNA duplexes were added to a solution containing Oligofectamine (3 µl) and Opti-MEM (100 µl) (Invitrogen) and incubated at room temperature for 25 min. The mixture was then added to HL-5 or 293 cells and incubated at 37 °C for 24 h. The cells were washed twice with serum-free medium and then incubated with the conditioned medium containing recombinant pro-ANP at 37 °C for 4 h. Pro-ANP and its derivatives in the conditioned medium were analyzed by immunoprecipitation and Western blotting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Corin mRNA in HL-5 Cells-- The murine HL-5 cell line was derived from the AT-1 atrial cardiomyocyte tumor lineage developed in transgenic mice overexpressing simian virus 40 large T antigen under the control of the ANP promoter (29, 30). The AT-1 cells were maintained by serial subcutaneous grafts in C57BL/6J mice and used to establish cardiac muscle cell lines that are stable under culture conditions. Previous studies showed that a similar cell line, HL-1, derived from the same AT-1 tumor lineage, exhibited electrophysiological and pharmacological features that were characteristic of adult cardiac myocytes (31). To determine if HL-5 cells also maintain a cardiac-specific gene expression profile, the expression of a set of selected cardiac-specific genes was examined by RT-PCR. As shown in Fig. 1, transcripts of the ANP, alpha -MHC, connexin43, and alpha -cardiac actin genes were detected in RNA samples from HL-5 cells and the adult mouse heart. By Northern hybridization (Fig. 2), a corin transcript of ~5 kb was detected in mRNA samples from HL-5 cells and the adult heart. In contrast, ANP, alpha -MHC, connexin43, alpha -cardiac actin, and corin mRNAs were not detected in samples from the mouse lung by the RT-PCR or Northern analyses. As a control, alpha -skeletal actin mRNA was detected in samples from the lung but not HL-5 cells and the heart (Figs. 1 and 2). These results are consistent with the previous report that the AT-1 tumor-derived cells maintain characteristics of adult atrial cardiomyocytes (31) and demonstrate that corin mRNA is present in HL-5 cells.


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 1.   Analysis of cardiac gene expression in HL-5 cells by RT-PCR. RNA samples were prepared from HL-5 cells and mouse heart and lung tissues. RT-PCR experiments were performed using oligonucleotide primers derived from murine corin, ANP, alpha -MHC, connexin43, alpha -cardiac actin, and alpha -skeletal actin cDNA sequences. PCR products for corin (700 bp), ANP (491 bp), alpha -MHC (302 bp), connexin43 (221 bp), and alpha -cardiac actin (494 bp) mRNA were detected in samples from HL-5 (upper panel) and the heart (middle panel) but not the lung (lower panel). In contrast, PCR products for alpha -skeletal actin (562 bp) were detected in samples from the lung but not HL-5 cells and the heart. As a negative control, no PCR products were detected when RT-PCR reactions were performed in the absence of mRNA samples (lane 1, upper, middle, and lower panels).


View larger version (52K):
[in this window]
[in a new window]
  		Fig. 2.   Northern analysis of corin mRNA expression. Northern blots were prepared using mRNA samples derived from HL-5 cells or mouse heart and lung tissues, as described under "Experimental Procedures." Northern hybridization was performed with a mouse corin cDNA probe. Corin mRNA was detected in samples from HL-5 cells and the heart but not the lung (upper panel). As a control, the blot was re-probed with a mouse actin probe. Actin mRNA was detected in all three samples (lower panel).

Processing of Pro-ANP in HL-5 Cells-- To study the processing of pro-ANP in HL-5 cells, transfection studies were performed using plasmids expressing human pro-ANP and corin. As reported in our previous study (23) and shown here as a control (Fig. 3A), processing of recombinant pro-ANP to ANP in 293 cells was not detected unless a corin-expressing plasmid was co-transfected with the pro-ANP-expressing plasmid. In contrast, in HL-5 cells co-transfection of the corin-expressing plasmid was not required for the conversion of recombinant pro-ANP to ANP. Processing of pro-ANP was detected in HL-5 cells transfected with only the pro-ANP-expressing plasmid (Fig. 3B), suggesting that recombinant pro-ANP was processed by endogenous corin present in HL-5 but not 293 cells. Expression of recombinant corin further enhanced the processing of pro-ANP in HL-5 cells, as shown in Fig. 3B, when the HL-5 cells were co-transfected with the corin-expressing plasmid. In a separate experiment, recombinant pro-ANP was incubated with either HL-5 cells or transfected 293 cells expressing recombinant corin. Western analysis showed similar processing of recombinant pro-ANP by both cell types (Fig. 3C). These results are consistent with the previous findings that corin is a transmembrane protease present on the surface of cardiac myocytes (18, 23).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 3.   Processing of recombinant pro-ANP in HL-5 and transfected 293 cells. Transfection experiments were performed in human 293 cells (panel A) or murine HL-5 cells (panel B) using pro-ANP expression plasmid (pcDNAproANP) together with either a control plasmid (pcDNA) or corin expression plasmid (pcDNACorin). Recombinant pro-ANP and its derivatives in the conditioned medium were analyzed by Western blotting using an anti-V5 antibody (upper sections in panels A and B). To show that recombinant corin was expressed in transfected cells, Western analysis was performed using an anti-V5 antibody. Recombinant corin was detected in cell lysate from 293 and HL-5 cells transfected with the corin-expressing plasmid (lower sections in panels A and B). C, conditioned medium containing recombinant human pro-ANP was incubated with either HL-5 cells or 293 cells expressing recombinant corin. Processing of pro-ANP was analyzed by Western blotting using an anti-V5 antibody. At high resolution, two bands of pro-ANP were detected on the Western blots possibly caused by differences in glycosylation in transfected cells.

The Activity of Recombinant ANP-- The biological function of ANP is mediated through its receptor that is present on the surface of targeted cells. The binding of ANP to its receptor stimulates the intrinsic guanylyl cyclase activity of the receptor, leading to generation of intracellular cGMP. To determine if the corin-processed recombinant ANP is biologically active, a cell-based cGMP assay was performed. As shown in Fig. 4, cGMP-stimulating activity was detected in the conditioned medium from 293 cells transfected with both corin and pro-ANP-expressing plasmids. In contrast, little cGMP-stimulating activity was detected in the conditioned medium from 293 cells transfected with either corin or pro-ANP-expressing plasmids alone (Fig. 4). In a parallel study, low levels of the cGMP-stimulating activity were detected in the conditioned medium from parental HL-5 cells (Fig. 4). The activity was most likely due to the presence of native ANP produced in HL-5 cells being processed by the native corin. The cGMP-stimulating activity was significantly increased in the conditioned medium from HL-5 cells transfected with corin and pro-ANP-expressing plasmids, either independently or simultaneously (Fig. 4). These results are consistent with the Western analysis of the processing of recombinant pro-ANP in 293 and HL-5 cells (Fig. 3), and demonstrate that corin-processed recombinant ANP is biologically active.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Stimulation of intracellular cGMP production. BHK cells were cultured in 96-well plates. The conditioned medium from parental or transfected 293 (solid bars) or HL-5 (open bars) cells was added to each well and incubated at 37 °C for 10 min. The cells were lysed by addition of a lysis buffer containing 2% dodecyl trimethylammonium and 50 mM sodium acetate, pH 5.8. The intracellular concentration of cGMP in BHK cells was determined with the Biotrak EIA kit, as described under "Experimental Procedures." Each experimental condition was assayed in quadruplicate.

Sequence Specificity of Pro-ANP Cleavage in HL-5 Cells-- In the heart, pro-ANP is processed by proteolytic cleavage at residue Arg-98, generating a 28-amino acid mature C-terminal peptide that is the major circulating form of ANP. In other tissues, including brain, kidney, and testis, proteolytic cleavage of pro-ANP can occur at other residues such as Arg-101 and Arg-102 (32, 33). Earlier we had shown that corin specifically cleaved pro-ANP at residue Arg-98 in transfected 293 cells (23). To determine if pro-ANP is also processed at Arg-98 in HL-5 cells, transfection experiments were performed in HL-5 cells using plasmids expressing human wild-type and mutant pro-ANPs. Recombinant pro-ANP and its derivatives in the conditioned medium were analyzed by Western blotting. As shown in Fig. 5, recombinant wild-type pro-ANP and mutant pro-ANPs R101A and R102A were processed in HL-5 cells. In contrast, no cleavage was detected when mutant pro-ANP R98A was expressed in HL-5 cells, indicating that mutation at Arg-98 prevented specific processing of pro-ANP by endogenous corin in the cardiac myocytes.


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 5.   Sequence specificity of pro-ANP cleavage. A control vector (pcDNA) or expression vectors for wild-type pro-ANP (pcDNAproANP) or mutant pro-ANPs R98A (pcDNAproANPR98A), R101A (pcDNAproANPR101A) and R102A (pcDNAproANPR102A) were transfected into HL-5 cells. Recombinant pro-ANP and its derivatives in the conditioned medium were analyzed by Western analysis using an anti-V5 antibody.

Effects of Protease Inhibitors-- Previous studies showed that the processing of pro-ANP by a partially purified protease present in membrane fractions from cardiac myocytes was inhibited by high concentrations (100 mg/ml) of benzamidine, leupeptin, and aprotinin but not soybean trypsin inhibitor (15). Similar effects of the protease inhibitors were observed on recombinant corin-mediated processing of pro-ANP in 293 cells (23). To examine effects of the protease inhibitors on the processing of pro-ANP in HL-5 cells, recombinant pro-ANP was incubated with HL-5 cells in the presence of these protease inhibitors. Western analysis showed that benzamidine, leupeptin, and aprotinin but not soybean trypsin inhibitor inhibited the processing of pro-ANP by HL-5 cells (Fig. 6). The results further support the hypothesis that corin is the endogenous pro-ANP convertase in the cardiac myocytes.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of serine protease inhibitors. Recombinant pro-ANP was expressed in 293 cells. The conditioned medium containing recombinant pro-ANP was incubated with HL-5 cells at 37 °C for 4 h in the absence or presence of one of the following serine protease inhibitors: benzamidine (20 mg/ml), soybean trypsin inhibitor (12.5 mg/ml), leupeptin (25 mg/ml), or aprotinin (2.5 mg/ml). Processing of pro-ANP was analyzed by SDS-PAGE and Western blotting using an anti-V5 antibody.

Effects of Overexpression of an Active Site Mutant Corin-- If corin is indeed the endogenous pro-ANP convertase, it may be possible to inhibit corin-mediated pro-ANP processing in HL-5 cells by overexpression of active site mutant corin S985A, in which the active site serine residue has been replaced by Ala. To test this hypothesis, an experiment was performed in which plasmids expressing pro-ANP and mutant corin S985A were co-transfected in HL-5 cells. As shown in Fig. 7, the processing of recombinant pro-ANP was inhibited when HL-5 cells were co-transfected with increasing concentrations of the plasmid expressing mutant corin S985A. In controls, co-transfection of plasmids expressing active site mutant human hepsin S353A or active site mutant human prothrombin S205A did not inhibit the processing of recombinant pro-ANP in HL-5 cells (data not shown). These results indicate that active site mutant corin S985A competed with endogenous corin, thereby inhibiting the processing of recombinant pro-ANP in HL-5 cells.


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of overexpression of mutant corin S985A. A plasmid expressing wild-type pro-ANP (pcDNAproANP) was co-transfected into HL-5 cells with either a control vector (5 µg, pcDNA) or increasing concentrations (0.5, 5, and 20 µg) of an expression vector for mutant corin S985A (pcDNACorinS985A). The conditioned medium was collected, and recombinant pro-ANP and its derivatives were analyzed by Western analysis using an anti-V5 antibody (upper panel). To show that recombinant mutant corin S985A was expressed in transfected HL-5 cells, Western analysis was performed using an anti-V5 antibody. Recombinant mutant corin S985A was detected in cell lysate from the transfected HL-5 cells (lower panel).

Effects of siRNA-- To further demonstrate the requirement of corin for the pro-ANP processing in cardiac myocytes, we used the RNA interference (RNAi) technique to block endogenous corin expression in HL-5 cells. This powerful oligonucleotide siRNA-mediated gene-silencing technique has been used successfully to prevent gene expression in cultured mammalian cells (34). In HL-5 cells, co-transfection of the plasmid expressing pro-ANP with increasing concentrations of oligonucleotide siRNA duplexes directed specifically against the mouse corin gene inhibited the pro-ANP processing in a dose-dependent manner (Fig. 8). In contrast, the siRNA duplexes had no inhibitory effects on the pro-ANP processing in 293 cells expressing recombinant human corin, demonstrating the sequence specificity of the siRNA duplexes (Fig. 8). Although we were unable to verify the down-regulation of mouse corin protein expression in the transfected HL-5 cells due to lack of antibodies against mouse corin, these results are consistent with the notion that inhibition of endogenous corin expression prevented the pro-ANP processing in the cardiac myocyte.


View larger version (59K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of siRNA duplexes against the mouse corin gene. Recombinant pro-ANP was expressed in 293 cells. The conditioned medium containing recombinant pro-ANP was collected and incubated at 37 °C for 4 h with HL-5 cells transfected with increasing concentrations (0.1, 1, and 10 µg) of oligonucleotide siRNA duplexes against the mouse corin gene. As controls, mock transfected HL-5 cells (HL-5 + mock transfection) were included in the experiments. To show the sequence specificity of siRNA, 293 cells expressing recombinant human corin (293/corin) were also transfected with siRNA (10 µg) against the mouse corin gene. The processing of pro-ANP in the conditioned medium was analyzed by Western analysis using an anti-V5 antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteolytic cleavage of propeptides is a fundamental process in the activation of polypeptide hormones. For most of the peptide hormones, the activation cleavage is mediated by a family of subtilisin-like serine proteases, known as precursor convertases (PCs). These convertases are located in the secretory pathway inside of the cell and cleave proproteins at specific sequences commonly composed of single or paired basic amino acids (35, 36). Processing of pro-ANP, however, appears to be mediated by a different mechanism. Following removal of its signal peptide, pro-ANP is stored in the dense granules of cardiac myocytes. The activation cleavage occurs on the cell surface when the pro-hormone is secreted from the cells. Earlier biochemical studies have indicated that a high-molecular-weight trypsin-like protease present on the cell surface is responsible for the processing of pro-ANP in cardiac myocytes (14-16).

Our discovery of the cardiac transmembrane serine protease, corin, and subsequent characterization of its function indicate that corin is probably the pro-ANP convertase. Corin mRNA was detected in tissues such as heart, kidney, testis, uterus, and bone where pro-ANP and other natriuretic peptides are known to be expressed (17). Biochemical studies showed that the corin protein had a molecular mass of ~150 kDa and was associated with the cell membrane (23). In transfected 293 cells, recombinant corin cleaved pro-ANP to produce a small peptide that was indistinguishable from the mature ANP by SDS-PAGE and Western analysis. Mutagenesis analysis showed that the corin-mediated cleavage of pro-ANP was highly sequence-specific. It remained, however, to show that corin is indeed the endogenous pro-ANP convertase in cardiac myocytes, because other serine proteases such as thrombin and kallikrein have also been reported in the past to cleave pro-ANP in vitro (37, 38).

In this study, we characterized the corin-mediated pro-ANP processing in a murine cardiac myocyte cell line, HL-5, that was derived from the murine AT-1 atrial cardiomyocyte tumor lineage. Previous studies have shown that AT-1-derived cells maintain a cardiac-specific phenotype (31). AT-1-derived cells, for example, exhibit a gene expression pattern that is very similar to that of adult atrial cardiac myocytes. AT-1-derived cells possess pro-ANP-containing dense granules and are capable of processing the prohormone when it is secreted from the cells. In RT-PCR and Northern analyses (Figs. 1 and 2), we showed that both corin and pro-ANP genes were expressed in HL-5 cells. In a cell-based cGMP assay, ANP activity was detected in the conditioned medium derived from HL-5 cells (Fig. 4), demonstrating endogenous processing of pro-ANP and constitutive secretion of active ANP from these cells. These results indicate that HL-5 cells are an excellent model for studying the role of corin in pro-ANP processing in cardiac myocytes.

In transfection studies, we showed that recombinant pro-ANP was processed by an endogenous enzyme present in HL-5 cells. Characteristically, the processing of pro-ANP in HL-5 cells was indistinguishable from the corin-mediated processing of pro-ANP in 293 cells. In both cell types, for example, the proteolytic activity was present on the cell surface (Fig. 3C), the cleavage in pro-ANP was sequence-specific (Fig. 5), and serine protease inhibitors had similar effects (Fig. 6). We also showed that transfection of a plasmid expressing corin enhanced pro-ANP processing in HL-5 cells and that the corin-processed ANP was biologically active as measured in a cell-based cGMP assay (Fig. 4). Moreover, overexpression of the active site mutant of corin S985A in HL-5 cells completely prevented the pro-ANP processing in HL-5 cells (Fig. 7). The dominant effect of mutant corin S985A in HL-5 cells appeared to be specific, because expression of two other active site mutant trypsin-like serine proteases, hepsin S353A and prothrombin S205A, did not inhibit the pro-ANP processing in HL-5 cells. In RNAi-mediated gene silencing experiments, oligonucleotide siRNA duplexes directed against the mouse corin gene inhibited the pro-ANP processing in HL-5 cells (Fig. 8). The specificity of the oligonucleotide siRNA duplexes for mouse corin and not for other sequences was demonstrated by the lack of effect on pro-ANP conversion in 293 cells expressing human corin (Fig. 8). Together, these results strongly indicate that corin is the physiological pro-ANP convertase in cardiac myocytes.

At the present time, the mechanism by which mutant corin S985A exhibited its dominant negative effect in HL-5 cells is not known, but several mechanistic possibilities exist. It is unlikely that the inhibitory effect of mutant corin S985A was caused by dimerization with the endogenous corin, because there was no evidence in Western analysis that corin formed dimers (23). Similarly, competition for occupancy of a limited number of sites in the plasma membrane is possible but unlikely. A more plausible mechanism is that the mutant corin may compete with the endogenous corin for substrate binding through either its protease domain or its distinct motifs in the propeptide domain such as frizzled-like cysteine-rich motifs, low density lipoprotein receptor repeats and macrophage scavenger receptor-like domain. The frizzled-, low density lipoprotein receptor-, and macrophage scavenger receptor-like motifs are known to be important in protein-protein interactions (39-41). In addition to competition for substrate binding, the mutant corin may also compete with the endogenous corin for an activator. The corin amino acid sequence indicates that corin, like most other trypsin-like serine proteases, is synthesized as a zymogen and that a proteolytic cleavage at residue Arg-801 by a trypsin-like enzyme is required for its activation (17). At this time, the identity of such a protease activator is completely unknown. Further study of corin will help to better understand corin-mediated pro-ANP processing and may also lead to discovery of other proteins that regulate corin activity.

    ACKNOWLEDGEMENT

We thank Dr. W. Dole for his support and encouragement.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804. Tel.: 510-669-4737; Fax: 510-669-4246; E-mail: qingyu_wu@berlex.com.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201503200

    ABBREVIATIONS

The abbreviations used are: ANP, atrial natriuretic peptide; ATCC, American Type Culture Collection; BHK, baby hamster kidney; BNP, brain-type natriuretic peptide; EIA, enzyme immunoassay; FBS, fetal bovine serum; alpha -MHC, alpha -cardiac myosin heavy chain; PCs, precursor convertases; pro-ANP, pro-atrial natriuretic peptide; RNAi, RNA interference; RT-PCR, reverse transcriptase-polymerase chain reaction; siRNA, small interfering RNA; MEM, minimal essential medium; AT-1, atrial myocyte tumor lineage; pc DNA, mammalian expression vector.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Levin, E. R., Gardner, D. G., and Samson, W. K. (1998) N. Engl. J. Med. 339, 321-328[Free Full Text]
2. Wilkins, M. R., Redondo, J., and Brown, L. A. (1997) Lancet 349, 1307-1310[CrossRef][Medline] [Order article via Infotrieve]
3. John, S. W., Krege, J. H., Oliver, P. M., Hagaman, J. R., Hodgin, J. B., Pang, S. C., Flynn, T. G., and Smithies, O. (1995) Science 267, 679-681[Abstract/Free Full Text]
4. Lopez, M. J., Wong, S. K., Kishimoto, I., Dubois, S., Mach, V., Friesen, J., Garbers, D. L., and Beuve, A. (1995) Nature 378, 65-68[CrossRef][Medline] [Order article via Infotrieve]
5. Burnett, J. C., Jr., Kao, P. C., Hu, D. C., Heser, D. W., Heublein, D., Granger, J. P., Opgenorth, T. J., and Reeder, G. S. (1986) Science 231, 1145-1147[Abstract/Free Full Text]
6. Gottlieb, S. S., Kukin, M. L., Ahern, D., and Packer, M. (1989) J. Am. Coll. Cardiol. 13, 1534-1539[Abstract]
7. Colucci, W. S., Elkayam, U., Horton, D. P., Abraham, W. T., Bourge, R. C., Johnson, A. D., Wagoner, L. E., Givertz, M. M., Liang, C. S., Neibaur, M., Haught, W. H., and LeJemtel, T. H. (2000) N. Engl. J. Med. 343, 246-253[Abstract/Free Full Text]
8. Hayashi, M., Tsutamoto, T., Wada, A., Maeda, K., Mabuchi, N., Tsutsui, T., Horie, H., Ohnishi, M., and Kinoshita, M. (2001) J. Am. Coll. Cardiol. 37, 1820-1826[Abstract/Free Full Text]
9. Allgren, R. L., Marbury, T. C., Rahman, S. N., Weisberg, L. S., Fenves, A. Z., Lafayette, R. A., Sweet, R. M., Genter, F. C., Kurnik, B. R., Conger, J. D., and Sayegh, M. H. (1997) N. Engl. J. Med. 336, 828-834[Abstract/Free Full Text]
10. Schwartz, D., Geller, D. M., Manning, P. T., Siegel, N. R., Fok, K. F., Smith, C. E., and Needleman, P. (1985) Science 229, 397-400[Abstract/Free Full Text]
11. Thibault, G., Garcia, R., Gutkowska, J., Bilodeau, J., Lazure, C., Seidah, N. G., Chretien, M., Genest, J., and Cantin, M. (1987) Biochem. J. 241, 265-272[Medline] [Order article via Infotrieve]
12. Shields, P. P., and Glembotski, C. C. (1988) J. Biol. Chem. 263, 8091-8098[Abstract/Free Full Text]
13. Ito, T., Toki, Y., Siegel, N., Gierse, J. K., and Needleman, P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8365-8369[Abstract/Free Full Text]
14. Seidah, N. G., Cromlish, J. A., Hamelin, J., Thibault, G., and Chretien, M. (1986) Biosci. Rep. 6, 835-844[CrossRef][Medline] [Order article via Infotrieve]
15. Imada, T., Takayanagi, R., and Inagami, T. (1988) J. Biol. Chem. 263, 9515-9519[Abstract/Free Full Text]
16. Sei, C. A., Hand, G. L., Murray, S. F., and Glembotski, C. C. (1992) Mol. Endocrinol. 6, 309-319[Abstract]
17. Yan, W., Sheng, N., Seto, M., Morser, J., and Wu, Q. (1999) J. Biol. Chem. 274, 14926-14935[Abstract/Free Full Text]
18. Hooper, J. D., Scarman, A. L., Clarke, B. E., Normyle, J. F., and Antalis, T. M. (2000) Eur. J. Biochem. 267, 6931-6937[Medline] [Order article via Infotrieve]
19. Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857-860[Free Full Text]
20. Leytus, S. P., Loeb, K. R., Hagen, F. S., Kurachi, K., and Davie, E. W. (1988) Biochemistry 27, 1067-1074[CrossRef][Medline] [Order article via Infotrieve]
21. Wu, Q. (2001) Front. Biosci. 6, D192-D200[Medline] [Order article via Infotrieve]
22. Kitamoto, Y., Yuan, X., Wu, Q., McCourt, D. W., and Sadler, J. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7588-7592[Abstract/Free Full Text]
23. Yan, W., Wu, F., Morser, J., and Wu, Q. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8525-8529[Abstract/Free Full Text]
24. Tomita, Y., Kim, D. H., Magoori, K., Fujino, T., and Yamamoto, T. T. (1998) J Biochem. (Tokyo) 124, 784-789[Abstract/Free Full Text]
25. Seidman, C. E., Bloch, K. D., Klein, K. A., Smith, J. A., and Seidman, J. G. (1984) Science 226, 1206-1209[Abstract/Free Full Text]
26. Robbins, J., Gulick, J., Sanchez, A., Howles, P., and Doetschman, T. (1990) J. Biol. Chem. 265, 11905-11909[Abstract/Free Full Text]
27. Beyer, E. C., Paul, D. L., and Goodenough, D. A. (1987) J. Cell Biol. 105, 2621-2629[Abstract/Free Full Text]
28. Alonso, S., Minty, A., Bourlet, Y., and Buckingham, M. (1986) J. Mol. Evol. 23, 11-22[CrossRef][Medline] [Order article via Infotrieve]
29. Field, L. J. (1988) Science 239, 1029-1033[Abstract/Free Full Text]
30. Borisov, A. B., and Claycomb, W. C. (1995) Ann. N. Y. Acad. Sci. 752, 80-91[Medline] [Order article via Infotrieve]
31. Claycomb, W. C., Lanson, N. A., Jr., Stallworth, B. S., Egeland, D. B., Delcarpio, J. B., Bahinski, A., and Izzo, N. J., Jr. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2979-2984[Abstract/Free Full Text]
32. Pandey, K. N., and Orgebin-Crist, M. C. (1991) Biochem. Biophys. Res. Commun. 180, 437-444[CrossRef][Medline] [Order article via Infotrieve]
33. Shiono, S., Nakao, K., Morii, N., Yamada, T., Itoh, H., Sakamoto, M., Sugawara, A., Saito, Y., Katsuura, G., and Imura, H. (1986) Biochem. Biophys. Res. Commun. 135, 728-734[CrossRef][Medline] [Order article via Infotrieve]
34. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Nature 411, 494-498[CrossRef][Medline] [Order article via Infotrieve]
35. Barr, P. J. (1991) Cell 66, 1-3[CrossRef][Medline] [Order article via Infotrieve]
36. Seidah, N. G., and Chretien, M. (1999) Brain Res. 848, 45-62[CrossRef][Medline] [Order article via Infotrieve]
37. Corthorn, J., Cantin, M., and Thibault, G. (1991) Mol. Cell. Biochem. 103, 31-39[CrossRef][Medline] [Order article via Infotrieve]
38. Moreau, T., Brillard-Bourdet, M., Chagas, J., and Gauthier, F. (1995) Biochim. Biophys. Acta 1249, 168-172[CrossRef][Medline] [Order article via Infotrieve]
39. Bhanot, P., Brink, M., Samos, C. H., Hsieh, J. C., Wang, Y., Macke, J. P., Andrew, D., Nathans, J., and Nusse, R. (1996) Nature 382, 225-230[CrossRef][Medline] [Order article via Infotrieve]
40. Brown, M. S., Herz, J., and Goldstein, J. L. (1997) Nature 388, 629-630[CrossRef][Medline] [Order article via Infotrieve]
41. Matsumoto, A., Naito, M., Itakura, H., Ikemoto, S., Asaoka, H., Hayakawa, I., Kanamori, H., Aburatani, H., Takaku, F., Suzuki, H., Kobari, Y., Miyai, T., Takahashi, K., Cohen, E. H., Wydro, R., Housman, D. E., and Kodama, T. (1990) Proc. Natl. Acad. Sci. U. S. A.  87, 9133-9137[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Circ. Res.Home page
W. Wang, X. Liao, K. Fukuda, S. Knappe, F. Wu, D. L. Dries, J. Qin, and Q. Wu
Corin Variant Associated With Hypertension and Cardiac Hypertrophy Exhibits Impaired Zymogen Activation and Natriuretic Peptide Processing Activity
Circ. Res., August 29, 2008; 103(5): 502 - 508.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
D. Enshell-Seijffers, C. Lindon, and B. A. Morgan
The serine protease Corin is a novel modifier of the agouti pathway
Development, January 15, 2008; 135(2): 217 - 225.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Liao, W. Wang, S. Chen, and Q. Wu
Role of Glycosylation in Corin Zymogen Activation
J. Biol. Chem., September 21, 2007; 282(38): 27728 - 27735.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
J. E. Rame, M. H. Drazner, W. Post, R. Peshock, J. Lima, R. S. Cooper, and D. L. Dries
Corin I555(P568) Allele Is Associated With Enhanced Cardiac Hypertrophic Response to Increased Systemic Afterload
Hypertension, April 1, 2007; 49(4): 857 - 864.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Y.-F. Xiao, E. M. TenBroek, J. J. Wilhelm, P. A. Iaizzo, and D. C. Sigg
Electrophysiological characterization of murine HL-5 atrial cardiomyocytes
Am J Physiol Cell Physiol, September 1, 2006; 291(3): C407 - C416.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
I. Giuliani, F. Rieunier, C. Larue, J.-F. Delagneau, C. Granier, B. Pau, M. Ferriere, M. Saussine, J.-P. Cristol, A.-M. Dupuy, et al.
Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood
Clin. Chem., June 1, 2006; 52(6): 1054 - 1061.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
D. L. Dries, R. G. Victor, J. E. Rame, R. S. Cooper, X. Wu, X. Zhu, D. Leonard, S.-I. Ho, Q. Wu, W. Post, et al.
Corin Gene Minor Allele Defined by 2 Missense Mutations Is Common in Blacks and Associated With High Blood Pressure and Hypertension
Circulation, October 18, 2005; 112(16): 2403 - 2410.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. C. Y. Chan, O. Knudson, F. Wu, J. Morser, W. P. Dole, and Q. Wu
Hypertension in mice lacking the proatrial natriuretic peptide convertase corin
PNAS, January 18, 2005; 102(3): 785 - 790.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
L. B. Yap, D. Mukerjee, P. M. Timms, H. Ashrafian, and J. G. Coghlan
Natriuretic Peptides, Respiratory Disease, and the Right Heart
Chest, October 1, 2004; 126(4): 1330 - 1336.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. H. Langenickel, I. Pagel, J. Buttgereit, K. Tenner, M. Lindner, R. Dietz, R. Willenbrock, and M. Bader
Rat corin gene: molecular cloning and reduced expression in experimental heart failure
Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1516 - H1521.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. L. Tran, X. Lu, M. Lei, Q. Feng, and Q. Wu
Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium
Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1625 - H1631.
[Abstract] [Full Text] [PDF]

Home page