Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors

Lingling Jiang,1 Jiafang Wang,1 R. Sergio Solorzano-Vargas,2 Hugh V. Tsai,1 Edgar M Gutierrez,1 Luis O. Ontiveros,1 Pawel R. Kiela,3 S. Vincent Wu,4 and Martín G. Martín1

Departments of 1Pediatrics and 4Medicine, Division of Gastroenterology and Nutrition, Mattel Children's Hospital, David Geffen School of Medicine at University of California Los Angeles, Los Angeles 90095; 2Department of Biology, California State University Northridge, Northridge, California 91330; and 3Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724

Submitted 20 March 2003 ; accepted in final form 13 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor (FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at –157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.

passive immunity; development; ontogeny; immunoglobulin


MAMMALS ARE BORN WITH A SYSTEMIC and mucosal adaptive immune system that is immature and incapable of providing sufficient protection against the wide assortment of antigens and bacteria that are encountered after birth (34). Many mammals, including humans, acquire passive immunity by transplacental flow of maternal IgG and through breast milk, which is a rich source of both IgG and secretory IgA. In those species that obtain IgG in utero, the most abundant immunoglobulin in breast milk is secretory IgA, which is unabsorbed and represents the main component of the humoral mucosal immune system during the suckling period of development. The in utero transfer of IgG is absent in other species including rodents, cattle, pigs, sheep, and horses, whereas as neonates, serum levels of IgG in these species are entirely dependent on passive transfer from breast milk (7).

The receptor that transfers maternally derived IgG to the systemic circulation of suckling animal is the Fc receptor of the neonate (FcRn) (28). FcRn is expressed by syncytiotrophoblast of the placenta, where it mediates the transplacental transfer of IgG (14). In the small intestine of suckling mammals, FcRn is expressed by enterocytes located along the entire length of the crypt-to-villus axis and is responsible for the uptake of luminal IgG across the epithelial layer (1). In rodents, FcRn mRNA levels are most abundant in the duodenum, and steadily decline along the horizontal axis of the small bowel (10, 18). In humans, FcRn has been detected in intestinal epithelial cells, where it may play a role in sampling luminal antigens and possibly mediate the transfer of IgG during breast feeding (27).

FcRn is a heterodimer that is noncovalently bound to {beta}2-microglobulin and is related to the family of nonclassic major histocompatibility complex (MHC) class Ib proteins (23). FcRn transports IgG, in a process termed transcytosis, from apical (intestinal lumen) to basolateral membranes (systemic circulation) (2). At the apical membrane, IgG-FcRn complexes form at an acidic pH and are endocytosed in clathrin-coated vesicles, which subsequently sort to intermediate compartments before trafficking to the basolateral membrane. At the basolateral membrane, the complex dissociates at the neutral pH, and IgG is released into the systemic circulation (25). Overall, FcRn serves an essential role in controlling the transfer of IgG from the maternal to the fetal/newborn circulation of all mammals.

In rodents and other mammals, the systemic humoral IgG immune system matures shortly after the cessation of breast feeding, and steady-state FcRn mRNA levels in the small bowel decline dramatically in weaned animals to nearly undetectable levels (10, 17, 18). The FcRn mRNA transcript has also been detected at much lower quantities in liver and vascular endothelial cells compared with levels seen in the small bowel of suckling animals (2). In vascular endothelial cells, FcRn has been shown to represent the "Brambell receptor" and control the extremely low rate of IgG catabolism (3, 11). Interestingly, hypogammaglobulinemia attributable to rapid IgG catabolism has been described in myotonic dystrophy and in a rare autosomal recessive disorder (OMIM 241600 [OMIM] ) (31, 32). Therefore, in humans, the FcRn protein controls serum IgG levels and may sample luminal antigens for presentation to the mucosal adaptive immune system.

Insufficient information is currently available regarding the regulation of the FcRn expression. Corticosteroids, thyroxine, EGF, and insulin have all been shown to decrease IgG transfer via the intestinal enterocyte of suckling animals (30). We have reported that the administration of either corticosteroids or thyroxine to suckling pups abolishes both IgG absorption and FcRn mRNA expression in a dose- and time-dependent manner (16). The reduction of FcRn expression in endothelial cells may explain the benefit of steroids and intravenous immunoglobulins in the management of various autoimmune disorders (16, 33).

Whereas the sequences of the human and murine FcRn genes have been reported, the elements that are critical in directing FcRn promoter activity have not been identified (12, 22). In a recent analysis of the human promoter, several elements were identified that form complexes with nuclear extracts; however, the study failed to adequately define the important elements that drive FcRn gene expression (22). To further characterize the mechanism of how the FcRn gene is regulated, we cloned the upstream region of the rat gene and identified the cis-acting elements that control its basal expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and Characterization of Rat Genomic Clones

Approximately 106 recombinant phage clones from a rat (Sprague Dawley) genomic library (Stratagene) were plated and blotted in duplicate on nitrocellulose filters. The filters were screened with a PCR-derived cDNA corresponding to nucleotides 500 to 1000 by standard methods (19, 28). Liquid phage stocks were produced of the phage clones that remained positive after tertiary screening, and we performed genomic mapping by digesting DNA with partial or double restriction enzymes that included BamHI, EcoRI, HindIII, and SmaI (New England Biolaboratories). DNA was fractionated on a 0.4% agarose gel, transferred to nitrocellulose filters, and hybridized to [{gamma}-P32]dATP (New England Nuclear Research Products) end-labeled oligonucleotides including T3, T7, and regions internal to the FcRn cDNA.

Intron/Exon Mapping and DNA Sequence Analysis

Genomic DNA containing each of the exons was digested, subcloned into Bluescript SKI (–), and purified by the standard Qiagen method. Each exon, the intron/exon boundary and 2.9 Kb of the 5' upstream region, was sequenced by the dideoxy method with [{alpha}-35S]dATP (New England Nuclear Research Products) and Sequenase 2 (United States Biochemical).

RNase Protection and Northern Blot Analysis

RNase protection assay was performed according to previously described methods (10). Specifically, total RNA was isolated by standard guanidine-phenol extraction methods and hybridized to radiolabeled {alpha}-sense riboprobe. After overnight incubation in a specific buffer, the mixture will be digested with RNaseA/T1 mixture for 30 min and electrophoresed on a polyacrylamide gel. Northern blot analysis was performed with 20 µg of total RNA isolated as previously described from Caco-2, HT-29, IEC-6, and endothelial SV40 cells (29). The human (Caco-2 and HT-29) and rodent (IEC-6, endothelial SV40) RNA samples were hybridized to the human or rat cDNA probe, respectively.

DNA Cloning and Substitution Mutagenesis

The FcRn #1 phage clone was digested with EcoRI, and the 3.3-Kb fragment was subcloned into a similarly digested pBluescript vector. The vector was then digested with BamHI, and a 2.9-Kb fragment was subcloned into the BglII site of the reporter clone pGL3-basic (Promega). The orientation was confirmed by sequencing, and the pGL3-basic 2.9-Kb FcRn clone was used to generate nested deletion clones whose size was confirmed by sequencing. Six sense, –450/–430 (5'-gca gat ctT CCT GAC AAG ATC TTG GGT TG), –300/–283 (5'-gca gat ctG AGA GTG GGC TGC AGC AGG), –216/–198 (5'-gca gat ctT GCT CAG AAT GAG TAA ACA C), –157/–139 (5'-gca gat ctT GCT CAG AAT GAG TAA ACA C), –101/–95 (5'-gca gat ctC CCC AGG AGG CTT CCA GA), and –58/–40 (5'-gca gat ctT GCT TAA GAG CTC GTG GGG T) and an antisense +113/+80 (5'-ata agc ttC CCT CTT CCT CAC AGA AGC C) oligonucleotides were developed to amplify various shorter-length products from rat genomic DNA. The fragments were digested with HindIII/BglII (restriction site indicated in lowercase lettering) and subcloned into the pGL3-enhancer luciferase reporter vector (Promega). Clones rFcRn–450/+135/E-Luc, rFcRn–300/+135/E-Luc, rFcRn–216/+135/E-Luc, rFcRn–157/+135/E-Luc, rFcRn–101/+135/E-Luc, and rFcRn–58/+135/E-Luc were sequenced to define their size and authenticity.

To define the nucleotides within the core promoter that were critical to maintain the gene's basal level of expression, 20 clones containing 10-bp mutations were produced by standard scanning mutagenesis. The location of each mutation is shown in Fig. 1. Mutagenizing oligonucleotides that contain 10-bp central transversion mutations (A-C, G-T) were designed and used to perform PCR with the antisense (+113/+80) oligonucleotide (Table 1). The PCR products were gel purified and with the addition of a sense (–157/–139) oligonucleotide, a second PCR reaction was performed to generate clones with 10-bp mutations. The full-length fragments were cut with BglII/HindIII, gel purified, and subcloned into the pGL3-E vector. All clones were sequenced to verify the presence of only the desired mutation.



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 1. Comparison of the 5'-upstream region of the mouse and rat Fc receptor (FcRn). The conserved rat (r) and mouse (m) nucleotides are displayed with the shaded rectangles. Nucleotides are labeled relative to the start of transcription shown as a vertical arrow labeled –1. The location of each experimental mutation is labeled, and its boundaries are defined by parallel vertical bars. The locations of the cis-acting elements identified in this study are labeled (Sp-A to -F) with a thin horizontal rectangle positioned above the DNA element. The start of translation is indicated by the ATG site at position 729.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Mutagenizing oligonucleotides

 
Drosophila SL2 cells were used to assess the role of the Sp family of transcription factors in controlling promoter activity. Because Sp1 alters the promoter activity of promoterless pGL3-basic, the wild-type FcRn's minimal promoter (–157/+135) was subcloned into p{beta}-Galactosidase ({beta}-Gal)-basic (Clontech) (13).

Cell Transfection Procedure

Caco-2, 3T3, Chinese hamster ovary (CHO), HeLa, and m-ICcl2 (kindly provided by Alain Vandewalle) cells were transfected with a calcium phosphate reagents as previously described (21). IEC-6 cells were electroporated with a BioRad RF module. Each pulse had 10 µg of luciferase construct and 1 µg of renilla vector for 4 x 106 cells. This mixture was then pulsed at 0% modulation, 0.2 kV, 50 Hz, for 2 ms, with 1-s intervals for five bursts. Cells were incubated for 48 h, rinsed with PBS, and lysed in SDS containing buffer (Analytical Luminescence Laboratory), and extracts were stored at –80°C. Both luciferase and renilla were measured as previously described (29).

Drosophila SL2 cells were maintained in Schneider's insect medium supplemented with 10% fetal bovine serum and antibiotics and were grown at 25°C without CO2. Transfection of SL-2 cells was performed as previously described (13). Specifically, 200 ng of rFcRn–157/+135/{beta}Gal were transfected with various amounts of Sp1 or Sp3 expression plasmids containing the Drosophila actin 5 promoter (pPacSp1 and pPacUSp3 were generously provided by Drs. R. Tjian and G. Suske, respectively). {beta}-Gal activity was expressed as relative light units per milligram of protein. The protein concentration was determined by bicinchoninic acid protein assay (Pierce).

DNase I Footprint Analysis

DNase I footprint analysis was performed to define the approximate region of protein-DNA interactions as previously described (20). The rFcRn–216/+135/E-Luc vector was cut with HindIII, kinased with [{gamma}-P32]dATP (6,000 Ci/mol) and then cut with BglII. Samples were electrophoresed on a 5% polyacrylamide gel, purified, and diluted to 6,000 counts/min (cpm)/µl. Footprints were performed with Caco-2 nuclear extracts that were dialyzed with 0.1 M KCl. Either nuclear extracts (6 µg) or BSA was mixed with probe (6,000 cpm), and serial dilution of DNase I was added to the reaction buffer [in mM: 40 CaCl2, 10 HEPES (pH 7.9), 0.1 EDTA, 0.05 KCl, 28 2-{beta}-mercaptoethanol, and 0.1 BSA, with 10% glycerol]. Samples were purified with phenol and chloroform, precipitated with ethanol, and run on a 6% polyacrylamide gel for 100 min at 50 W. The locations of the protein/DNA binding sites were determined by running a DNA ladder sequenced by the Maxam-Gilbert method.

EMSA and Preparation of Nuclear Extract

Nuclear extracts were obtained from both Caco-2 cells by standard methods as previously described, and recombinant Sp1 was also used for a limited number of studies (Promega) (21). EMSA was performed using a series of double-stranded oligonucleotides, which were labeled with Klenow fragments. The identities of each oligonucleotide used in this study are included in results. Approximately 2 x 104 cpm of probe and 2.5–5 µg nuclear extracts were used for each reaction. Standard competition studies were run using excess unlabeled oligonucleotide duplexes. Antisera against Sp1, Sp2, and Sp3 were purchased from Santa Cruz Biotechnology and were used to perform supershift studies.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and structure of the rat FcRn gene. Three overlapping bacteriophage plaques were confirmed by screening and ranged in size from 11 to 19 Kb. The clones were isolated and restriction mapped with various 5', midregion, and 3' probes, and various regions (including 1.6-Kb SmaI, 6.8-, 3.3-, and 1.6-Kb EcoRI, and 8-Kb HindIII) were subcloned into pBluescript. The entire upstream region of the gene (2.6 Kb) along with each exon and intron-exon boundary was sequenced bidirectionally using standard dideoxy chain-termination reaction. The gene is composed of seven exons and is similar to other MHC class I genes with each domain encoded by a separate exon. In addition, the transmembrane and cytoplasmic domains are represented by two individual exons, and the exact intron-exon boundaries have been solved (data not shown). The transcriptional initiation site of the rat FcRn gene was determined by standard primer extension (data not shown) and RNase protection assays (Fig. 2A), and its location is depicted as a downward arrow in Fig. 1.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2. A-C: identification of FcRn's transcriptional start site, mRNA abundance in several cell lines, and DNase I footprints. A: total RNA from the proximal intestine of a 15 day-old rat was hybridized with 32P-labeled S2 and FcRn cRNA probes and precipitated without RNase digestion (lane 2). In contrast, lane 1 consists of the same RNA that was hybridized with the S2 cRNA probe and RNase digestion, whereas lane 4 was with an FcRn cRNA probe that was RNase digested. B: Northern blot analysis containing 2 independent samples of 20 µg of RNA hybridized with either human (Caco-2, HT-29) or rat (IEC-6 and SVEC) cDNA probes. C: footprint analysis was performed with nuclear extracts isolated from Caco-2 cells (lanes 4–6) or in the presence of BSA (lanes 1–3). DNA was labeled in the sense orientation in the presence of increasing concentrations of DNase I. The filled rectangles on the right depict the location of the footprint, and * represents the location of the hypersensitivity sites.

 
Abundant Steady-State Levels of FcRn mRNA are Detectable in Caco-2, HT-29, IEC-6, and an Endothelial Cell Line

We performed Northern blot analysis for the FcRn mRNA transcript in several cell lines including Caco-2, HT-29, IEC-6, and a rodent endothelial (SVEC) cell line. As shown in Fig. 2B, the FcRn transcript was readily abundant in each of the cell lines tested, and these data were interpreted to suggest that these cells lines would be good candidates to perform functional analysis of the FcRn promoter.

DNase I Footprint Analysis of Core Promoter Identifies Several DNA-Protein Complexes

To determine the approximate location of the cis-elements that bind to nuclear extracts, Caco-2 extracts were used to perform DNase I footprint analysis of the core promoter region. These data demonstrate that as many as two distinct footprints (–26 to –61 and –90 to –121) and five hypersensitivity sites were identified within the gene's immediate promoter region in the presence of Caco-2 nuclear extracts (Fig. 2C).

Transient Transfection of Clones Containing Various Lengths of the Upstream Region of the Rat FcRn Gene in Intestinal Cell Lines Identified the Core Promoter

To begin assessing the role of the various regions of the 5'-upstream portion of the FcRn gene, chimeric-luciferase reporter clones were transiently transfected into both Caco-2 and IEC-6 cells. Transfection of the full-length (rFcRn–2940/+14) and sequentially shorter reporter constructs resulted in reporter activity in both cell lines that ranged from 100- to 40-fold higher than the empty pGL3-basic vector (data not shown). Comparable data were obtained with several clones that contain a shorter length of the 5' region of the FcRn promoter region subcloned upstream of the reporter vector pGL3-enhancer (Fig. 3A). Reporter activity declined abruptly with clones shorter than FcRn–157/+135, suggesting that the core promoter region of the FcRn gene is located within nucleotides –157 and +135.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. A and B: transient transfection of various rat FcRn 5'-upstream deletion clones into intestinal and other cell lines. A: each clone is named by the size of the subcloned 5'-upstream region relative to the start of transcription. Chimeric promoter-reporter constructs were precipitated with a Renilla luciferase construct, transfected into either IEC-6 or Caco-2 cells, and processed 48 h later. B: the clone containing the minimal promoter (FcRn–157/+135/E-Luc) was transfected into various intestinal (Caco-2, IEC-6, m-ICcl2) and other [3T3, Chinese hamster ovary (CHO) and HeLa] cell lines. The data are shown as fold elevation of either empty pGL3-enhancer vector. Values are means ± SE of sextuplet data from 2 different experiments.

 
Transient Transfection of the Clone Containing the Core Promoter Region of FcRn in Intestinal and Other Cell Lines Suggests that the Promoter Drives Primarily Intestinal-Specific Gene Expression

To assess the cell specificity of the core promoter region of the FcRn gene, we transiently transfected several intestinal (Caco-2, IEC-6, and m-ICcl2) and nonintestinal (3T3, CHO, and HeLa) cell lines. Maximum expression was obtained in the three intestinal cell lines, with the most abundant expression occurring in Caco-2 cells (Fig. 3B). Whereas 3T3 cells supported the expression of the reporter, promoter activity in both CHO and HeLa cells was only marginal. These data suggest that the core promoter region (nt –157 and +135) of the FcRn gene is sufficient to drive abundant expression in primarily cells of intestinal origin.

Site-Directed Mutagenesis of the Core Promoter Revealed Two Critical Regions that Control Promoter Activity

To assess the critical regions within the core promoter (–157 to +135) that are responsible for basal expression, a series of 20 clones was developed that contained 10 base pair mutations (Table 1 and Fig. 1). When these clones were transfected into either Caco-2 or IEC-6 cells, mutations in two specific regions were shown to dramatically influence promoter activity (Fig. 4). In Caco-2 cells, promoter activity of the wild-type –157/+135 clone was more than 120-fold greater than the empty pGL3-enhancer vector. Clones with mutations in the M8–M11 regions had promoter activities that were ~25% of the wild-type rFcRn–157/+135 clone. In contrast, promoter activities of the clones containing mutations in M15–M17 region were only 30% of the empty pGL3-enhancer vector. Similar albeit less dramatic results were obtained with IEC-6 cells.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 4. Transient transfection of clones containing scanning mutations within the minimal promoter of FcRn. A series of clones containing 10 nucleotide mutations (shown in Fig. 1) was cotransfected with Renilla luciferase expressing vector into either Caco-2 or IEC-6 cells. The relative location of each mutation is represented by a black box on left. Data are presented as firefly luciferase per unit of Renilla luciferase and shown relative to the wild-type FcRn–157/+135/E-Luc clone. Values are means ± SD of sextuplet data from 2 separate experiments.

 
These data were interpreted to suggest that the M8–M11 and the M15–M17 regions interact with proteins that control basal expression of the FcRn promoter. The location of the DNA-protein footprints also corresponds to the approximate location of these two large transcriptional active sites (Fig. 2C). Specifically, the footprint between –26 and –61 corresponds to the region mutated in the M8–M11 clones, whereas the –90 and –121 complex overlaps the location of the M15–M17 mutations (Figs. 1, 2C, and 4). To assist in the identification of putative cis-acting elements in the core promoter region of the FcRn gene, we used the TRANSFAC database (8). The analysis showed that the promoter was highly enriched with GC residues and identified as many as five potential Sp sites (Sp-A to Sp-E) within the immediate upstream region of the promoter and an additional Sp site (Sp-F) just upstream of the minimal promoter (Figs. 1 and 5). Taken together, these data suggest the presence of at least two large distinct regions that are capable of binding to nuclear proteins and altering basal expression of the FcRn's core promoter region. Our subsequent analysis focused on exploring the potential role that the Sp family of proteins has in controlling basal expression of the rat FcRn minimal promoter.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5. The Sp1 binding site located in the immediate promoter region of the FcRn gene. The name and location of the specific sites are shown. The consensus Sp1 binding site is shown above, and the score on the right shows the extent to which the element conforms to the consensus Sp1 sequence.

 
EMSA's Identified Six Sp cis-Elements with Various Affinities to the Sp Family of Proteins

Sp-A, Sp-B, and Sp-C sites. To further evaluate the proteins responsible for controlling the basal promoter activity of the FcRn gene, we developed a series of double-stranded oligonucleotides to the M8–M13 region of the promoter. EMSA performed with the Sp-A oligonucleotide that spans from –47 to –22 forms two specific complexes that supershift with antiserum to various members of the Sp family of transcriptional factors including Sp1, Sp2, and Sp3 (Fig. 6, lanes 1–9). Specifically, the slower migrating complex is formed by binding of Sp1, Sp2, and other unidentified nuclear proteins, whereas the faster complex was entirely supershifted with an anti-Sp3 antibody. The –47/–22 probe was also capable of specifically binding recombinant Sp1 (Fig. 6, lanes 10 and 11), and competition with a group of duplex oligonucleotides containing mutations in regions that span –47 to –22 confirmed that the complexes form by interacting with regions located between –28 to –39 (data not shown). This Sp1 site was named Sp-A, and it corresponds to a classic Sp1 consensus sequence (Figs. 5 and 6).



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 6. The Sp-A element binds to members of the Sp family proteins. EMSA was performed using Caco-2 nuclear extracts and a labeled duplex oligonucleotide (–47/–22). Shown is a supershift analysis performed with different possible combinations of Sp1, Sp2, and Sp3 antiserum (lanes 1–9). Recombinant Sp1 also bound and could be competed off with 100x unlabeled –47/–22 oligonucleotide duplexes (lanes 10 and 11). The element in this region is labeled as Sp-A and is shown as a rectangle in the top portion of the figure. The location of mutants M9 and M8 are also shown relative to Sp-A for comparison.

 
The gel shift assay performed with the Sp-B probe that spans nt –66 to –22 also forms two complexes with Caco-2 nuclear extracts (Fig. 7, lane 1). Competition with 50-fold excess of a duplex wild-type oligonucleotide (–66 to –22) entirely competed for binding of the complexes (Fig. 7, lane 2), whereas competition with oligonucleotides that contained mutations in the Sp-B site ({Delta}–61 to –47), failed to compete for any of the complexes (Fig. 7, lanes 4-6). The identities of these complexes were confirmed using antiserum to Sp1, Sp2, and Sp3 (Fig. 7, lanes 8–16).



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 7. The Sp-B element binds to members of the Sp family proteins. EMSA was performed with Caco-2 nuclear extracts, and a duplex oligonucleotide that spans –66 to –42 was labeled. Competition was performed with 100-fold excess of either wild-type or mutant unlabeled oligonucleotides (lanes 2-7). The nucleotides mutated in each mutant oligonucleotide are indicated by {Delta}, and their location is depicted below the nucleotide sequence. Supershift results using specific antiserum to Sp1, Sp2, and Sp3 are shown in various combinations (lanes 8–15). Nonreactive (NR) serum is used for comparison (lane 16). The location of the Sp-B element is displayed in the top region of the figure, and the location of the M10 and M11 mutations is shown for comparison.

 
To identify the potential role of the more upstream Sp1 site, named Sp-C, we performed EMSA with a duplex oligonucleotide that spans –96 to –60 and contains only the Sp-C site. This Sp-C site forms two complexes that resemble the Sp family of proteins based on size as well as an intermediate smudge (Fig. 8, lanes 5 and 6). These complexes are in contrast to the two predominant complexes seen when either the Sp-A or Sp-B probe was used alone (Fig. 8, lanes 1–4). When a longer duplex oligonucleotide that spans –96 to –22 and contained the Sp-A, Sp-B, and Sp-C sites was labeled, three high-affinity complexes were detected (Fig. 8, lanes 11 and 12). It should be noted that the slower migrating complex was faintly visible with labeled oligonucleotides that contained individual Sp-A and Sp-B sites (Fig. 8, lanes 1–4), and we hypothesize that it may represent a multimerized form of the Sp family of proteins. Alternatively, the large complex (Fig. 8, lane 11) may represent simultaneous binding of Sp proteins to adjacent cognate Sp elements. Interestingly, when a duplex oligonucleotide that spans –96 to –22 and contained a mutation in the Sp-C site (Sp-A-B-c; {Delta}–78 to –69) was used as a probe, the two slower migrating complexes (1 and 2) were more abundant, suggesting that an intact Sp-C site impeded with the formation of these complexes (Fig. 8, lanes 13 and 14). Furthermore, the slower migrating complex failed to form when an oligonucleotide that spans –96 to –22 and contained a mutation in the Sp-A site (Sp-a-B-C; {Delta}–41 to –22) was used (Fig. 8, lanes 15 and 16), suggesting that the presence of both the Sp-A and Sp-B sites is required for the formation of the complex.



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 8. A size comparison of each of the complexes that interact with Sp-A to Sp-D. EMSA was performed with Caco-2 nuclear extracts and 9 duplex oligonucleotides that span from Sp-A to Sp-D, and their identities are discussed in the text. Competition was performed with 50-fold excess of the unlabeled wild-type oligonucleotide. Some oligonucleotides span several Sp sites, as indicated by the name of the probe. The uppercase letters that follow Sp is indicative of an intact element, whereas lowercase letters indicate that the probe contains a mutation within the particular Sp site.

 
To investigate the relative affinity of the Sp-A, Sp-B, and Sp-C elements, EMSA was performed with 50-fold competition of unlabeled duplex oligonucleotides. The Sp-A element has the highest affinity site, because it competes with itself and the Sp-B site (Fig. 9, lanes 1–4 and 7–10). In contrast, whereas the Sp-C site forms specific complexes, the site is only marginally capable of competing with Sp-A, while competing slightly better for the Sp-B site (Fig. 9, lanes 1–18). Interestingly, neither the Sp-A nor Sp-B site was capable of competing for the intermediate smudge complex (Fig. 9, lanes 13–16). Taken together, these data were interpreted to suggest the Sp-A and Sp-B sites bind to the Sp family of proteins (Sp1, Sp2, and Sp3) with high affinity, whereas the Sp-C site is a low-affinity Sp-element that is capable of interacting with a complex that is distinct from Sp at higher affinity. Moreover, the formation of the multimerized form of Sp requires the simultaneous presence of Sp-A and Sp-B, and that presence of Sp-C dampened the formation of this slow migrating complex.



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 9. The relative ability of each Sp site to compete with one another. EMSA was performed with Caco-2 nuclear extracts and 6 duplex oligonucleotides that span from Sp-A to Sp-D. Competition was performed with 50-fold excess of the unlabeled oligonucleotides as specified.

 
Sp-D and Sp-E sites To analyze the region of the promoter that was most adversely affected by the scanning mutagenesis of the promoter, duplex oligonucleotides (–125 to –87) were developed to the region spanning M15–M17, and it was named Sp-D-E (Figs. 1 and 4). EMSA performed with this probe formed two complexes that resemble the Sp family of proteins and a considerably fainter and slower migrating complex (Fig. 8, lanes 17–18). The Sp family of proteins appeared to bind the Sp-E site, because a probe that contained exclusively the Sp-D site (with mutations in the Sp-E site; Sp-D-e) fails to form the Sp-proteins (Fig. 8, lanes 7–8 and 17–18). In contrast, the slow migrating complex is specific for the Sp-D site, because the probe that contains Sp-E (with mutations in the Sp-D site; Sp-d-E) is incapable of forming this complex while retaining the ability to form complexes with Sp-proteins, albeit at lower affinity (Fig. 8, lanes 9–10 and 17–18). These data demonstrate that the cognate Sp-E element appears to form an interaction with proteins that resemble Sp-proteins (based on size), whereas the Sp-D site interacts with a component of the nuclear extract that forms a large complex that appears to facilitate binding of Sp-proteins to the Sp-E element.

To further assess the characteristics of the Sp-D and Sp-E complexes, we performed EMSA-competition experiments with the unlabeled Sp-A, Sp-B, and Sp-C oligonucleotides. Competition of the Sp-D-E probe with either Sp-A or Sp-B entirely competed for binding of the Sp family of proteins while failing to compete for the slower migrating complex (Fig. 9, lanes 31–33). Moreover, competition with the Sp-C and Sp-d-E oligonucleotides only partially competed for the Sp family of complexes, whereas the Sp-D-e oligonucleotides failed to compete entirely (Fig. 9, lanes 34–36). In contrast, removal of the Sp-D site (using probe Sp-d-E) interfered with the ability of even the high-affinity Sp-A site to compete for the Sp-complexes, although still retaining the ability to compete with the unlabeled Sp-d-E oligonucleotide (Fig. 9, lanes 25–30). Moreover, the slower migrating complex was completed by excess of either the Sp-d-E or Sp-D-e oligonucleotides but not by Sp-A, Sp-B, or Sp-C (Fig. 9, lanes 19–24).

The identities of these complexes were assessed by supershift assay using antiserum specific for Sp1, Sp2, and Sp3. Labeling of the Sp-D-E oligonucleotides formed complexes that were supershifted with each of the three Sp antibodies confirming the identity of each complex (Fig. 10, lanes 1–9). Similar results were obtained when the Sp-E element was assessed using the Sp-d-E oligonucleotide (Fig. 10, lanes 19–27). However, none of the antibodies was capable of interacting with the slower migrating protein that interacts with the Sp-D site as shown with the Sp-D-e oligonucleotides (Fig. 10, lanes 10–18). Overall, these data were interpreted to suggest that the Sp-D element binds an unidentified protein complex that is neither Sp1, Sp2, nor Sp3 and that this complex facilitates the binding of Sp-proteins to the Sp-E element.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 10. The Sp-D and Sp-E elements interact with specific complexes. EMSA was performed with Caco-2 nuclear extracts, and a duplex oligonucleotide that spans from –125 to –87 was labeled (Sp-D-E). Oligonucleotides that contained mutations in the Sp-E site (indicated Sp-D-e) or Sp-D site (indicated Sp-d-E) were also labeled. Supershift results using specific antiserum to Sp1, Sp2, and Sp3 are shown in various combinations. NR serum is used for comparison. The locations of the Sp-D and Sp-E elements are displayed in the top region of the figure, whereas the location of the M10 and M11 mutations is shown for comparison.

 
Sp-F sites. An additional Sp site was identified with the use of duplex oligonucleotides that spans from nt –296 to –187 (Fig. 1). The two complexes supershifted with antibodies that recognize the Sp family of proteins and resemble the pattern seen with the Sp-A site, and a mutation of the Sp-F site significantly reduced luciferase reporter activity (Fig. 1, data not shown).

Drosophila SL2 Cells were Used to Confirm the Role of Sp1 and Sp3 in Driving Activity of FcRn's Minimal Promoter

To further assess the role of the Sp family of proteins in inducing FcRn's promoter activity, the rFcRn–157/+135/{beta}-Gal clone was cotransfected with various amounts of Sp1 (pPacSp1) and Sp3 (pPacUSp3) expression vectors in Sp-deficient Drosophila SL2 cells. The data demonstrate that increasing concentrations of Sp1 augments FcRn promoter activity more than fourfold, whereas Sp3 increases activity ninefold (Fig. 11, A and B).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 11. A and B: transfection of Drosophila SL-2 cells with FcRn's minimal promoter and transactivation with Sp1 and Sp3. SL-2 cells were cotransfected with 200 ng of rFcRn–157/+135/{beta}-Galactosidase ({beta}-Gal) and various amounts of Sp1 or Sp3 expression plasmids (pPacSp1 and pPacUSp3). {beta}-Gal activity was expressed as relative light units (RLU) per milligram of protein. Values are means ± SD of sextuplet data from 2 separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we describe the initial characterization of the promoter region of the rat FcRn gene. Initial analysis of basal transcription using transfected reporter assays demonstrated that intestinal epithelial cells Caco-2 and IEC-6 support promoter activity of the 5'-upstream region of the FcRn gene, and deletion analysis revealed that the location of the gene's minimal promoter is approximately between nt –157 and +135 relative to the start of transcription. Evidence of the formation of DNA-protein complexes was identified in the gene's minimal promoter by DNase footprint analysis, and site-directed mutagenesis confirmed that the complexes interact with specific cis-elements to control basal expression in intestinal cell lines.

A previous analysis of the human FcRn gene suggested the role of GC elements in the control of promoter activity; however, selective mutations that assessed the function of these sites were not performed (22). In the current study, we took an unbiased approach and assessed the entire minimal promoter region for potentially active cis-elements by performing scanning mutagenesis. The immediate upstream region of the rat promoter contains several GC elements that were mutated in the M8–M11 clones, and transcriptional activities of these clones were significantly reduced in both Caco-2 and IEC-6 cells (Fig. 4). These mutations disrupt binding of members of the Sp family of proteins to the Sp-A (–40/–29) and -B (–59/–50) sites and decrease activity to ~25% of the –157/+135 wild-type clone (Fig. 4). EMSA and supershift assays confirmed that both sites bind to members of the Sp family of proteins, including Sp1, Sp2, and Sp3 (Figs. 6 and 7) (24). Specifically, the slower migrating complex was composed of Sp1, Sp2, and other unidentified complexes, whereas the faster migrating complex represents Sp3.

Although Sp1 is considered a ubiquitously expressed transcription factor, protein levels vary dramatically in different organs and cell lines (26). Therefore, members of the Sp family of proteins play an important role in the regulation of many tissue and developmentally specific genes (21). Moreover, the various members of the Sp-multigene family function as both transcriptional activators and repressors and may also interact directly with other transcriptional factors to control gene expression (6, 13, 26). Phosphorylation of the DNA-binding domain of Sp1 and other zinc finger transcriptional factors has recently been shown to inactivate protein-DNA interactions and to inhibit transactivation (5). Therefore, it is plausible that the multiple GC elements identified in the immediate promoter region of the FcRn gene may influence both the developmental and tissue expression of this gene.

Two additional Sp sites were also identified just downstream of Sp-A, which were labeled Sp-B (–59/–50) and Sp-C (–78/–69). In this region, the Sp-A element has the highest affinity to members of the Sp family of proteins, whereas the Sp-C site has lower affinity while also interacting with a yet unidentified complex. These findings are consistent with the transfection studies that showed disruption of the Sp-A site inhibited promoter activity to 25% of wild type, whereas mutation of the Sp-C site had luciferase levels that were 50% of wild type (Fig. 4). The slowest migrating complex identified with the Sp-A-B-c probe in Fig. 8 most likely represents multimerzation of the Sp family of proteins binding to the Sp-A and -B elements. The three Sp elements (Sp-A, Sp-B, and Sp-C) are on the sense strand and separated by 14 nucleotides (from the center of each element). The adjacent Sp sites should be capable of simultaneously binding Sp proteins because spatial constraints are minimized if the distance between the center of adjacent elements is greater than 10 nucleotides (4).

We also determined that the Sp-E site forms a complex with the Sp family of proteins, whereas the Sp-D element forms a complex with a slower migrating protein that appears to facilitate binding of Sp-proteins to the Sp-E element (Figs. 8 and 9). Interestingly, mutagenesis of these sites inhibited basal promoter activity more than 200-fold. Our interpretation of this data would be that disruption of either site (Sp-D and Sp-E) may either remove very potent positive transacting factors or, alternatively, could augment the binding of factors that suppress promoter activity. The current studies failed to decipher between these two possibilities. Whereas we confirmed that the Sp-D complex is neither Sp1, Sp2, nor Sp3, the data suggest that in its absence, the binding affinity of the Sp-E site for the Sp proteins declines considerably (Fig. 9). Moreover, the Sp complexes that form with the Sp-d-E probe could not be competed with an excess of even the high-affinity Sp-A and Sp-B elements, suggesting that a protein other than Sp1, Sp2, and Sp3 could be binding to Sp-E in the absence of the Sp-D complex (Fig. 9). The identity of the Sp-D complex remains unclear, and whether its removal augments the binding of other transcription factors to the Sp-E site has not been addressed in the current study.

Nevertheless, the transfection data do suggest that the absence of either the Sp-D or Sp-E sites does dramatically alter the activity of the core promoter region. Moreover, we hypothesize that the promoter activity of the FcRn gene may be controlled by either changes in the zinc concentrations, abundance of various Sp-proteins, or alteration of the phosphorylation state of zinc finger proteins (5). Each of these manipulations will influence binding of these various Sp proteins to the gene's core promoter and alter activity. The physiological relevance of these sites in controlling the developmental repression expression of the FcRn gene in the intestine of weaned mice is currently under investigation.

Overall, this study represents the first detailed analysis of the elements that control the expression of the FcRn gene in any species. We have identified six specific elements located in the immediate upstream of the rat FcRn promoter that interact with members of the Sp family of proteins (Fig. 5). The Sp proteins that interact with the Sp-A, -B, or -C sites moderately augment basal expression of the gene core promoter. In contrast, the complexes that form with the Sp-D and -E sites appear to dramatically repress basal expression of the gene. Nevertheless, the role that these elements have in controlling the expression of the FcRn gene in the developing animals has yet to be identified.

Whereas many genes that are expressed by small intestinal enterocytes are developmentally regulated at about the time of weaning, the FcRn and lactase-phlorizin hydrolase genes are the only well-characterized genes that decline in postweaned animals (9, 15). Whereas lactase and FcRn expression are most abundant in the proximal intestine of suckling rodents, the FcRn transcript declines at weaning to undetectable levels throughout the intestine. In contrast, lactase expression in the proximal bowel of postweaned mice declines 50% compared with their preweaned littermates, whereas expression levels in the distal intestine decline to undetectable levels (10). Therefore, FcRn appears to be rather unique among the various developmentally regulated genes of the small intestine, and further analysis of the mechanism that underlies its transcriptional control may lead to further insights into understanding the process of enterocyte development and differentiation.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Grants HD-34706 and HD-41034 from National Institute of Child Health and Human Development, Crohn's and Colitis Foundation of America Grant 016714, and fellowships from the Robert Wood Johnson Foundation and the Warren-Whitman-Richardson grant from Harvard Medical School.


    ACKNOWLEDGMENTS
 
We thank Yuhua Wang for providing technical support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. G. Martín, UCLA School of Medicine, Dept. of Pediatrics, Division of Gastroenterology and Nutrition, 10833 Le Conte Ave., 12–383 MDCC, Los Angeles, CA 90095 (E-mail: mmartin{at}mednet.ucla.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Berryman M and Rodewald R. {beta}2-Microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat. J Cell Sci 108: 2347–2360, 1995.[Abstract/Free Full Text]
  2. Blumberg RS, Koss T, Story CM, Barisani D, Pollschuk J, Lipin A, Pablo L, Green R, and Simister NE. A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes. J Clin Invest 95: 2397–2402, 1995.[ISI][Medline]
  3. Brambell FW, Hemmings WA, and Morris IG. A theoretical model of gamma-globulin catabolism. Nature 203: 1352–1354, 1964.[ISI]
  4. Courey AJ and Tjian R. Mechanisms of transcriptional control as revealed by studies of human transcription factor Sp1. Transcriptional Regulation 743–769, 1992.
  5. Dovat S, Ronni T, Russell D, Ferrini R, Cobb BS, and Smale ST. A common mechanism for mitotic inactivation of C2H2 zinc finger DNA-binding domains. Genes Dev 16: 2985–2990, 2002.[Abstract/Free Full Text]
  6. Hagen G, Muller S, Beato M, and Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13: 3843–3851, 1994.[Abstract]
  7. Halliday R. The absorption of antibody from immune sera and from mixtures of sera by the gut of the young rat. Proc R Soc Med Sect B 148: 92, 1996.
  8. Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, and Kolchanov NA. Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26: 362–367, 1998.[Abstract/Free Full Text]
  9. Henning SJ, Rubin DC, and Shulman RJ. Ontogeny of the intestinal mucosa. In: Physiology of the Gastrointestinal Tract. New York, Raven, 1994, p. 571–610.
  10. Jenkins SL, Wang J, Vazir M, Vela J, Sahagun O, Gabbay P, Hoang L, Diaz RL, Aranda R, and Martín MG. Role of passive and adaptive immunity in influencing enterocyte-specific gene expression. Am J Physiol Gastrointest Liver Physiol 285: G714–G725, 2003.[Abstract/Free Full Text]
  11. Junghans RP and Anderson CL. The protection receptor for IgG catabolism is the {beta}2-microglobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci USA 93: 5512–5516, 1996.[Abstract/Free Full Text]
  12. Kandil E, Noguchi M, Ishibashi T, and Kasahara M. Structural and phylogenetic analysis of the MHC class I- like Fc receptor gene. J Immunol 154: 5907–5918, 1995.[Abstract/Free Full Text]
  13. Kiela PR, LeSueur J, Collins JF, and Ghishan FK. Transcriptional regulation of the rat NHE3 gene. Functional interactions between GATA-5 and Sp family transcription factors. J Biol Chem 278: 5659–5668, 2003.[Abstract/Free Full Text]
  14. Leach JL, Sedmak DD, Osborne JM, Rahill B, Lairmore MD, and Anderson CL. Isolation from human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal antibody transport. J Immunol 157: 3317–3322, 1996.[Abstract]
  15. Lee SY, Wang Z, Lin CK, Contag CH, Olds LC, Cooper AD, and Sibley E. Regulation of intestine-specific spatiotemporal expression by the rat lactase promoter. J Biol Chem 277: 13099–13105, 2002.[Abstract/Free Full Text]
  16. Martín MG, Wu SV, and Walsh JH. Hormonal control of intestinal Fc receptor gene expression and immunoglobulin transport in suckling rats. J Clin Invest 91: 2844–2849, 1993.[ISI][Medline]
  17. Martín MG, Wu SV, Ohning G, Wong H, and Walsh JH. Parenterally or enterally administered anti-somatostatin antibody induces increased gastrin in suckling rats. Am J Physiol Gastrointest Liver Physiol 266: G417–G424, 1994.[Abstract/Free Full Text]
  18. Martín MG, Wu SV, and Walsh JH. Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine. Dig Dis Sci 42: 1062–1069, 1997.[CrossRef][ISI][Medline]
  19. Martín MG, Gutierrez EM, Lam JT, Li TW, and Wang J. Genomic cloning and structural analysis of the murine polymeric receptor (pIgR) gene and promoter region. Gene 201: 189–197, 1997.[CrossRef][ISI][Medline]
  20. Martín MG, Wang J, Li TW, Lam JT, Gutierrez EM, Solorzano-Vargas RS, and Tsai AH. Characterization of the 5'-flanking region of the murine polymeric IgA receptor gene. Am J Physiol Gastrointest Liver Physiol 275: G778–G788, 1998.[Abstract/Free Full Text]
  21. Martín MG, Wang JF, Solorzano-Vargas RS, Lam JT, Turk E, and Wright EM. Regulation of the human Na+-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1. Am J Physiol Gastrointest Liver Physiol 278: G591–G603, 2000.[Abstract/Free Full Text]
  22. Mikulska JE and Simister NE. Analysis of the promoter region of the human FcRn gene. Biochim Biophys Acta 1492: 180–184, 2000.[ISI][Medline]
  23. Obata Y, Satta Y, Moriwaki K, Shiroishi T, Hasegawa H, Takahashi T, and Takahata N. Structure, function, and evolution of mouse TL genes, nonclassical class I genes of the major histocompatibility complex. Proc Natl Acad Sci USA 91: 6589–6593, 1994.[Abstract]
  24. Pascal E and Tjian R. Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev 5: 1646–1656, 1991.[Abstract]
  25. Praetor A, Ellinger I, and Hunziker W. Intracellular traffic of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells. J Cell Sci 112: 2291–2299, 1999.[Abstract/Free Full Text]
  26. Saffer JD, Jackson SP, and Annarella MB. Developmental expression of Sp1 in the mouse. Mol Cell Biol 11: 2189–2199, 1991.[ISI][Medline]
  27. Shah U, Dickinson BL, Blumberg RS, Simister NE, Lencer WI, and Walker WA. Distribution of the IgG Fc receptor, FcRn, in the human fetal intestine. Pediatr Res 53: 295–301, 2003.[Abstract/Free Full Text]
  28. Simister NE and Mostov KE. An Fc receptor structurally related to MHC class I antigens. Nature 337: 184–187, 1989.[CrossRef][ISI][Medline]
  29. Solorzano-Vargas RS, Wang J, Jiang L, Tsai HV, Ontiveros LO, Vazir MA, Aguilera RJ, and Martín MG. Multiple transcription factors in 5'-flanking region of human polymeric Ig receptor control its basal expression. Am J Physiol Gastrointest Liver Physiol 283: G415–G425, 2002.[Abstract/Free Full Text]
  30. Svendsen LS, Westrom BR, Svendsen J, Ohlsson BG, Ekman R, and Karlsson BW. Insulin involvement in intestinal macromolecular transmission and closure in neonatal pigs. J Pediatr Gastroenterol Nutr 5: 299–304, 1986.[ISI][Medline]
  31. Waldmann TA and Terry RJ. Familial hypercatabolic hypoproteinemia: a disorder of endogenous catabolism of albumin and immunoglobulin. J Clin Invest 86: 2093–2098, 1990.[ISI][Medline]
  32. Wochner RD, Drews G, Strober W, and Waldmann TA. Accelerated breakdown of immunoglobulin G (IgG) in myotonic dystrophy: a hereditary error of immunoglobulin catabolism. J Clin Invest 45: 321–329, 1966.[ISI][Medline]
  33. Yu Z and Lennon VA. Mechanism of intravenous immune globulin therapy in antibody-mediated autoimmune diseases. N Engl J Med 340: 227–228, 1999.[Free Full Text]
  34. Zinkernagel RM. Maternal antibodies, childhood infections, and autoimmune diseases. N Engl J Med 345: 1331–1335, 2001.[Free Full Text]




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Jiang, L.
Articles by Martín, M. G.
Articles citing this Article
PubMed
PubMed Citation
Articles by Jiang, L.
Articles by Martín, M. G.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.