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(Received for publication, December 29, 1995, and in revised form, May 2, 1996)
From the Hagedorn Research Institute, Niels Steensensvej 6, DK-2820 Gentofte, Denmark
ABSTRACT Development of a high capacity multiplex reverse
transcriptase-polymerase chain reaction protocol has allowed us to
screen lineage related rat islet tumors classified as INTRODUCTION The developmental maturation of precursor cells into more
specialized phenotypes during ontogeny is characterized by highly
specific changes in gene expression. Thus, a detailed picture of
mRNA levels in developmentally related cells may provide insight
into both mechanisms and pathways of differentiation. The four distinct
cell types ( In this study we have: 1) determined the degree by which these
transformed phenotypes reflect the gene expression of their normal
islet counterparts, 2) identified islet tumor phenotype-specific
expression of selected genes and thereby predicted those involved in
normal islet cell specialization.
The genes selected comprise those coding for products known or thought
to be involved in specialized functions of the individual islet cells
as well as transcription factors, which might be involved in specifying
the process of islet cell differentiation. Since transcription factors
regulating key differentiation steps are often involved in activating
the genes that confer the specialized functions of terminally
differentiated cells (11, 12, 13, 14), we have included all cloned factors,
which have been implicated in insulin gene regulation.
Analysis of the mRNA levels of 54 selected genes in the different
tumor phenotypes has led to the identification of several candidate
genes with a predicted cell-specific expression in the normal islet.
Importantly, we show by immunohistochemistry on sections of adult rat
pancreas that the newly identified homeodomain protein Nkx 6.1 is
expressed specifically in EXPERIMENTAL PROCEDURES Isolated,
neonatal rat islets of Langerhans were cultured as described (15). The
transformed rat islet cell line MSL-G2 (5), the somatostatin-expressing
rat cell line Tu-6 (10), the rat insulinoma RIN-5AH (6), the mouse
insulinoma RNA isolation was carried out by lysing cells
or tissue in guanidinium thiocyanate/phenol buffer (RNAzol, Cinna
Biotecx) according to manufacturer's instructions. The RNA was
resuspended in diethyl pyrocarbonate-treated water, reprecipitated with
2.5 volumes of EtOH, 0.1 volume of 4 M NaCl, recovered by
centrifugation, and washed with 70% ice-cold EtOH.
Total RNA was diluted in diethyl
pyrocarbonate-treated water to 0.2 µg/µl, denatured at 85 °C for
3 min, and quickly chilled on ice. 5 µl of the total RNA was mixed
with 20 µl of RT-mix (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 units of Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.), 40 units of
RNAsin (Promega), 3 µg of random hexamers (Life Technologies, Inc.),
and 0.9 mM dNTPs (Pharmacia Biotech Inc.) all in final
concentrations) and left 10 min at room temperature and subsequently
incubated at 37 °C for 1 h. After cDNA synthesis the
reaction was diluted with 50 µl of H2O.
Primer sequences are given in Table
I. In order to obtain as identical amplification
kinetics for individual amplicons as possible, we set rules for the
design of primers as follows. The length of the oligonucleotides should
be within 19-23 bases, with a GC content around 50%. Furthermore, the
length of the products was restricted to be between 150 and 320 base
pairs. When possible primers were synthesized based on published rat
sequences. In cases where no rat sequences were available, primers were
synthesized based on published mouse or hamster sequences. All of these
amplify the homologous rat genes with the expected size in exact base
pairs. However, due to possible mismatched bases, it remains a
possibility that the ratio to the internal standard is reduced, as a
result of less efficient amplification during the first cycles. This
does not, however, affect the relative difference in expression between
the samples tested, and will still allow a comparative expression
analysis.
Data of the amplicons analyzed
Volume 271, Number 31,
Issue of August 2, 1996
pp. 18749-18758
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Cell-specific Homeodomain Transcription Factor*
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-, 
-, and
-like as judged by their hormone profile for differential expression
of more than 50 selected genes. We find that in addition to insulin the
insulinoma express the normal -cell markers Pdx-1, IAPP, and Glut-2,
and that these markers are absent from the glucagonoma: a reflection of
the normal -cell. Furthermore, this study suggests that the GLP-1,
glucagon, GIP, IGF-1, and insulin receptors as well as E-cadherin,
R-cadherin, Id-1, and Id-2 are differentially expressed within the
islet of Langerhans. Importantly, insulinoma-specific expression of the
recently cloned homeodomain protein Nkx 6.1 predicted -cell-specific
expression in the normal islet. Immunohistochemistry using antibodies
raised against recombinant Nkx 6.1 did indeed localize Nkx 6.1 expression exclusively to the nuclei of normal islet -cells. Apart
from pancreatic islets only the antral part of the stomach contained
Nkx 6.1 mRNA. We conclude that multiplex reverse
transcriptase-polymerase chain reaction-based mRNA profiling is a
powerful tool to identify differentially expressed genes within
phenotypically related cells and propose that Nkx 6.1 is involved in
specifying the unique characteristics of the -cell.
- - -, and PP-cells) present in the pancreatic
islet of Langerhans are thought to derive from common precursor cells
through stages of transient phenotypes expressing multiple islet as
well as gut and brain peptide hormones (1, 2). Such multi-hormonal and
presumably immature phenotypes are also hallmarks of pancreatic
endocrine tumors (3, 4). From the clonal heterogeneous rat islet tumor
culture MSL-G2 (5) of identical clonal origin as the widely used
RIN-5AH insulinoma cell line (6, 7), several distinct and more
homogeneous phenotypes comprising transplantable insulinomas (referred
to later as ``IN insulinoma'') (8), glucagonomas (``AN
glucagonoma'') (9), as well as a somatostatin-producing in
vitro culture (``Tu-6'') (10) have been derived.
-cells as predicted from the tumor
analysis. The tissue distribution of Nkx 6.1 mRNA showed a
remarkable tissue restriction (comparable to that of insulin and IAPP),
suggesting that this factor plays a crucial role in specifying the
unique nature of the pancreatic islet -cell.
-TC3 (16), the mouse glucagonoma culture -TC1 (17) and
its derivative -TC1.9 (18) were cultured in RPMI 1640 supplemented
with 10% fetal calf serum as described (5). The insulinoma, MSL-G2-IN
(8), and the glucagonoma, MSL-G-AN (9), were propagated in NEDH rats as
described (8, 9).
Multiplex PCR was carried out in 50-µl reactions
using 3 µl of the diluted cDNA reaction as template mixed with 47 µl of PCR mix (50 mM KCl; 10 mM Tris-HCl, pH
9.0 at 25 °C; 0.1% Triton X-100; 1.5 mM
MgCl2; 40 µM dATP, TTP, and GTPs, and 20 µM dCTP, all in final concentrations; 10 pmol of each
primer; 2.5 units of Taq polymerase (Promega); and 2.5 µCi
of 3000 Ci/mmol [-32P]dCTP (Amersham Corp.)). Mineral
oil (50 µl) was added to each tube. Standard thermal cycle profile
was as follows. A single denaturing step at 94 °C/1 min was followed
by the chosen number of cycles as given: 94 °C/30 s; 55 °C/1 min;
72 °C/1 min, 30 s, with the exception of reactions amplifying
GC-rich amplicons (>60% GC content) where a thermal cycle profile of
95 °C/1 min; 55 °C/1 min; 72 °C/1 min, 30 s was required
for reproducible amplification.
Reaction products were separated on 0.4-mm 7 M urea, 1 × TBE (0.13 M Tris base, 80 mM boric acid,
0.25 mM EDTA), 6% polyacrylamide gels. These were dried
and exposed overnight to a phosphorimage storage screen. Screens were
scanned by a Molecular Dynamics PhosphorImager series 400, and band
intensities were calculated using the Imagequant software by the use of
rectangle mode/local background/volume integration. All amplicons were
measured against internal standards. -Tubulin was used at 18 cycles,
glucose-6-phosphate dehydrogenase at 22 and 25 cycles, and TATA-binding
protein at 26 cycles. Expression data are given as ratios to the
co-amplified internal standard, and normalized for dC content. Negative
controls were always included. Second, by the use of primers specific
for genomic sequences, we do not observe genomic DNA amplified even at
high (> 30) cycle numbers (not shown). Positive controls of all primer
sets were performed on cDNA prepared from tissue known to express
the gene.
The present multiplex RT-PCR method allows the co-amplification of
several cDNA products from total RNA preparations in a single tube.
To avoid biased results due to potential interference between
individual amplicons, we analyzed the amplification kinetics of
individual amplicons in reactions where several products are
co-amplified. Representative experiments of simultaneous amplification
of either high or low abundance cDNAs (Fig. 1, A and
B, respectively) directly show the non-competitive
amplification of individual products, and also that these share an
almost identical rate of amplification, highlighted by the almost
identical slopes observed from a linear regression analysis within the
exponential phase (Fig. 1C). However, as data obtained
during the exponential phase may fail to detect an interference
occurring during the first cycles, we combined multiplex sets with
increasing numbers of products (from 1 to 5) co-amplified. Two
representative sets are shown in Fig. 1D. No
initial interference is detected, in any combinations, as no decrease
in absolute product yield is observed for any product. Thus, all of
these amplicons are independent of the presence of other products.
However, as interference detection based on these assays is
time-consuming, we devised a cross-combination assay (not shown) as a
mean to detect amplicon interference. Principally, 25 amplicons were
randomly grouped in 5 combinations (A-E) of 5 different amplicons,
avoiding any co-migration, and co-amplified with an internal standard.
These combinations are termed vertical (as A1-A5, B1-B5; etc.). All
the amplicons were additionally combined in a horizontal manner,
(i.e. A1-E1, A2-E2, etc.). If a significant difference in
product yield of a given amplicon were observed between the vertical
and the horizontal combinations, we accredited this to interference,
which allowed us to avoid unsuitable combinations of particular primer
sets. Furthermore, the cross-combination assay allows identification of
products being amplified with combinations of primers in between sets.
If any such significant products were detected, co-amplification of
these primer sets was not allowed.
Fig. 1. Validation of multiplexed amplification of several cDNAs. A, multiplex amplification of high abundance cDNAs; B, low abundance cDNAs. Reactions were assembled in triplicate and aliquots removed at indicated cycle numbers. Data points represent average band intensity, with standard deviation. Volume is the arbitrary unit of band intensity obtained from the phosphoimager. C, linear regression analysis of reaction efficiencies. D, progressive inclusion of amplicons in multiplex RT-PCR assembly, 2 representative sets (A and B) are shown. Reactions were assembled with 1-5 sets of primers in ascending order from left to right. Asterisk denotes a combination product arising from the CREB and GLP-1-R amplicons. A 1:1:1 mix of Tu-6, IN and AN cDNA was used as template in A, whereas in B and D, a 1:1 mix of Tu-6 and IN cDNA was used.
Antibodies, Immunohistochemistry, and Western Blotting
GST-Nkx 6.1 fusion protein was generated by insertion of
a PCR fragment amplified from rat islet cDNA encoding 66 C-terminal
Nkx 6.1 amino acids in-frame with GST in the pGEX-4T-1-plasmid
(Pharmacia, Uppsala, Sweden). The sequence of the primers used was:
upstream 5
-tccgaattcATGGCCACCGCCAAGAAGAAGCAG-3 and downstream
5tgccaagtgcggccgcTCAGGAGCCCTCGGCCTCGG-3. Lowercase letters indicate
nucleotides included for cloning purposes. Fusion protein was purified
using the RediPack GST purification module (Pharmacia) according to
manufacturer's instructions. Immunization of rabbits was performed as
described (19). Immunohistochemistry and Western blotting was performed
as detailed elsewhere (5, 20). Monoclonal antibodies to insulin
(HUI-18), glucagon (GLU-001), and somatostatin (SOM-018) were from
Novo-Nordisk Biolabs, Bagsvaerd, Denmark and used as described
previously (21). Rabbit antiserum 1856 raised against rat Pdx-1 (as a
recombinant GST fusion protein; Ref. 22), a nuclear marker for all
adult islet -cells as well as of few -cells (23) was used in
comparative analyses to characterize the expression profile of
immunoreactive Pdx-1 versus Nkx 6.1 in pancreatic sections
and islet cell cultures.
RESULTS
The RT-PCR assay showed expression of the islet hormones
insulin and IAPP in the IN insulinoma, glucagon in the AN glucagonoma,
and somatostatin in Tu-6, selectively (Fig.
2A and Table II), concordant
with previously described Northern and immunocytochemistry analyses (8,
10, 21, 24). Furthermore, all three major islet hormones were
simultaneously detected in islets. The fourth islet hormone, pancreatic
polypeptide (PP), was detected in islets and unexpectedly also in the
AN glucagonoma.
Fig. 2. Selected multiplex-RT-PCR combinations. A, major pancreatic polypeptide hormone mRNAs (18 cycles, insulin out of the exponential phase in islets); B and C, other polypeptide hormone mRNAs (25 cycles). Pit, pituitary (positive control for pituitary adenylate cyclase activating peptide); Br, brain (positive control for oxytocin). D-H, analysis of low abundance mRNAs of various types (25 cycles), marker lane (M) shown for set F. I, low abundance mRNAs using TATA-binding protein as internal control, 26 cycles. C, negative control lane. Lane 1, MSL-G2; lane 2, Tu-6 somatostatinoma; lane 3, IN insulinoma; lane 4, AN glucagonoma; lane 5, RIN 5AH; lane 6, neonatal rat islets.
|
We then determined the expression profile of several neuroendocrine
polypeptide hormones reported to be expressed during islet ontogeny. In
agreement with previous reports we find that neuropeptide Y, peptide
YY, and thyrotropin-releasing hormone are all highly expressed in
newborn rat islets (25, 26, 27), and that CGRP- but not CGRP- is
expressed in islets (25, 28). Neither peptide YY nor CGRP- were
detected in any of the tumor clones, whereas thyrotropin-releasing
hormone was detected at very low levels. In contrast, secretin was
absent from newborn islets but found at low levels in all tumor
phenotypes including RIN cells. Neuropeptide Y, however, is selectively
expressed in the AN glucagonoma, which in addition also selectively
expressed rather high levels of calcitonin. GH, GH-releasing hormone,
oxytocin, pituitary adenylate cyclase activating peptide, and
vasoactive intestinal peptide mRNA were detected neither in islets
nor in the islet tumors. Finally, we confirmed previously reported
expression levels of gastrin and cholecystokinin in the different
MSL-derived tumor phenotypes and islets (5, 9).
Glucose-sensing, which is an essential character of
the mature -cell, has been shown to rely on expression of a specific
form of hexokinase (29) and possibly also a specific glucose
transporter (30). We analyzed for potential tumor phenotype-specific
expression levels of Glut-1, -2, -3, -4, glucokinase (hexokinase IV),
and the low Km hexokinase II. In summary, we
observed Glut-1 to be expressed at low levels in all tumor phenotypes
as well as in islets. In contrast, Glut-2 was expressed at high levels
in MSL-G2, IN insulinoma, and islets, at lower levels in Tu-6 and
completely absent in AN glucagonoma and RIN-5AH cells (Fig.
2D). In these samples, we only detected very low levels of
Glut-3 and no Glut-4 (data not shown). Glucokinase was expressed at
lower levels in islets as well as in the various tumor phenotypes,
although with a slightly reduced expression in RIN-5AH and Tu-6 cells.
Furthermore, the exon-1a-containing glucokinase transcript initiated
from the upstream promoter, which has been shown previously to be
expressed in pancreatic -cells, was the predominant form, as we
observed similar quantitative data when using primers common to both
the liver- and the -cell-specific forms (Fig. 2D, product
at 161 base pairs). Surprisingly, the AN glucagonoma showed the highest
level of glucokinase expression. The hexokinase II gene expression
level was vastly elevated in all tumor cells compared to normal islets
(Fig. 2G).
Glucagon, GLP-1, and GIP have all been suggested to act as incretins, potentiating glucose-induced insulin release. We therefore analyzed the mRNA expression profile of their plasma membrane receptors belonging to the seven-transmembrane G-protein-coupled receptor family in order to search for a heterogeneous distribution among the MSL clonal variants. The GLP-1-R, GIP-R, and the glucagon-R were detected in islets. Interestingly, expression of these receptors segregated predominantly with a particular tumor phenotype: GLP-1-R in the IN insulinoma and RIN-5AH cells (Fig. 2E), glucagon-R in Tu-6, and the expression of the GIP-R was 5-fold elevated in the AN glucagonoma (Fig. 2G) compared to the IN insulinoma. Several other members of the seven-transmembrane G-protein-coupled receptor and tyrosine kinase-R families were tested (see Table II). Particularly the IGF-1-R and insulin-R showed differential expression among the tumor phenotypes. Although insulin-R was expressed in all samples, a variation in splicing was observed. Using primers designed to distinguish between insulin-R splice variants (with or without exon 11), all tissues showed a balanced splicing yielding both forms, except the AN glucagonoma, which selectively expressed insulin-R mRNA without exon 11, similar to the splice pattern found in the brain. IGF-1-R expression was selectively absent from AN glucagonoma (Fig. 2D) while detected in other MSL-derived clones as well as in islets. In contrast, the GH-R was homogeneously expressed in islets as well as all the tumor phenotypes.
Finally, we analyzed the expression of the cell-adhesion molecules, NCAM and R-, N-, and E-cadherin (uvomorulin/L-CAM). These are all previously reported to be present in islets and thought to be involved in the specification of the islet architecture. In agreement with our earlier observations (20), we did not detect E-cadherin in the AN glucagonoma (Fig. 2I), whereas it was present in all other tumors and cell lines. R-cadherin was expressed in all lines but only at very low levels in Tu-6 and in RIN-5AH. In contrast, NCAM and N-cadherin (Fig. 2G) was homogeneously expressed in all tumor cells.
Tumor Phenotype-specific Expression Profiles of Hormone Gene Transcription FactorsTo analyze for correlation between
phenotype-specific markers and presence of specific transcription
factors, we next determined the expression profile of several gene
regulatory proteins implicated in islet hormone gene expression.
Transcription factors from the helix-loop-helix (HLH) family have been
shown to be involved in islet hormone gene activation. We analyzed
expression of the HLH factors Pan-1/Pan-2 (rat homologues of human
E47/E12, respectively), BETA-2 (hamster)/NeuroD (mouse), and also the
inhibitory factors Id-1, -2, -3, and -4. Both the Pan-1 and -2 splice
forms were detected at low levels in all samples (Fig. 2I),
although with a predominant (2-5-fold) expression of Pan-1. In
comparison, NeuroD was similarly homogeneously expressed between
samples (Fig. 2I), but found at much higher levels than the
pan-mRNAs. In contrast, distribution of the Id factors was more
heterogeneous, with Id-1 expressed at high levels in Tu-6 and RIN-5AH
cells (Fig. 2H). Id-2 was found at high levels in Tu-6 and
at lower levels in all other samples (Fig. 2E). Id-3 was
only detectable in Tu-6, while Id-4 was selectively expressed in the IN
insulinoma and Tu-6 (Fig. 2H). Within the islets, only Id-1
and Id-2 were expressed at significant levels. We also analyzed
expression of factors belonging to the basic-leucine zipper family.
None of the basic-leucine zipper family factors including CREB,
CREB, and c-Jun, as well as CREB-binding protein (Fig. 2,
E-G), showed marked differential expression within the
tumor phenotypes, and all were detected at comparable levels in newborn
islets.
The homeodomain protein Pdx-1, an insulin gene transcription factor
that also is required for pancreas formation (14), was found to be
expressed at comparable levels in the MSL-G2 stem-cell culture, the IN
insulinoma, and the Tu-6 culture (from which it was originally cloned
(31)) (Fig. 2F) Importantly, this factor was absent from the
AN glucagonoma as in the normal islet -cell. Several other
homeodomain proteins (Isl-1, cdx-3, lmx-1) cloned from islet cell lines
and able to interact with the insulin promoter (32, 33) were analyzed.
Isl-1 was found at low levels in RIN-5AH cells but homogeneously
expressed in all MSL-G2-derived tumor phenotypes (Fig. 2H)
as well as in islets, while cdx-3 and lmx-1 were only found in RIN-5AH
cells but not in islets (Fig. 2F, note that the narrow bands
in lanes 1-4 migrating slightly faster than the doublet
lmx-1 band in lane 5 are nonspecific). The LIM domain
proto-oncogene, rhombotin (Lmo-1), was
homogeneously expressed in all tumor phenotypes.
-Cell
Differentiation
When analyzing the expression profile of the
homeodomain factor Nkx 6.1, previously cloned by degenerate PCR from
HIT cells (34), we found that expression of Nkx 6.1 was induced in the
transition from MSL-G2 to the IN insulinoma (Fig. 2H).
Additionally, Nkx 6.1 expression was detected in Tu-6. This expression
profile marks the Nkx 6.1 gene as one of the few genes whose activity
segregates differentially between the MSL-G2 culture and the
insulinoma: a profile shared only by insulin, Id-4, and GLP-1-R genes.
As Nkx 6.1 mRNA was readily detected in islets, we wanted to
address the question whether the tumor data would predict a
-cell-specific expression pattern in the normal islet. We therefore
raised antibodies to a GST-Nkx 6.1 fusion protein that included the 66 C-terminal amino acids of the rat Nkx 6.1 protein. Two distinct rabbit
antisera, -Nkx 6.1-173 and -Nkx 6.1-174, reacted identically in
Western blotting to a doublet band migrating at 44 and 46 kDa. We could
thus confirm the expression profile of Nkx 6.1 at the protein level
between the MSL-G2-derived variants (Fig. 3). The
staining of both bands were completely abolished when antisera were
preincubated with the recombinant Nkx 6.1 fusion protein (Fig. 3).
Moreover, when analyzing sections of mouse (data not shown) and rat
pancreas, Nkx 6.1-like immunoreactivity was strictly confined to
-cell nuclei (Fig. 4, A and B).
No glucagon-positive or somatostatin-positive cells co-expressed Nkx
6.1 as evidenced by co-immunostaining performed on dispersed rat islet
cells in monolayer (Fig. 4, D and E).
Additionally, we observed that all insulin-positive cells were Nkx
6.1-positive as well (Fig. 4C). The specificity control
experiment shown in Fig. 5 demonstrates that the
observed nuclear staining is due to the presence of Nkx 6.1-like
immunoreactivity. Furthermore, when testing the expression profile in a
large number of tissues by RT-PCR, Nkx 6.1 mRNA was selectively
detected at high levels in pancreatic islets and in very low amounts in
antrum (Fig. 6). Lack of Nkx 6.1 mRNA in MSL-G-AN is
in accordance with absence of immunoreactive Nkx 6.1 in normal
-cells (Fig. 4A) and in mouse -TC1 (Fig. 3);
nonetheless, in accordance with Rudnick et al. (34), we do
detect mRNA for Nkx 6.1 in the -TC1.9 subclone derived from
-TC1 cells.
Fig. 3. Western blot analysis of Nkx 6.1.
-Nkx-6.1-173 (1:1000) was used as primary antisera. 15 µg of
nuclear extract was applied per lane. NHI-6F is a MSL-G2 subclone
stably transfected with the human insulin gene (8). NHI-6F-28 is an
insulinoma culture derived from the NHI-6F line after -cell
maturation during in vivo passage. The NHI-6F/NHI-6F-28
cells are identical to the MSL-G2/IN system (8). The NHI-6F-28 nuclear
extract is slightly degraded. GH3 is a rat pituitary cell
line. -TC1 and -TC3 are large T-antigen transformed cell lines
derived from transgenic mice with heritable glucagonomas (17) and
insulinomas (16), respectively. Absorption control is shown to the
right. The anti-Nkx 6.1 staining pattern (two left
lanes) was not affected by preabsorption to GST-Pdx-1 (data not
shown) but completely blocked by preabsorption to GST-Nkx 6.1 (100 µg/ml).
Fig. 4. Distribution of Nkx 6.1 in normal islet cells. Double immunofluorescence labeling with
-Nkx-6.1-174
(green) and pancreatic hormones (red) on frozen
pancreatic sections (A and B, scale bar 100 µm)
and NRI-cells (dispersed newborn rat islet cells) (C-E,
scale bar 20 µm). A, islet staining of Nkx 6.1 and
glucagon; B, islet staining of Nkx 6.1 and somatostatin.
Arrows in A and B point to glucagon
(A) and somatostatin-positive (B) cells, both Nkx
6.1-negative. C-E, NRI-cells stained with Nkx 6.1 combined
with either insulin (C), glucagon (D), or
somatostatin (E). Arrow in E points to
a Nkx 6.1-negative cell, presumably either glucagon or PP-positive.
Note that all insulin-positive cells are Nkx 6.1-positive as
well.
Fig. 5. Specificity control of the anti-Nkx 6.1 antisera for immunocytochemistry. NHI-6F-28 insulinoma cells (positive for Nkx 6.1 and Pdx-1 mRNA) were stained using antisera against Nkx 6.1 and Pdx-1. Both antisera were preabsorbed with either antigen (GST-Nkx 6.1, GST-Pdx-1) as indicated. As expected, only the corresponding antigen abolished staining. Identical results were obtained on pancreatic sections (not shown). Photomicrographs (A and D) were overexposed to visualize unstained nuclei.
Fig. 6. RT-PCR (25 cycles) analysis of Nkx 6.1 expression in extrapancreatic tissues. Using glucose-6-phosphate dehydrogenase as internal standard, 22 different rat tissues were analyzed for Nkx 6.1 expression. Various tumor lines (first seven lanes) served as control. High level expression was confined to pancreatic islets, with antrum as the only other tissue showing detectable expression.
DISCUSSION
By multiplex RT-PCR, we have screened a large number of genes for
differential expression in phenotypically distinct islet tumor
phenotypes using neonatal rat islets of Langerhans as control tissue.
Importantly, we find that the expression of the previously identified
islet -cell restricted genes IAPP, GLP-1-R, and Glut-2 and the
transcription factor Pdx-1 segregate with insulin gene activity in the
insulinoma phenotype. Derivation of the glucagonoma from pluripotent
MSL-cells is associated with a shutdown of insulin, Glut-2, and Pdx-1
gene activity. These observations reflects the nature of normal islet
- and -cells. We therefore conclude that these phenotypically
distinct transformed islet cells are useful representatives of the
corresponding cell types in the normal islet.
Of the genes tested, a substantial fraction showed differential
expression among the different tumor phenotypes, of which the most
important are summarized in Fig. 7. It is striking that
the MSL-G2 line is almost identical to the -cell-like IN insulinoma
in expression of most of the selected genes except for the inverse
correlation between the two major pancreatic hormones insulin and
glucagon and the presence of Nkx 6.1, Id-4, and GLP-1-R in the
insulinoma. Several otherwise -cell restricted factors, such as
Glut-2, IAPP, and, most importantly, the Pdx-1 gene, are expressed
prior to insulin gene activation. This supports our previous proposal
that the MSL-G2 culture is representing a pre--cell phenotype
(21).
Fig. 7. Changes in gene expression as a result of phenotype maturation by the MSL-G2 cells. Only genes showing profound changes in expression levels between clonal variants are included (see Table II). One arrow represents a significant change in level of expression (up or down), two arrows up denotes a marked induction from undetectable expression, and two arrows down denote a marked reduction to undetectable levels.
In contrast, the in vivo derivation of glucagonomas is associated with profound changes in the gene expression profile (Fig. 7). Several hormone genes (neuropeptide Y, PP, and calcitonin) as well as the GIP receptor gene are activated, while the expression of the calcitonin-R, IGF-1-R, R-, and E-cadherin, as well as Glut-2 and Pdx-1, are completely turned off. From these results we predict that these genes are differentially expressed in normal islet cells.
Glucokinase expression in the insulinoma as well as in the glucagonoma
is interesting and suggests a role of the enzyme in sensing low glucose
levels, which might be coupled to the regulated secretion of glucagon
in the normal -cell. Agreeing with the glucagonoma expression, the
glucokinase promoter has also been found active in -cells of
transgenic mice (35).
During the -cell-like maturation seen in Tu-6 cells, which proceeded
through the establishment of an in vitro culture from the
insulinoma tumor cells (10), we observe down-regulation of several
hormonal genes (gastrin, cholecystokinin, IAPP, and insulin), whereas
somatostatin is activated in most cells of the Tu-6 line. This is
additionally accompanied by the reduction of Glut-2, GLP-1-R, and
R-cadherin expression, and a concomitant activation of the glucagon
receptor and the Id-1 and -2 genes. However, both Pdx-1 and Nkx 6.1 are
still expressed in the Tu-6 line, indicating that some features of the
-cell phenotype are retained in this somatostatinoma culture. The
fact that the Id factors are expressed in this material is interesting,
in light of the HLH-protein-dependent hormone gene
activation, e.g of the insulin gene. A possible role of
these factors could be to evoke tissue-specific functions of the (in
the islet) homogeneously expressed complex IEF-1 (36, 37), consisting
of the Pan1/2 and NeuroD factors. That is, these factors may act to
negatively regulate the IEF-1 complex through binding to the individual
components of IEF-1, inactivating these in non-DNA-binding
heterodimeric complexes. In this respect, the low level insulin gene
activation in Tu-6, despite presence of the known insulin gene
transactivating proteins (Pdx-1, NeuroD, and the pan-factors) could be
explained by the high level expression of the Id-1 and Id-2 genes
(38).
Together, our results show that the maturation pathways of the MSL-G2
tumor cells closely resembles the hypothetical model of islet ontogeny
(1, 39, 40) proposing that cells positive for Pdx-1 (40) are precursors
for all four islet cell types, and that -cell maturation involves
inactivation of the Pdx-1 gene. The Pdx-1/glucagon double-positive
cells constituting the majority of the cells of the MSL-G2 culture may
reflect an endocrine precursor phenotype as previously suggested (2,
40). Furthermore, Glut-2-positive and hormone-negative cells in the
developing pancreas have been proposed as pre--cells (39). The fact
that -cell-like maturation of the Tu-6 somatostatinoma culture
occurred through the insulinoma phenotype is in accordance with the
cell lineage model of islet ontogeny as -cell maturation was
proposed to proceed through an insulin/somatostatin double-positive
phenotype (1). PP-cell maturation has earlier been suggested to proceed
through insulin and somatostatin-expressing precursor cells with
terminal differentiation occurring late in islet development (1, 40).
This is contrasted by our finding that, within the tumor system, only
the AN glucagonoma expresses PP, suggesting that PP- and -cell types
could be linked more closely. Supporting this hypothesis are findings
of a cell type co-expressing glucagon and PP in man (41), and that a
high fraction of the earliest PP-positive cells during rat pancreatic
development are co-expressing
glucagon.2
An important observation of this study is the selective co-activation
of the homeodomain protein Nkx 6.1 with insulin in the IN insulinoma.
This suggests a role for Nkx 6.1 in the specification of the normal
-cell, substantiated by our finding that Nkx 6.1 immunoreactivity
within the islet is confined to the nuclei of pancreatic -cells
exclusively. Several otherwise -cell restricted genes, such as
Glut-2, IAPP, and Pdx-1 were found to be expressed in the MSL-G2
culture prior to insulin and GLP-1-R gene activation. It thus appears
that differentiation of the -cell can be divided into discrete
steps, each requiring the action of appropriate key regulatory factors.
Both Pdx-1 and Nkx 6.1 are likely mediators of these processes.
However, although this study may suggest that Nkx 6.1 is playing a role
late in -cell development, its action is probably not limited to the
specification of the -cell phenotype, as developmental studies have
shown that Nkx 6.1 protein is expressed in the majority of the
endodermal pancreatic precursor cells (Pdx-1- and
Glut-2-positive-cells), during early stages of pancreatic
organogenesis, and at subsequent stages segregates to -cells
exclusively.2
In addition to the MSL-G2-derived cells, we analyzed the commonly used
rat insulinoma cell line, RIN-5AH, which is syngeneic to MSL cells. In
many respects, the RIN-cells appear to represent much less
differentiated -cells when compared to the MSL-G2-derived IN tumor.
This is most notably exemplified by the low insulin production, and
lack of IAPP, Glut-2, Nkx 6.1, R-cadherin, and IGF-1 receptors.
Additionally, RIN-5AH cells express the homeodomain proteins cdx-3 and
lmx-1, which we do not find in islets. We believe that the prolonged
in vitro culture of RIN-5AH cells combined with their fast
growth rate are major determinants of these differences. From this
comparison, we suggest that the MSL-G2-IN phenotype in many respects is
superior to RIN-5AH cells when addressing aspects of -cell biology
by the use of transformed cells.
In summary, we have, by the use of mRNA profiling, successfully
predicted the -cell-specific expression of Nkx 6.1. We also predict
a number of other genes to be expressed in particular islet cell types.
Whether these predictions are accurate is currently being investigated
using in situ hybridization and immunohistochemistry.
FOOTNOTES
To whom correspondence should be addressed. Tel.: 45-31680860;
Fax: 45-44438000; E-mail: odm{at}hrl.dk.
1 The abbreviations used are: RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polmerase chain reaction; CGRP, calcitonin gene-related peptide; CREB, cAMP-responsive element-binding protein; GH, growth hormone; GIP, gastric inhibitory peptide; GLP-1, glucagon like peptide-1; Glut, glucose transporter; GST, glutathione S-transferase; HLH, helix-loop-helix; IAPP, islet amyloid polypeptide; IEF-1, insulin enhancer factor-1; IGF-1, insulin like growth factor-1; NCAM, neural cell adhesion molecule; Pdx-1, pancreatic duodenal homeodomain factor-1; PP, pancreatic polypeptide; -R (suffix), receptor.
2 A. Øster, J. Jensen, P. Serup, O. D. Madsen, and L.-I. Larsson, manuscript in preparation.
Acknowledgments
We are thankful for the excellent technical assistance of Heidi I. Jensen and Erna E. Petersen. We also thank Dr. Christian Bjørbæk for technical advice and Dr. Lars-Inge Larsson and Dr. Allan E. Karlsen for critical reading of this manuscript. Hagedorn Research Institute is an independent research component of Novo Nordisk A/S.
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B. Tyrberg, J. Ustinov, T. Otonkoski, and A. Andersson Stimulated Endocrine Cell Proliferation and Differentiation in Transplanted Human Pancreatic Islets: Effects of the ob Gene and Compensatory Growth of the Implantation Organ Diabetes, February 1, 2001; 50(2): 301 - 307. [Abstract] [Full Text]
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C. E. Pierreux, F. J. Nicolás, and C. S. Hill Transforming Growth Factor beta -Independent Shuttling of Smad4 between the Cytoplasm and Nucleus Mol. Cell. Biol., December 1, 2000; 20(23): 9041 - 9054. [Abstract] [Full Text]
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T. Kakuma, Y. Lee, M. Higa, Z.-w. Wang, W. Pan, I. Shimomura, and R. H. Unger Leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets PNAS, July 18, 2000; 97(15): 8536 - 8541. [Abstract] [Full Text] [PDF]
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P. Jacquemin, S. M. Durviaux, J. Jensen, C. Godfraind, G. Gradwohl, F. Guillemot, O. D. Madsen, P. Carmeliet, M. Dewerchin, D. Collen, et al. Transcription Factor Hepatocyte Nuclear Factor 6 Regulates Pancreatic Endocrine Cell Differentiation and Controls Expression of the Proendocrine Gene ngn3 Mol. Cell. Biol., June 15, 2000; 20(12): 4445 - 4454. [Abstract] [Full Text]
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R. G. Mirmira, H. Watada, and M. S. German beta -Cell Differentiation Factor Nkx6.1 Contains Distinct DNA Binding Interference and Transcriptional Repression Domains J. Biol. Chem., May 5, 2000; 275(19): 14743 - 14751. [Abstract] [Full Text] [PDF]
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A. E. Karlsen, D. Pavlovic, K. Nielsen, J. Jensen, H. U. Andersen, F. Pociot, T. Mandrup-Poulsen, D. L. Eizirik, and J. Nerup Interferon-{gamma} Induces Interleukin-1 Converting Enzyme Expression in Pancreatic Islets by an Interferon Regulatory Factor-1-Dependent Mechanism J. Clin. Endocrinol. Metab., February 1, 2000; 85(2): 830 - 836. [Abstract] [Full Text]
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M Sander, L Sussel, J Conners, D Scheel, J Kalamaras, F Dela Cruz, V Schwitzgebel, A Hayes-Jordan, and M German Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas Development, January 12, 2000; 127(24): 5533 - 5540. [Abstract] [PDF]
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V. Schwitzgebel, D. Scheel, J. Conners, J Kalamaras, J. Lee, D. Anderson, L Sussel, J. Johnson, and M. German Expression of neurogenin3 reveals an islet cell precursor population in the pancreas Development, January 8, 2000; 127(16): 3533 - 3542. [Abstract] [PDF]
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E. D. Galsgaard, J. H. Nielsen, and A. Moldrup Regulation of Prolactin Receptor (PRLR) Gene Expression in Insulin-producing Cells. PROLACTIN AND GROWTH HORMONE ACTIVATE ONE OF THE RAT PRLR GENE PROMOTERS VIA STAT5a AND STAT5b J. Biol. Chem., June 25, 1999; 274(26): 18686 - 18692. [Abstract] [Full Text] [PDF]
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J.-C. Jonas, A. Sharma, W. Hasenkamp, H. Ilkova, G. Patane, R. Laybutt, S. Bonner-Weir, and G. C. Weir Chronic Hyperglycemia Triggers Loss of Pancreatic beta Cell Differentiation in an Animal Model of Diabetes J. Biol. Chem., May 14, 1999; 274(20): 14112 - 14121. [Abstract] [Full Text] [PDF]
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J. B. Hansen, R. K. Petersen, B. M. Larsen, J. Bartkova, J. Alsner, and K. Kristiansen Activation of Peroxisome Proliferator-activated Receptor gamma Bypasses the Function of the Retinoblastoma Protein in Adipocyte Differentiation J. Biol. Chem., January 22, 1999; 274(4): 2386 - 2393. [Abstract] [Full Text] [PDF]
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 L. H. Hansen, B. Madsen, B. Teisner, J. H. Nielsen, and N. Billestrup Characterization of the Inhibitory Effect of Growth Hormone on Primary Preadipocyte Differentiation Mol. Endocrinol., August 1, 1998; 12(8): 1140 - 1149. [Abstract] [Full Text]
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T. L. Jetton, J. M. Moates, J. Lindner, C. V. E. Wright, and M. A. Magnuson Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells PNAS, July 21, 1998; 95(15): 8654 - 8659. [Abstract] [Full Text] [PDF]
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U. Ahlgren, J. Jonsson, L. Jonsson, K. Simu, and H. Edlund beta -Cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta -cell phenotype and maturity onset diabetes Genes & Dev., June 15, 1998; 12(12): 1763 - 1768. [Abstract] [Full Text]
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C. M. Larsen, K. A. W. Wadt, L. F. Juhl, H. U. Andersen, A. E. Karlsen, M. S.-S. Su, K. Seedorf, L. Shapiro, C. A. Dinarello, and T. Mandrup-Poulsen Interleukin-1beta -induced Rat Pancreatic Islet Nitric Oxide Synthesis Requires Both the p38 and Extracellular Signal-regulated Kinase 1/2 Mitogen-activated Protein Kinases J. Biol. Chem., June 12, 1998; 273(24): 15294 - 15300. [Abstract] [Full Text] [PDF]
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A. Øster, J. Jensen, P. Serup, P. Galante, O. D. Madsen, and L.-I. Larsson Rat Endocrine Pancreatic Development in Relation to Two Homeobox Gene Products (Pdx-1 and Nkx 6.1) J. Histochem. Cytochem., June 1, 1998; 46(6): 707 - 716. [Abstract] [Full Text]
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A. Øster, J. Jensen, H. Edlund, and L.-I. Larsson Homeobox Gene Product Nkx 6.1 Immunoreactivity in Nuclei of Endocrine Cells of Rat and Mouse Stomach J. Histochem. Cytochem., June 1, 1998; 46(6): 717 - 722. [Abstract] [Full Text]
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L Sussel, J Kalamaras, D. Hartigan-O'Connor, J. Meneses, R. Pedersen, J. Rubenstein, and M. German Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells Development, January 6, 1998; 125(12): 2213 - 2221. [Abstract] [PDF]
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A. Sharma, E. Henderson, L. Gamer, Y. Zhuang, and R. Stein Analysis of the Role of E2A-Encoded Proteins in Insulin Gene Transcription Mol. Endocrinol., October 1, 1997; 11(11): 1608 - 1617. [Abstract] [Full Text]
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K.-M. Yao, M. Sha, Z. Lu, and G. G. Wong Molecular Analysis of a Novel Winged Helix Protein, WIN. EXPRESSION PATTERN, DNA BINDING PROPERTY, AND ALTERNATIVE SPLICING WITHIN THE DNA BINDING DOMAIN J. Biol. Chem., August 8, 1997; 272(32): 19827 - 19836. [Abstract] [Full Text] [PDF]
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M Sander, A Neubuser, J Kalamaras, H C Ee, G R Martin, and M S German Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development. Genes & Dev., July 1, 1997; 11(13): 1662 - 1673. [Abstract] [PDF]
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R. Gasa, P. B. Jensen, H. K. Berman, M. J. Brady, A. A. DePaoli-Roach, and C. B. Newgard Distinctive Regulatory and Metabolic Properties of Glycogen-targeting Subunits of Protein Phosphatase-1 (PTG, GL, GM/RGl) Expressed in Hepatocytes J. Biol. Chem., August 18, 2000; 275(34): 26396 - 26403. [Abstract] [Full Text] [PDF]
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Y. Lee, M.-Y. Wang, T. Kakuma, Z.-W. Wang, E. Babcock, K. McCorkle, M. Higa, Y.-T. Zhou, and R. H. Unger Liporegulation in Diet-induced Obesity. THE ANTISTEATOTIC ROLE OF HYPERLEPTINEMIA J. Biol. Chem., February 16, 2001; 276(8): 5629 - 5635. [Abstract] [Full Text] [PDF]
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A. E. Allen-Jennings, M. G. Hartman, G. J. Kociba, and T. Hai The Roles of ATF3 in Glucose Homeostasis. A TRANSGENIC MOUSE MODEL WITH LIVER DYSFUNCTION AND DEFECTS IN ENDOCRINE PANCREAS J. Biol. Chem., July 27, 2001; 276(31): 29507 - 29514. [Abstract] [Full Text] [PDF]
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H. Wang, P. Maechler, B. Ritz-Laser, K. A. Hagenfeldt, H. Ishihara, J. Philippe, and C. B. Wollheim Pdx1 Level Defines Pancreatic Gene Expression Pattern and Cell Lineage Differentiation J. Biol. Chem., June 29, 2001; 276(27): 25279 - 25286. [Abstract] [Full Text] [PDF]
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J.-C. Jonas, D. R. Laybutt, G. M. Steil, N. Trivedi, J. G. Pertusa, M. Van de Casteele, G. C. Weir, and J.-C. Henquin High Glucose Stimulates Early Response Gene c-Myc Expression in Rat Pancreatic beta Cells J. Biol. Chem., September 14, 2001; 276(38): 35375 - 35381. [Abstract] [Full Text] [PDF]
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A. E. Karlsen, S. G. Ronn, K. Lindberg, J. Johannesen, E. D. Galsgaard, F. Pociot, J. H. Nielsen, T. Mandrup-Poulsen, J. Nerup, and N. Billestrup Suppressor of cytokine signaling 3 (SOCS-3) protects beta -cells against interleukin-1beta - and interferon-gamma -mediated toxicity PNAS, October 9, 2001; 98(21): 12191 - 12196. [Abstract] [Full Text] [PDF]
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