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Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia
Address all correspondence and requests for reprints to: Dr. David Cram, Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia. E-mail: cram{at}wehi.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TC1. Following three successive subtractions of
TC1 complementary DNA from ßTC3 complementary DNA, difference
products were cloned into pUC19 and nucleotide sequences determined.
Comparison of 91 sequences against the databases identified 11 known
and 8 novel genes. Known genes included previously reported ß
cell-specific genes, insulin I/II and islet amyloid polypeptide, as
well as other non-ß cell-specific genes such as those for
insulin-like growth factor II, selenoprotein P, neuronatin, prohormone
convertase, and type 1 protein kinase A regulatory subunit. By Northern
blot hybridization, expression of the majority of known and novel genes
was restricted to ßTC3 cells. Novel genes BA-12, -13, -14, and -18
were expressed not only in ßTC3 cells, but also in normal pancreatic
islets and a limited number of other tissues. The deduced amino acid
sequence of BA-14 showed significant homology with members of the
cadherin superfamily indicating that BA-14 may encode a cadherin-like
molecule potentially involved in ß cell adhesion events during islet
ontogeny. In ßTC3 cells, none of the novel genes were regulated at
the RNA level by high glucose. However, in parallel studies,
transcription of BA-12 was significantly increased by both sodium
butyrate and nicotinamide, suggesting that this gene may play a role in
pancreatic ß cell growth and/or differentiation. In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate ß cell growth and differentiation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
, and PP endocrine cells that secrete the hormones
glucagon, insulin, somatostatin, and pancreatic polypeptide,
respectively. The predominant ß cells are the sole source of insulin,
and their selective destruction by autoimmune T cells leads to
insulin-dependent diabetes mellitus (IDDM) (1). The identification of
ß cell-specific molecules involved in growth and differentiation,
insulin biosynthesis, or autoimmunity could lead to new approaches to
the treatment of IDDM.
Many studies have attempted to characterize ß cell molecules using
traditional bio- or immuno-chemical techniques. However, only a few
have attempted to systematically and comprehensively analyze gene
expression in ß cells. In one study, Tanaka et al. (2)
sequenced randomly selected clones from a human islet complementary DNA
(cDNA) library and catalogued several hundred genes. Velculescu
et al. (3) analyzed large numbers of pancreatic transcripts
by serial analysis of sequence tags (SAGE) and identified 13 novel
genes. Recently, Neophytou et al. (4) identified
differentially expressed mouse ß cell genes by subtraction of a
glucagonoma ( cell tumor) from an insulinoma (ß cell tumor).
Despite these studies, the genes uniquely expressed in ß cells have
not been reported.
With the advent of PCR-based subtraction techniques, it is now possible
to rapidly map unique differences in gene expression between related
cells. As a model system for defining genes expressed specifically in
endocrine ß cells, we have applied cDNA representational difference
analysis (cRDA) (5) to the well characterized SV40-transformed mouse
cell lines, ßTC3 (6), and TC1 (7), that secrete the hormones
insulin and glucagon, respectively. These cell lines are relatively
differentiated and are thought to be derived from a common endocrine
precursor. In this study, subtraction of TC1 from ßTC3 cDNA by
cRDA identified both known and novel difference products expressed
uniquely in ßTC3 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TC1 cell lines were kindly provided by Professor
D. Hanahan (University of California, San Francisco, CA). Both cell
lines were cultured in DMEM (5 mM glucose) containing 10%
FCS under a 10% CO2 atmosphere at 37 C. For functional
studies, ßTC3 cells were exposed for 6 h to either 25
mM glucose, 1 mM sodium butyrate or 2
mM nicotinamide. Brain, liver, kidney, lung, heart,
skeletal muscle, spleen, and pancreas were immediately excised from
8-week-old CBA mice killed by cervical dislocation, rapidly washed in
ice-cold phosphate buffered saline (PBS), snap-frozen in a dry
ice/hexane bath, and stored at -70 C before RNA extraction. Islets
were dissociated from the pancreas of 8 week old CBA mice by
collagenase digestion and purified on BSA gradients (8).
Oligonucleotides and enzymes
Oligonucleotides for RDA were as follows: R-Bgl-12,
5'-GATCtgcggtga-3'; R-Bgl-24, 5'-AGCACTCTCCAGCCTCtcaccgca-3';
J-Bgl-12, 5'-GATCtgttcatg-3'; J-Bgl-24,
5'-ACCGACGTCGACTATCcatgaaca-3'; N-Bgl-12, 5'-GATCttccctcg-3';
N-Bgl-24, 5'-AGGCAACTGTGCTATCcgagggaa-3' Lower case letters signify
regions of complementarity between the oligonucleotide pairs. All
enzymes for DNA manipulations except Taq polymerase (Perkin
Elmer, Roche Molecular Systems Inc., Branchburg, NJ) were purchased
from New England Biolabs, Beverly, MA.
Generation of amplicons and RDA PCR subtraction
Cytoplasmic RNA was purified from ßTC3 and TC1 cells grown
to 50% confluency and directly lyzed with RNAzol B (Biotecx
Laboratories, Inc., Friendswood, TX). The polyA+ RNA
fraction of cytoplasmic RNA was isolated by oligo dT chromatography
using the PolyATtract messenger RNA (mRNA) isolation system (Promega,
Madison, WI), and cDNA was synthesized from polyA+ RNA
using the Riboclone cDNA Synthesis System (Promega) according to the
manufacturer’s instructions.
The cDNA RDA method was essentially as described by Hubank and Schatz (5), except for minor modifications. Double-stranded cDNA (2 µg) was digested with DpnII and purified by phenol extraction and ethanol precipitation. Digested cDNA (1.2 µg) was annealed with R-Bgl-24 (8 µg) and R-Bgl-12 (4 µg) and ligated with 1,200 U T4 DNA ligase at 14 C for 12–16 h in a 50-µl volume. Aliquots of the ligation mix (adjusted to 6 µg/ml) were amplified in multiple PCR reactions using R-Bgl-24. Diluted ligation mix (1 µl) was added to 100 µl of PCR buffer containing 67 mM Tris-HCl (pH 8.8), 4 mM MgCl2, 16 mM (NH4)2SO4, 20 mg/ml BSA, 0.25 mM dNTPs, and 10 µg/ml R-Bgl-24 and heated at 72 C for 3 min to melt away the adapter R-Bgl-12. Following the addition of 5 U Taq polymerase and further incubation at 72 C for 5 min to fill the 5' ends, the mixtures were amplified by 20 cycles of PCR (95 C, 1 min; 72 C, 3 min) with a final extension at 72 C for 10 min. Products were combined, phenol extracted, and resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8.0) at a final concentration of 0.5 µg/ml. To generate the driver amplicon, R adapters were removed from the representation by DpnII digestion followed by phenol extraction and ethanol precipitation. Twenty micrograms of this representation were subjected to agarose gel electrophoresis and purified using Qiaex resin (Qiagen) to yield tester amplicon, which was finally ligated to the J-Bgl-12/24 combination as described above.
The first round of subtraction hybridization and amplification was performed using 0.4 µg of J-ligated tester and 40 µg of driver (tester:driver = 1:100). The mixture was phenol extracted, ethanol precipitated, and resuspended in 4 ml EE x 3 buffer [30 mM N-(2-hydroxy-ethyl) piperazine-N'-3-propane sulfonic acid (Sigma Chemical Co., St. Louis, MO); 3 mM EDTA (pH 8.0)]. The solution was overlaid with mineral oil at 98 C for 5 min to denature the DNA and the salt concentration adjusted by the addition of 1 µl of 5 M NaCl. After annealing for 20 h at 67 C, the reaction mixture was diluted to 400 µl with TE containing transfer RNA carrier. For every subtraction, eight 100-µl PCR reactions were set up as before with 10 µl of diluted hybridization mixture. Reactions were incubated at 72 C for 3 min to melt away J-Bgl-12 adapter and a further 5 min with 5 U Taq polymerase to extend the ends. Products from ten cycles of amplification (95 C, 1 min; 70 C, 3 min) were pooled after phenol extraction and isopropanol precipitation, resuspended in 400 µl of 0.2 x TE, and digested with 40 U mung bean nuclease for 35 min at 30 C. Reactions were terminated by addition of 160 µl of 50 mM Tris-HCl (pH 8.9), heated at 98 C for 5 min, and chilled on ice. The first difference product (DP1) was reamplified in eight separate PCR reactions containing 10 µl mung bean nuclease digestion reaction, 2 µg J-Bgl-24, and 5 U Taq polymerase for 18 cycles (95 C, 1 min; 70 C, 3 min).
DpnII-digested PCR cDNA products were ligated to new adapters, either N-Bgl-12 after the first subtraction or J-Bgl-12 after the second subtraction. The RDA process was repeated as before using this amplicon as tester with N-Bgl-24 or J-Bgl-24 as the PCR amplification primers. To generate the second difference products (DP2), 50 ng of N-ligated DP1 was hybridized with 40 µg driver (tester:driver = 1:800). Likewise, to generate the third difference products (DP3), 100 pg J-ligated DP2 was mixed with 40 µg driver (tester:driver = 1:400,000). Amplification, however, was performed for 22 cycles to maximize the yield of difference product for cloning. Difference product cDNA was digested with DpnII and subcloned into the BamHI site of the pUC19 vector. Double-stranded plasmid DNA was prepared from each clone, and sequence determined by dideoxy terminator cycle sequencing using an ABI DNA sequenator (Applied Biosystems, Foster City, CA). Nucleotide sequences were compared to GenBank and dbEST databases and protein translation sequences were compared with SWISSPLOT Protein and GenBank Protein Translation databases using the FASTA search program (W.R. Pearson, University of Virginia).
Northern blotting
Total RNA was fractionated on formaldehyde agarose gels and
transferred to Hybond-N nylon membrane (Amersham, Buckinghamshire, UK).
DNA probes extracted from polyacrylamide gels were labeled with
[-32P] dATP using the Megaprime DNA labeling system
(Amersham) and unincorporated 32P removed using nick
translation columns (Pharmacia Biotech, Uppsala, Sweden).
Prehybridizations (4 h) and hybridizations with labeled cDNAs (16 h)
were performed at 65 C in 6 x SSC, 2 x Denhardt’s
solution, 0.1% SDS, and 200 µg/ml salmon sperm DNA. Membranes were
rinsed in 2 x SSC, 0.1% SDS, washed in 2 x SSC, 0.1% SDS
for 30 min at 65 C, and, if necessary, washed further in 0.2 x
SSC, 0.1% SDS for 30 min at 65 C to reduce background before
autoradiography.
RT-PCR
DNase I-treated mouse islet RNA (1 µg) was reverse-transcribed
with 200U of MMLV RT (Life Technologies, Gaithersburg, MD) in the
presence of 0.5 µM random hexanucleotides (Bresatec,
Australia) and 200 µM dNTPs. Aliquots (one-tenth volumes)
of the first-strand synthesis reactions were amplified by PCR in PCR
buffer (Perkin Elmer) containing 200 µM dNTPs, 1
µM each of the gene-specific oligonucleotides and 1 U
Taq polymerase. PCR reactions were performed for 25–30
cycles (95 C, 45 sec; 50–60 C, 1 min; 72 C, 1 min) and amplified
products analyzed in 1.5% agarose gels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TC1 and ßTC3 cellsTC1 and
ßTC3 cells, Northern blots of total RNAs from cells grown to mid-log
phase were hybridized with insulin, glucagon, and somatostatin cDNA
probes (9). ßTC3 cells expressed high levels of mRNA for insulin, but
not for glucagon or somatostatin, whereas TC1 cells expressed only
mRNA for glucagon (Fig. 1
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TC1 polyA+ RNA
were of similar size and concentration as judged by ethidium bromide
staining (Fig. 2A) and provided the templates for
generating representative amplicons. The ßTC3 and TC1 amplicons
both contained cDNAs ranging from 200–800 bp in size (Fig. 2B),
indicating that there was no discernible bias in the initial
amplification step. To generate ß cell differences, the tester
(ßTC3) and the driver (TC1) amplicons were subjected to three
successive rounds of subtraction. Difference products, DP1, DP2, DP3,
were generated by using hybridization tester:driver ratios at 1:100,
1:800 and 1:400,000, respectively. After three subtraction steps,
indistinct amplicon bands evident in DP1 were significantly resolved as
individual cDNA species in DP3 (Fig. 2C). The bands were excised from
the gel and subcloned into the BamHI site of the vector
pUC19.
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). Eighty-two DP3 clones
contained sequences of known cDNAs that represented a total of 11
different genes that included insulin I/II, islet amyloid polypeptide
(IAPP), type I protein kinase A (PKA) regulatory subunit, insulin-like
growth factor II (IGF II), selenoprotein P, and neuronatin. The
remaining eight DP3 clones contained cDNA sequences that lacked
similarity with any database sequences and in total comprised seven
potentially novel genes. In some cases, several of the known DP3 clones
were found to contain contiguous DpnII fragments of the same
gene. For example, two subfragments each of IAPP, type I PKA regulatory
subunit, selenoprotein P, insulin I/II and IGF II cDNAs were
identified.
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TC1 and ßTC3 cells were compared with
Northern blotting (Fig. 3). In all cases, except BA-10
(vacuolar H+-ATPase), BA-11 (ATP citrate lyase) and BA-19
(novel), a signal representing mRNA for each DP3 gene was only detected
in ßTC3 cells. Even when autoradiographs were overexposed or TC1
cell RNA loading increased 2-fold over ßTC3 cell RNA, no expression
of these DP3 genes was observed in TC1 cells (data not shown). These
results indicate that the majority of DP3 genes identified are
exclusively expressed in ßTC3 cells.
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).
Each clone displayed a different tissue-restricted pattern. Transcripts
for BA-14 appeared to be expressed exclusively in ßTC3 cells, whereas
those for BA-12, -13, -17, and -18 were expressed in at least one other
tissue. Conversely, transcripts for BA-15 and BA-16 were ubiquitously
expressed. None of these genes were, however, expressed in the pancreas
(Fig. 4) or in isolated islets (data not shown). To confirm the
restricted tissue mRNA expression for genes BA-12, -13, -14, -17, and
-18, and further investigate their expression in pancreas and islet
tissue, the RNA samples analyzed by Northern blotting were subjected to
the more sensitive technique of RT-PCR using gene-specific
oligonucleotides (Fig. 5). Overall, the tissue
distribution of mRNA for BA-12, -13, -14, and -18 determined by RT-PCR
was similar to that seen by Northern blotting, although ubiquitous
expression for BA-17 was detected by RT-PCR. Furthermore, by RT-PCR,
mRNA for all these five novel genes was detected in both pancreas and
islet tissue. These results indicate that these genes are expressed not
only in ßTC3 cells but also in normal islets.
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. Within the
BA-14 sequence, nine residues were identical with P- and R-cadherin and
eight with N- and E-cadherin, and the remaining nonidentical residues
were largely conserved. No significant homology with known protein
sequences was found in the predicted amino acid sequences of the other
novel genes.
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). None of the three agents induced
significant changes in the levels of BA-13, -14, -17, or -18 mRNA (data
not shown). However, sodium butyrate and nicotinamide stimulated 2.6-
and 2.8-fold increases, respectively, in mRNA expression for BA-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TC1 cell lines. These
cell lines, representative of pancreatic islet ß cells and cells,
respectively, were established from SV40 T antigen transgenic mice
derived insulinomas (ßTC3) and glucagonomas (TC1). Thus, we
expected that most, if not all, difference products between ßTC3 and
TC1 cells would probably be involved in ß cell-specific functions.
Apart from the type I PKA regulatory subunit that has been reported to
be up-regulated in tumor cell lines (18), we could find no evidence in
the literature that the expression of any of the identified difference
products is altered following cellular transformation events. The majority of DP3 genes were expressed uniquely in ßTC3 cells. Insulin and IAPP, which are known to be ß cell specific, were present among the difference products, indicating that the RDA procedure selectively amplified authentic differences in gene expression between the two endocrine cells. Of the ßTC3 cell-unique genes identified, IAPP, insulin and type I PKA regulatory subunit were the most frequent. However, by Northern blot analysis of ßTC3 RNA, significantly higher levels of IAPP and insulin mRNA than type I PKA regulatory subunit mRNA were detected, indicating that cRDA has efficiently selected difference products regardless of gene mRNA expression levels.
Although cRDA identified a set of genes expressed in ß but not
cells, the majority of the known genes, except insulin and IAPP, have
been previously reported to be expressed in other tissues.
Nevertheless, it is interesting to speculate on the possible roles of
several of these genes in the ß cell, for example, selenoprotein P
and IGF II. Selenoprotein P is a secreted glycoprotein that contains
the essential trace element selenium in the form of selenocysteine
residues. Although its physiological role has not been clarified, it
has been postulated that selenoprotein P may be involved in regulating
the redox state of the cell by incorporating and releasing selenium
(19). Free selenium in the cell is readily converted into
selenodiglutathione, a highly efficient oxidant of reduced thioredoxin
(20). ß cells modify proinsulin posttranslationally to biologically
active insulin by the formation of two interchain and one intrachain
disulfide bonds, whereas glucagon synthesis in cells has no
requirement for disulfide bond formation. Selenoprotein P might
therefore contribute to the formation and maintenance of insulin
disulfide bonds by regulating the redox state of the insulin-secreting
granules.
Insulin-like growth factors IGF-I and IGF-II are expressed in the developing pancreas (21, 22). Our finding of IGF II mRNA in ßTC3 cells is consistent with previous immunohistochemical studies (22) that localize IGF II mRNA and protein to ß cells in human and animal islets. Although a direct role of IGF II in islet cell growth and differentiation has not been demonstrated directly (21), IGF II produced by ß cells could potentially act as an autocrine or paracrine factor during islet neogenesis. Interestingly, the IGF II gene in human (23), rat (24) and mouse (25), is physically located near the 3' end of the insulin (human) or insulin II (rat and mouse) gene and transcribed in the same direction. It has been postulated that this arrangement may allow these genes to share common regulatory elements (26) and raises the possibility that coordinate expression of insulin and IGF II may be important for ß cell development.
Apart from these known genes, eight novel genes were also found among
the difference products. Expression of BA-12 to -18 was demonstrated in
ßTC3 cells and in adult pancreatic islets that predominantly contain
ß cells. From database searches, one gene, BA-14, encoded a protein
with the molecular characteristics of a cadherin-like molecule. It is
well documented that cadherins play a central role in cellular
differentiation and organ morphogenesis by mediating calcium-dependent
homotypic intercellular adhesion (27, 28). In the pancreas, E-, N-, and
R-cadherins are expressed predominantly in islet tissue (29, 30, 31).
In vitro studies of cultured rat islets suggest that
E-cadherin mediates most of the cell adhesion events during islet
ontogeny (29). Recently, transgenic expression of a dominant negative
mutant of E-cadherin in ß cells has demonstrated directly that this
molecule is important in the regulation of ß cell aggregation into
islets (30). By analogy, it is conceivable that the cadherin-like
molecule encoded by BA-14 may have a similar function in the developing
islet. As it appears to be expressed in ß but not cells, it could
potentially play a role in the central clustering of ß cells within
the islet.
From regulation studies of novel gene expression in ßTC3 cells, mRNA for one gene BA-12 was significantly up-regulated by both sodium butyrate and nicotinamide. Sodium butyrate is a lipophilic acid that induces differentiation of pluripotent RIN cell lines (14, 15). Nicotinamide, an inhibitor of poly ADP-ribose synthetase, has been shown to stimulate ß cell differentiation of fetal islets (16, 17). The increase in transcription of BA-12 in ßTC3 cells exposed to these agents leads us to speculate that this gene may have a potential role in the growth and/or differentiation of ß cells. When ßTC3 cells were exposed to 25 mM glucose for 6 h, there was no significant change in the levels of mRNA for any of the novel genes or insulin. The inability of high glucose to alter insulin mRNA levels has also been reported in the closely related cell line ßTC1(6). Thus, under these experimental conditions, we have no evidence as yet that any of these novel genes are regulated by glucose.
Another possibility is that one or more of these gene products could be a target of ß cell autoimmunity in IDDM. Of the recognized autoantigens insulin (32), glutamic acid decarboxylase (33), and tyrosine phosphatase IA-2 (34), only the former is expressed uniquely in ß cells. The molecular and biological characterization of these novel genes may reveal if they are relevant to ß cell development, function, and autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Supported by Kaneka Corporation (Japan).
Received September 9, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells. Neuron 1:605–613[CrossRef][Medline]
in
developing human fetal pancreatic islets. J Endocrinol 138:127–136[Abstract]
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