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Transplantation Laboratory, Haartman Institute and Children’s Hospital, University of Helsinki, Helsinki FIN-00014, Finland
Address all correspondence and requests for reprints to: Dr. Mari-Anne Huotari, Transplantation Laboratory, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. E-mail: mari.huotari{at}helsinki.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TGF-), neu
differentiation factor, and TGF-ß were inactive. None of these
factors affected the insulin content of INS-1 cells. In contrast,
certain differentiation factors, including nicotinamide, sodium
butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited
the DNA synthesis and increased the insulin content. Also
all-trans-retinoic acid had an inhibitory effect on cell
DNA synthesis but no effect on insulin content. From these findings
betacellulin emerges as a novel growth factor for the ß-cell.
Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25
pM betacellulin. Interestingly, betacellulin had no effect
on RINm5F cells, whereas both EGF and TGF- were slightly mitogenic.
These effects may possibly be explained by differential expression of
the erbB receptor tyrosine kinases. In RINm5F cells a
spectrum of erbB gene expression was detected (EGF
receptor/erbB-1, erbB-2/neu, and
erbB-3), whereas INS-1 cells showed only expression of EGF
receptor. Expression of the erbB-4 gene was undetectable in
these cell lines. In summary, our results suggest that the INS-1 cell
line is a suitable model for the study of ß-cell growth and
differentiation because the responses to previously identified ß-cell
mitogens were essentially similar to those reported in primary cells.
In addition, we have identified betacellulin as a possible modulator of
ß-cell growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the adult, the proliferative capacity of the islet cells is very limited, but islet mass can nevertheless increase through two possible mechanisms: replication of preexisting ß-cells or differentiation from ductal precursor cells. ß-cell replication can be readily demonstrated in fetal and neonatal islets both in rodents (4) and man (5). This activity decreases with aging, but a low degree of ß-cell proliferation is present even in adult islets (6). It has been shown that islet-cell neogenesis can be reactivated in adult rodents, and that this is a major pathway responsible for the increased ß-cell mass (7, 8).
The extracellular factors regulating islet differentiation and growth remain poorly understood. It is obvious that this requires the synergistic function of several genes and their products. Several growth factors have been associated with the regulation of ß-cell differentiation, their replication and maintenance of ß-cell mass (9). A recently identified exocrine pancreatic protein, INGAP (10), and other members of pancreas-associated lectin-type proteins, including reg (11), may be important for the initiation of ductal cell stimulation resulting in islet neogenesis. Some well-known factors including insulin-like growth factors (IGF-1 and -2), GH and related factors, particularly PRL (4, 12), are ß-cell mitogens. Hepatocyte growth factor (HGF) is capable of increasing the proliferation of fetal human ß-cells through its receptor, c-met (13). HGF also stimulates the growth of pancreatic duct cells, and may thus participate in the regenerative process at various levels (14). Vascular endothelial growth factor (VEGF) may have a role in ß-cell maturation from ductal precursor cells because its receptor (Flk-1) is found in the ductal cells and exogenous VEGF stimulates their proliferation (15, 16).
Some members of the epidermal growth factor (EGF) family are proposed
to have a role in the development of the endocrine pancreas.
Transforming growth factor- (TGF-) is abundantly present in the
developing pancreas (17), and its overexpression together with gastrin
in transgenic mice increases the islet mass significantly (18). The EGF
receptor (EGFR) is expressed throughout the human fetal pancreas, and
mice lacking EGFR show disturbed formation of pancreatic islets (19).
Betacellulin, a novel member of the EGF family, can together with
activin A convert exocrine AR42J cells to insulin expressing cells
(20). All growth factors of the EGF family are ligands for one or more
of the four receptor tyrosine kinases encoded by the erbB
gene family (EGFR/erbB-1, erbB-2/neu,
erbB-3, and erbB-4) (21).
Rodent insulinoma cell lines are useful in studies of ß-cell biology. Though these cells are often dedifferentiated, they share important characteristics with normal ß-cells. The INS-1 cell line retains a relatively high level of insulin synthesis and release which is responsive to glucose (22). In the present study, we wanted to obtain a more complete picture of the growth factor mediated regulation of ß-cells. For this purpose, we characterized the responses of INS-1 cells to several growth and differentiation factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
recombinant human VEGF were obtained from R & D Systems Europe, Ltd.
(Oxford, UK). Recombinant human HGF was a gift from Dr. Jeffrey Rubin
(Laboratory of Cellular and Molecular Biology, National Cancer
Institute, Bethesda, MD). Recombinant human nerve growth factor ß
(NGF-ß), all-trans-retinoic acid, nicotinamide (NIC), and
sodium butyrate (Sod.But.) were obtained from Sigma Chemical Co. (St.
Louis, MO). Recombinant human neu differentiation factor (NDF) was a
gift from Dr. Walter Birchmeier (MDC, Max Delbrück Centrum,
Berlin, Germany). Recombinant human activin A (lot no. 15365–36) and
recombinant porcine PRL were obtained through the National Hormone and
Pituitary Program (NIDDK, NIH, Bethesda, MD). 1,25-dihydroxyvitamin
D3 (1, 25 -(OH)2D3) was a product
of Leo Pharmaceutical Products (Ballerup, Denmark) and recombinant
human GH (Genotropin) was from Pharmacia AB (Uppsala, Sweden).
Cell cultures
INS-1 cells (passages 70 to 95, kindly provided by Prof. Claes
Wollheim, University of Geneva, Geneva, Switzlerland) were cultured in
tissue culture flasks (Corning, NY) in complete medium composed of
medium RPMI 1640 supplemented with 2 mM
L-glutamine, 10 mM HEPES, 1 mM
sodium pyruvate, 50 µM 2-mercaptoethanol, 100 U/ml
penicillin, 100 µg/ml streptomycin and 10% heat-inactivated FCS
(GIBCO, Life Technologies, Paisley, Scotland, UK) under a humified
condition of 95% air and 5% CO2 at 37 C (22). RINm5F
cells (also from Prof. Wollheim) were cultured in medium RPMI 1640
supplemented with 2 mM L-glutamine, 10
mM Hepes, 100 U/ml penicillin, 100 µg/ml streptomycin and
10% heat-inactivated FCS. For assays the cells were detached by a
brief incubation with trypsin/EDTA (GIBCO).
DNA synthesis and cell proliferation
For proliferation studies, the cells (3 x 104)
were plated in 96-well microtiter plates (Nunclon, Denmark) and allowed
to attach in complete medium for 24 h. After that the cells were
preincubated in medium supplemented with 0.5% FCS for another 24
h. The final 24 h incubation was carried out in medium
supplemented with 0.5% FCS and the growth factor to be tested. For the
measurement of DNA synthesis 1 µCi/ml of 3H-thymidine
(925GBq/mmol, 25Ci/mmol, Amersham) was added to each well for the last
4 h of culture. The cells were then harvested with trypsin/EDTA
and transferred on glass fiber filters using a cell harvester (Skatron
Combi, Skatron, Norway) and 3H-radioactivity was measured
in a liquid scintillation counter. The results are presented as percent
of control ± SEM from three individual experiments
done with six parallels.
To confirm the effect of mitogenic factors, the cells (1.5 x 105) were plated in 12-well plates (Falcon) in groups of three and incubated for either 4- or 7-day periods. After that the actual cell numbers were counted in a hemocytometer (Hawksley, UK). The results are expressed as cell numbers/well ± SEM from three experiments.
Measurement of insulin content
To determine the effect of the various factors on insulin
content of INS-1 cells, the cells (1.5 x 105) were
plated in 12-well plates and allowed to attach in complete medium for
24 h. After that the cells were incubated in complete medium with
the factor to be tested for 7 days. The cells were then detached from
the wells by cell dissociation solution (Sigma), washed with PBS (pH
7.4), resuspended in 300 µl of distilled water, and homogenized by
sonication. Cellular insulin content was measured by RIA (Diagnostic
Products Corp., Los Angeles, CA) in dilutions (1:20) of acid ethanol
extracts. DNA was measured from the sonicates fluorometrically (23).
The results are presented as percent of control ± SEM
from triplicates of three individual experiments.
Analysis of gene expression
Total RNA from both INS-1 and RINm5F cells, as well as from rat
pancreas, liver, and brain were isolated using guanidinium thiocyanate
extraction followed by CsCl gradient centrifugation (24). Poly(A)RNA
was purified from total RNA preparations with oligo(dT)-coated magnetic
microbeads (Dynabeads, Dynal A.S., Oslo, Norway). Messenger RNA (mRNA)
(5 µg/lane) was fractionated on a denaturating 1.2% agarose gel and
then transferred to a nylon membrane (Hybond-N, Amersham) via capillary
blotting. The complementary DNA (cDNA) probes were
32P-labeled by a random hexamer priming method (Pime-A-Gene
Labeling System, Promega). EGFR and erbB-2 (neu) cDNAs
corresponded to the extracellular domains of the mouse and rat
receptors, respectively. erbB-3 and erbB-4 cDNAs
represented the whole coding areas of human mRNAs (all receptor cDNAs
kindly provided by Dr. Päivi Miettinen and Prof. Kari Alitalo,
University of Helsinki). Mouse ß-actin cDNA used for loading control
and mouse betacellulin cDNA (synthesized in our laboratory by RT-PCR)
corresponded to the coding area of the mRNA. Hybridizations were
carried out according to the Hybrid-Ease hybridization chamber
instructions (Hoefer Scientific Instruments) at 65 C, buffer containing
1% SDS, 1 M NaCl, and 8% dextran sulfate. The blots were
washed at 65 C in 1 x SSC and finally in 0.5 x SSC.
Hybridization signals were visualized using a Bio-imaging analyzer
(Fuji Photo Film Co., Ltd).
Statistical analysis
Significances of differences in DNA synthesis and insulin
content were analyzed with one-way ANOVA and the Fisher’s PLSD test
using Statview 4.1 software (Abacus, Berkeley, CA) for the
Macintosh.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and NDF) had no
effect (Fig. 1
).
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). However,
the magnitude of the increase in cell number was less than might be
expected from the DNA synthesis results, suggesting that increased
proliferation might also induce increased cell death.
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),
whereas the other ligands (EGF, TGF-, and NDF) had no response.
Interestingly, the results were quite different in the RINm5F cells. In
this cell line, betacellulin did not affect the DNA synthesis, whereas
EGF and TGF- had a slight but significant stimulatory effect (Fig. 3B).
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). 1,25-(OH2)D3 also
had a morphogenic effect on the INS-1 cells indicative of
differentiation; it caused the cells to aggregate and detach from the
culture dish into three-dimensional islet-like structures. This effect
appeared within 48 h of stimulation and was maximal after 4
days.
Insulin content
The factors affecting proliferation were also tested for their
effects on the insulin content of INS-1 cells (Fig. 4). After 7-day incubation with 10
mM nicotinamide, the cellular insulin content per DNA had
increased 1.5-fold as compared with control. Incubation with 1
mM sodium butyrate had an almost 2-fold stimulatory effect.
The combination of these two agents increased the amount of insulin per
DNA 3.7-fold. Remarkably, 2 nM activin A was equally
potent. 1,25-(OH2)D3 also increased the insulin
content 1.8-fold. Retinoic acid did not affect the insulin content,
neither did any of the mitogenic factors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Betacellulin, which was first discovered from mouse tumoral ß-cells
based on its mitogenic activities (30), stimulated INS-1 cell
replication in picomolar concentrations. Betacellulin is a member of
the EGF family, which consist of several polypeptide growth factors
[e.g., EGF, TGF-, amphiregulin, heregulin (NDF) and
heparin-binding EGF] (31). They are ligands for one or more of the
erbB family receptor tyrosine kinases
(EGFR/erbB-1, erbB-2/neu, erbB-3,
erbB-4). It has been shown that betacellulin is a ligand for
EGFR and erbB-4 (32). Signaling through the
erbB-receptor tyrosine kinases is based on
heterodimerization and cross-phosphorylation after binding of the
various EGF ligands. Thus, although EGF has no affinity for
erbB-2, EGF stimulates tyrosine phosphorylation of
erbB-2/neu in cell lines bearing both EGFR/erbB-1
and erbB-2/neu. Likewise, betacellulin may cross-activate
erbB-3 (21, 33, 34).
In our study, betacellulin was mitogenic only for the INS-1 cells but
not for the more primitive RINm5F cells. On the other hand, EGF and
TGF-, which are well-known ligands for EGFR, did induce some
proliferation in RINm5F cells but not in INS-1 cells. We found that
these two cell lines express EGFR/erbB-1. However, only the
more primitive RINm5F cell line expressed both erbB-2/neu
and erbB-3 genes. erbB-4 expression was not
detected in either of the cell lines. Low level of endogenous
betacellulin expression was detected in both INS-1 and RINm5F cells. It
is difficult to unambiguously explain the different response of the
cell lines to the EGF family growth factors. One explanation might be
that, though INS-1 cells show endogenous expression of betacellulin,
the functional protein is not translated in these cells. The
differential expression of erbB family receptors might also
offer an explanation. erbB-2 has been shown to be the
preferred heterodimerization partner of all other erbB
proteins (35), and also erbB-3 tends to form heterodimers
with EGFR/erbB-1. The absence of these two receptors in
INS-1 cells would thus indicate that betacellulin activates only
EGFR/erbB-1 in these cells, whereas in RINm5F cells a
heterodimer of EGFR/erbB-1 with erbB-2 and/or -3
would be formed. Whether this is the explanation for the differential
activity of betacellulin on these two cell lines, is not clear at this
point. Alternatively, another unknown receptor for betacellulin could
be involved in EGFR/erbB-1 heterodimerization and thus, in
signal transmission (36).
The present observations on the specific mitogenic action of
betacellulin on the INS-1 cells adds further proof for a specific role
of this growth factor in islet development. This notion is based on the
pattern of betacellulin expression, which is particularly high in the
pancreas and small intestine (37). Additionally, betacellulin can
specifically convert clonal exocrine pancreatic cells of the AR42J line
into insulin-expressing cells (20). Betacellulin is also required for
the induction of insulin gene expression in clonal -cells
transfected with the pdx-1 gene (38).
Our results are in line with previous findings of GH and PRL being equally effective in increasing INS-1 DNA synthesis (28). Because the majority of hGH binding sites in both rat islets and in INS-1 cells are of lactogenic specificity (39), the effects of hGH may have been mainly mediated by PRL receptors.
VEGF was mitogenic for INS-1 cells in our experiments. Flk-1, a receptor for VEGF, has previously been identified in pancreatic ducts (15) and VEGF has been suggested to stimulate the proliferation of duct cells but not the ß-cells, and perhaps enhance the differentiation of islet cells (16). It appears that INS-1 cells share some characteristics of ß-cell precursors in this respect.
To our surprise, HGF did not have any effect on the proliferation of INS-1 cells. This negative result is likely not due to the use of human recombinant growth factor on rat cells because hHGF has been shown to be fully active on rodent cells (40). HGF has previously been shown to be mitogenic for human fetal (13) and adult (41) ß-cells. The data concerning localization of the HGF-receptor (encoded by c-met) has been conflicting. While it was found to be particularly high in ducts and ß-cells of the human pancreas (13), another study found it only in the ductal epithelium (14). Also in the adult rat, HGF receptors are found in ducts but not in ß-cells (S. Bonner-Weir, personal communication). Whereas fetal human ß-cells do proliferate in response to HGF (13, 42), it appears that HGF does not induce the replication of adult human ß-cells, but instead the proliferation of duct cells (43). Taken together, this suggests that HGF is mainly involved in the stimulation of duct cells, and possibly ß-cell progenitors but not the mature ß-cells. In this respect, the INS-1 cells represent a more mature phenotype.
Using the INS-1 cell line we were able to identify growth-inhibitory factors that are possibly involved in ß-cell differentiation. From these nicotinamide has previously been shown to act as an inducer of endocrine differentiation in fetal human pancreatic cells (44), and to increase islet neogenesis in models of pancreas regeneration (45). Sodium butyrate was another potent inhibitor of INS-1 cell DNA synthesis, which is in line with its well-known activity as a differentiation factor in RIN cells (46). Combining nicotinamide and sodium butyrate led to potentiated differentiative effects with a 4-fold increase in insulin content and decrease of proliferation down to 30% from control.
1,25-(OH)2D3 induced differentiation of INS-1 cells, indicated by decreasing cell replication and increasing cellular insulin content. 1,25-(OH)2D3 also stimulated the INS-1 cells to form islet-like clusters instead of growing as a monolayer. These observations are in agreement with previous findings showing that vitamin D analogs act as growth inhibitors on pancreatic adenocarcinoma cell lines (47) and also on RIN cells (48).
Activin A has previously been reported to disrupt epithelial lobulation in the developing mouse pancreas (49). It has also been shown to induce neurite-like processes from exocrine AR42J cells in culture (20). When exposed to activin A together with betacellulin or HGF, these cells are transformed into insulin-secreting cells (20). In accordance with this, we found that activin A was a potent inhibitor of INS-1 cell growth with a concomitant increase in insulin content. These results underline the role of activin A in ß-cell differentiation.
We conclude that the INS-1 cell line is a suitable model for the study of ß-cell growth and differentiation. It responds to several growth factors similar to primary rat islets. In addition, we have identified betacellulin as a novel possible mitogen for insulin-producing cells. It appears that the action of betacellulin is not entirely mediated through the known erbB receptor tyrosine kinases. We also found 1,25-dihydroxyvitamin D3, nicotinamide and sodium butyrate to be potent factors for the induction of the genes responsible for ß-cell differentiation.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Juvenile Diabetes Foundation International Career
Development Award.
Received October 10, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TGF-) and insulin gene expression in
human fetal pancreas. Development 114:833–840[Abstract]
-induced ductular precursor cells. J Clin Invest 92:1349–1356
-TC
1 clone 6 cells in the presence of betacellulin. Diabetes 45:1826–1831[Abstract]
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