(Received for publication, November 28, 1995; and in revised form, January 17, 1996)
From the
Pancreatic beta cells and neuronal cells show a large number of similarities. For example, functional receptors for nerve growth factor are present in beta cells. Here we investigate whether TrkC, a neuronal high affinity receptor for neurotrophin-3, is expressed in the insulin-secreting cell line INS-1. We demonstrate the expression in INS-1 cells of mRNAs coding for TrkC identical in size to those found in the brain. As in neuronal cells, different alternatively spliced forms of TrkC mRNA, differing by the insertion of an alternative exon in their kinase domain, were expressed in INS-1 cells. TrkC protein is also expressed in INS-1 cells and is functional. Indeed, when INS-1 cells were treated with neurotrophin-3, TrkC became phosphorylated on tyrosine residues, and the expression of early response genes was induced. This activation of the receptor was paralleled by a rapid and transient increase in cytosolic free calcium due to an influx of extracellular calcium. Functional receptors for NT-3 are thus expressed in INS-1 cells. This cell line provides a new model for the study of NT-3 signal transduction and should be useful in the understanding of the role of neurotrophins in insulin-secreting cells.
The insulin-secreting beta cells of the pancreas are similar in
many ways to neuronal cells(1, 2, 3) . We had
thus previously postulated that neurotrophic factors, which are
implicated in the differentiation, growth, and survival of neuronal
cells, could act on beta cells. We have shown that, indeed, both the
high and the low affinity (TrkA and p75) nerve growth
factor (NGF) (
)receptors are expressed in a number of
insulin-producing cell lines and in fetal and adult rat
islets(4, 5) . In different beta cell lines, these
receptors are functional, as demonstrated by binding studies using
iodinated NGF and by phosphorylation of TrkA on tyrosine residues and
induction of early response genes by NGF treatment(6) .
The
family of neurotrophic factors contains NGF, the first neurotrophin to
be identified(7) , but also brain-derived neurotrophic factor
(BDNF) (8) and neurotrophin-3 (NT-3)(9, 10) .
Signal transduction by these neurotrophins is initiated by high
affinity binding to and activation of specific tyrosine kinase
receptors. TrkA and TrkC bind NGF and NT-3, respectively, whereas TrkB
binds BDNF(11) . In addition, all neurotrophins bind to the low
affinity receptor p75(12) . The precise role of
p75
in NGF signal transduction is not fully understood.
Different roles have been recently attributed to NT-3 in the central
and peripheral nervous system. It has been shown to support the
survival of sensory neurons isolated from nodose ganglia, of
oligodendrocytes, and of noradrenergic neurons in
vivo(9, 10, 13, 14) , and to
promote the proliferation of neural crest cells (15) . NT-3 has
also been implicated in short term effects, such as potentialization of
neuronal activity(16, 17) . Moreover, NT-3 has been
shown to induce intracellular free calcium
([Ca]
) elevation in
hippocampal neurons within seconds following treatment(18) .
Whereas the expression of NGF and BDNF has been detected only in a limited number of peripheral tissues, NT-3 mRNAs have been detected in all the tissues tested, including brain, heart, skin, gut, muscle, lung, spleen, and liver(9, 10) . This ubiquitous pattern of expression suggests that NT-3 could act outside the nervous system. In fact, it has been shown recently that melanocytes constitutively express low levels of TrkC and that NT-3 prevents cell death of melanocytes (19) .
The aim of the present study was to investigate whether the insulin-producing cells, INS-1, express functional TrkC receptors and whether NT-3 plays a role in these cells, similar to that which has previously been described for neurons. We therefore studied 1) the expression of TrkC mRNA in INS-1 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis and determined which isoform of TrkC was expressed; 2) signal transduction steps, such as phosphorylation of TrkC after NT-3 treatment, and induction of the expression of early response genes, such as c-fos and NGFI-A; 3) cytosolic free calcium as it is known that, in hippocampal neurons, NT-3 increases cytosolic free calcium; and 4) finally, whether NT-3 affects insulin secretion in INS-1 cells. Our data demonstrate that INS-1 cells represent a new experimental system for studying the different steps of signal transduction by NT-3 in endocrine cells.
Figure 1:
Expression of different TrkC isoforms
in INS-1 cells. Top, ethidium bromide staining of 1% agarose
gels containing PCR products of reverse-transcribed total RNA in the
presence (+RT) or the absence (-RT) of
reverse transcriptase, using TrkC and TrkC
as primers that corresponded to part of the extracellular domain
of TrkC (a), or TrkC
and TrkC
as
primers that corresponded to part of the kinase domain of TrkC (b), or cyclophilin-specific primers as a control (c). Bottom, nucleotide and amino acid sequence of
part of TrkCK1 and TrkCK14 cDNAs cloned from INS-1 cells as described
under ``Experimental
Procedures.''
Various truncated forms
of Trk, lacking a kinase domain, and unable to transduce a signal into
the cell, have previously been
described(25, 29, 30, 31) . Thus, to
determine whether mRNA coding for the TrkC kinase domain was expressed
by INS-1 cells, PCR analysis was also performed using oligonucleotides
spanning the kinase domain. As shown in Fig. 1a, when
TrkC and TrkC
primers were used, the
major amplification product obtained using rat brain cDNA had the
predicted 685-base pair size. The same sized amplification product was
obtained when INS-1 cDNA was used. No amplification was obtained from
cDNA prepared from PC12 cells or when reverse transcriptase was
omitted. In addition, a second major product of higher molecular weight
was coamplified from INS-1 cDNA. In the brain, different TrkC tyrosine
kinase isoforms containing variable-sized amino acid insertions within
the tyrosine kinase domain have been recently
described(25, 31) . They were shown to differ in their
response to NT-3, the form without insert (TrkCK1) being the most
potent(25, 30, 31) . To determine whether the
TrkCK1 isoform was expressed in INS-1 cells, the PCR products
corresponding to the kinase domain were subcloned and representative
clones were submitted to sequence analysis. Two different sequences
with or without 42 bases encoding a 14-amino acid insert were detected
in INS-1 cells (Fig. 1b). Thus, the two products of
different molecular weight, which were coamplified from INS-1 cells
cDNA when oligonucleotide primers spanning the TrkC kinase domain were
used, are identical to TrkCK1 and TrkCK14 previously detected in the
brain.
TrkC expression was then examined by Northern blot analysis of total RNA extracted from INS-1 cells, PC12 cells, and adult rat brain. Two different probes, specific for the extracellular and kinase domain of TrkC, respectively, were used. Analysis with the TrkC extracellular probe (Fig. 2a) revealed a similar pattern of expression in rat brain and INS-1 cells with five major transcripts of different molecular sizes: 14, 7, 4.7, 4, and 1.1 kb. No signal was detected in PC12 cell RNA. Analysis with the TrkC kinase domain probe (Fig. 2b) showed the presence in both INS-1 cells and brain of four transcripts of different molecular sizes: 14, 10, 5, and 2.9 kb. A 2.9-kb band was also detected in RNA from PC12 cells. This band, which is absent when the extracellular probe is used, could represent cross-hybridization with TrkA. The 14-kb band is recognized by both probes in brain and INS-1 cells and represents thus a full-length TrkC receptor. In addition, a number of other transcripts in INS-1 cells, which could encode different truncated forms of TrkC, were revealed by Northern blot analysis.
Figure 2: Northern blot analysis of TrkC transcripts. Ten µg of total RNAs from adult rat brain, PC12 and INS-1 cells were hybridized using a part of extracellular domain of TrkC as probe (a) or a part of kinase domain of TrkC as probe (b). The arrows denote different transcripts detected with each probe. Ethidium bromide staining of the membranes probed with a part of extracellular domain of TrkC (c) and a part of kinase domain of TrkC (d), allowing comparison of the total amount of RNA per sample.
Figure 3: TrkC protein is expressed in INS-1 cells. INS-1 and PC12 cells were lysed and immunoprecipitated with an antibody recognizing the extracellular domain of TrkC. The resulting immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose filter, incubated using the same anti-TrkC antibody, and demonstrated using the ECL chemiluminescence system.
Figure 4: NT-3-induced tyrosine phosphorylation of TrkC in INS-1 cells. INS-1 and PC12 cells were treated with NT-3 (50 ng/ml) or NGF (50 ng/ml) for 5 min, lysed, and immunoprecipitated with an antibody recognizing the intracellular domain of TrkC. The resulted immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose filter, incubated using an anti-phosphotyrosine antibody, and demonstrated using the ECL chemiluminescence system.
Figure 5: Effect of NT-3 on c-fos and NGFI-A gene expression in INS-1 and PC12 cells. The cells were cultured in medium depleted in serum for 16 h and then treated for different periods of time with NT-3 (50 ng/ml), or 10% of fetal calf serum (FCS). Cytoplasmic RNAs were isolated and hybridized using c-fos, NGFI-A, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes as described under ``Experimental Procedures.''
Figure 6:
Effect of NT-3 on cytosolic free calcium
[Ca]
in INS-1 cells.
The concentrations of [Ca
]
were measured by dual emission microspectrofluorometry using
Indo 1 as the intracellular fluorescent calcium probe. A, the
effect of NT-3 on cytosolic free calcium was researched in the presence
of 1.5 mM CaCl
. B, the effect of NT-3 on
cytosolic free calcium was measured in the absence of extracellular
calcium. C, the cells were preincubated with 1 mM cobalt before NT-3 addition. D, the cells were
preincubated with K252a before NT-3 stimulation. E, the effect
of NGF on cytosolic free calcium was researched in the presence of 1.5
mM CaCl
.
Here, we report that the insulin-secreting cell line INS-1 expresses functional receptors to NT-3. By Northern blot analysis and RT-PCR, mRNAs coding for TrkC, a high affinity NT-3 receptor, are detected in INS-1 cells, identical in size to those found in the brain. Upon addition of NT-3, TrkC is phosphorylated on tyrosine residues, and the expression of early response genes is induced. This activation of the receptor is paralleled by a rise in intracellular free calcium.
Most of the data available concerning the expression of TrkC derive from the nervous system, in which it is expressed at its highest levels (25, 31) . In the brain, different forms of TrkC have been identified (25, 30, 31) . Some contain an intracytoplasmic kinase domain, while others lack a kinase domain and are thus thought to be unable to transduce an intracellular signal. This type of truncated form lacking a kinase domain has previously been demonstrated in the case of other members of the Trk family, such as TrkB, a high affinity receptor for BDNF and NT4(29, 33) . For TrkB receptors, both full-length and truncated forms are detected in the brain(29, 33) , whereas only truncated forms are detected in non-neuronal tissues, such as spleen, submaxillary gland, testes, kidney, or pituitary(33, 34) . In the case of TrkC, the situation is slightly more confusing. Indeed, when RNAs prepared from different tissues are analyzed, a weak signal is detected using a probe recognizing TrkC in the thymus, lung, kidney, stomach, and testes. Whereas some reports propose that, as in the case of TrkB, only truncated forms of TrkC are expressed in non-neuronal tissues (31) , others detect mRNA coding for full-length TrkC forms in non-neuronal tissues(25) . It was thus important to define whether full-length TrkC mRNA was expressed in INS-1 cells, which are thought to derive from the endoderm. Parallel analyses using probes that recognize the extracellular and the kinase domain of TrkC demonstrate the presence of a similar 14-kb transcript with each probe in total RNA prepared from brain and INS-1 cells, demonstrating the expression of full-length TrkC mRNA in the non-neuronal cell line INS-1. The other transcripts at 7.2, 4.7, and 4 kb, which were detected using the extracellular probe but not using the kinase domain probe, probably correspond to truncated forms of TrkC lacking a kinase domain as previously suggested by Valenzuela et al.(31) .
In addition to forms lacking an intracytoplasmic tyrosine kinase domain, other TrkC isoforms have recently been described in the brain (25, 30, 31) . These isoforms contain or not an insertion in their kinase domain of 14, 25, or 39 amino acids and have been shown to mediate different responses within the target cells. Whereas all of the TrkC tyrosine kinase isoforms have the ability to autophosphorylate after NT-3 treatment, the form with no insert is the only one able to mediate proliferation or neuronal differentiation in fibroblasts or PC12 cell lines that have been engineered to express the different forms of TrkC(25, 30, 31) . We demonstrate in the present study that the pancreatic beta cell line INS-1 expresses, in addition to other forms, TrkCK1, which is, as described above and according to different groups, the most potent receptor for NT-3(25, 30, 31) . Thus INS-1 cells express, in addition to other TrkC isoforms, the full-length NT-3 receptor TrkCK1, known to be functional in neuronal cells.
In INS-1 cells, NT-3 receptors are functional as demonstrated by tyrosine autophosphorylation of the receptor and induction of early response gene expression, such as c-fos and NGFI-A after NT-3 treatment. Thus, INS-1 cells represent a new experimental system for the study of TrkC-mediated signal transduction. In fact, whereas PC12 cells have been used for years to study NGF signal transduction, to our knowledge, no such system exists for the study of NT-3 signal transduction. The only available cell lines are fibroblasts or PC12 cells engineered to express TrkC. Whereas these cell lines are very useful for the study of some steps of neurotrophin signal transduction, Trk receptors behave differentially, depending on the cellular context. For example, NT-3 is comparable to BDNF in its ability to induce TrkB autophosphorylation in fibroblasts transfected with TrkB, but not in PC12 cells transfected with TrkB (35) . Moreover, according to the experimental system used, neurotrophins can act as growth factors and induce cell proliferation or act as differentiation factors and induce growth inhibition(36) . Thus the insulin-producing cell line INS-1, which constitutively expresses TrkC, will represent a new experimental system for the study of the effects of NT-3.
In INS-1
cells, cytoplasmic free Ca plays a fundamental role
in signal transduction. For example, short term variations in
[Ca
]
are implicated in insulin
release by glucose(32) . When glucose is taken up by beta
cells, there is a rise in ATP, inducing the closure of ATP-dependent
potassium channels. This leads to opening of voltage-dependent calcium
channels, increase in [Ca
]
, and
insulin release. Since it has been shown that NT-3 induces an elevation
in [Ca
]
(18) , we
investigated whether such a variation could be produced in INS-1 cells.
We demonstrated that NT-3 induces an elevation of cytosolic free
calcium mediated by Trk. This elevation is due to an influx of
extracellular calcium and requires the phosphorylation of TrkC on
tyrosine residues. No such calcium influx is seen when INS-1 cells are
treated with NGF. INS-1 cells represent thus an experimental system in
which two neurotrophins acting via two different receptors produce
different effects. It would now be interesting to compare the signal
transduction pathway of NGF and NT-3 via TrkA and TrkC, respectively,
in INS-1 cells and to define at which point they diverge.
Because
elevation in [Ca]
has been
implicated in insulin secretion, we tested whether NT-3, which induces
a raise in [Ca
]
, would induce
insulin release. In the experimental conditions tested, we were unable
to detect any effect of NT-3 on insulin release. There are different
possible explanations for the discrepancy between the rise in
[Ca
]
and its expected effect on
insulin secretion. Application of various peptides as well as
-adrenergic agonists have been shown to suppress
insulin exocytosis, despite sufficient high levels of intracellular
calcium(37) . Thus, in certain conditions, increase in
[Ca
]
and insulin secretion can
be dissociated. The absence of insulin release upon NT-3 treatment
could also be due to the experimental system itself; although it has
been reported that INS-1 cells secrete insulin after glucose
treatment(21) , in our hands, after a given number of passages,
we were not able to reproducibly observe a significant increase of
insulin secretion using the range of glucose concentrations reported
under ``Experimental Procedures.'' Therefore, caution is
necessary in concluding that NT3 has no effect on insulin secretion.
Obviously, more information is needed using mature islets of
Langerhans.
Whereas TrkC is a receptor for NT-3, it seems that, in
certain experimental systems, NT-3 can signal through TrkA. Indeed, in
fibroblasts engineered to express TrkA, both NGF and NT-3 induce the
rapid phosphorylation of TrkA and the transient expression of c-Fos
protein(36) . In PC12 subclones that express TrkA but not TrkC
and that have been engineered to express different levels of
p75, NT-3 is fully capable of inducing neurite outgrowth
in the clones deficient in p75
but not in the parental
clones expressing p75
. Finally, in PC12 cells, when an
anti-p75
antibody is used to block NGF and NT-3 binding
to the p75
, NT-3 induces tyrosine autophosphorylation of
TrkA, transcription of Zif268, and cellular
differentiation(38, 39) . Thus, in different cell
lines and in the absence of p75
expression, NT-3
transduces a signal via TrkA. The possibility for NT-3 to signal via
TrkA has also been studied in neurons, and it has been shown that, at
certain stages of development, NT-3 can signal through
TrkA(40) . Because we have previously shown that INS-1 cells
express functional TrkA receptors(4, 6) , it was
important to demonstrate that NT-3 signals through TrkC in INS-1 cells.
We have now accumulated much evidence demonstrating that, in INS-1
cells, NT-3 signal transduction is mediated by TrkC. First, full-length
TrkC mRNAs, identical to those present in the brain, are detected in
INS-1 cells; second, INS-1 cells express high levels of
p75
; third, TrkC is specifically phosphorylated upon
NT-3 treatment of INS-1 cells; and finally, NT-3 induces an elevation
in [Ca
]
in INS-1 cells, while
no effect of NGF on [Ca
]
is
detected in the same cell type. Taken together, the data demonstrate
that TrkC is implicated in NT-3 signal transduction in INS-1 cells.
Beta cell lines, such as INS-1, can thus respond to NGF via TrkA and to NT-3 via TrkC. These neurotrophin receptors belong now to the increasing list of molecules shared by both beta and neuronal cells. For example, enzymes such as tyrosine hydroxylase and glutamic acid decarboxylase(1, 2) , molecules of the cytoskeleton such as neurofilaments(3) , and type II sodium channels(41) , first thought to be specific of neuronal and neural crest-derived cells, are expressed by both beta and neuronal cells. These similarities suggest that identical tissue-specific transcription factors could be expressed in beta and neuronal cells. It is in fact interesting to note that isl-1, a homeobox gene which was originally identified for its ability to transactivate the insulin gene, is also expressed in motoneurons in the embryonic spinal cord, where it could be involved in motoneuron fate(42) . Pax-6, a homeodomain-containing protein, also belongs to the family of specific transcription factors shared by neuronal and islet cells; it is expressed in the neuroretina and in the neural tube, but also in insulin- and glucagon-producing cells(43) . Another hypothesis exists to explain the large number of similarities between neurons and beta cells; it is based on the possible common absence in these two cell types of a specific repressor. Such a repressor, present in all non-neuronal cells, would inhibit the expression of neuron-specific proteins. This is substantiated by the finding of a 25-base pair element in certain murine genes such as SCG10, synapsin I, and type II sodium channels(44) . This 25-base pair element is believed to play the role of silencer after binding a specific repressor expressed in non-neuronal cells. Recently, such a repressor has been cloned(44) . The demonstration of the lack of expression of this repressor in beta cells would help to understand the expression of a large number of neuronal markers within beta cells.