In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase−polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, β-galactosidase (β-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in β-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature β-cells expressed Kit, indicating that Kit is a marker of a subpopulation of β-cells. Finally, by following β-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.
It is now well established that in different organs, ligands of receptor tyrosine kinases (RTKs) control a large number of processes, such as proliferation, differentiation, survival, and metabolic homeostasis (1). This seems to also be the case in the pancreas, where signals mediated by few ligands of RTKs have been implicated in these specific processes. For example, such signals are thought to control pancreatic cell development (2,3,4,5,6), β-cell survival (2,7), and insulin secretion (8,9).
To further study the role of signals mediated by RTKs in pancreatic development and function, we previously randomly screened for RTKs that were expressed in the rat embryonic pancreas, with an approach that exploited sequence similarities in the kinase domain of these receptors. In this previous work, 30 different RTKs were identified (10). The Kit gene, which codes for the KIT ligand (KITL) receptor or stem cell factor, was one of the genes identified in this study. Both KIT and its ligand are known to be required for different key steps of development. For example, it has been shown that KIT and its ligand play crucial roles in the development of germ cells, hematopoietic cells, and melanocytes (11,12). Little information is available concerning the pattern of expression and potential role of KIT and its ligand in the pancreas. As a first step, we thus asked whether Kit is expressed in the pancreas, and then followed in detail its profile of expression during pancreatic development.
Here, we first used INS-1 cells to demonstrate the expression of Kit in insulin-producing cells. We also showed that the receptor was functional in terms of signal transduction in this cell line. We next extended these data and demonstrated that Kit was also expressed in islets prepared from adult rats. To study the pattern of expression of Kit at the cellular level, we used mice that carry the lacZ reporter gene inserted into the first exon of Kit. In these mice, lacZ expression reflects normal expression of the endogenous Kit gene (13). We demonstrated that in adult mouse islets, Kit is specifically expressed in β-cells. Interestingly, only a subpopulation of β-cells express Kit. Finally, by studying the ontogeny of Kit expression during pancreatic development, we showed that it followed a specific pattern of expression.
RESEARCH DESIGN AND METHODS
Cell culture conditions.
INS-1 cells were derived from a rat insulinoma (14). They were grown in RPMI-1640 supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum, 2 mmol/l glutamine, 1 mmol/l sodium pyruvate, 10 mmol/l HEPES, and 50 μmol/l 2-mercaptoethanol, as previously described (3). NIH 3T3 cells (mouse fibroblastic cell line ATCC CRL 1658) were grown in RPMI-1640 supplemented with 10% fetal calf serum. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2.
Islet cell preparation.
Rat islets were prepared according to the method of Gotoh et al. (15), purified on a Histopaque gradient (Histopaque-1077; Sigma), and directly used for RNA extraction.
Rat islets were prepared from fetuses (21 days of gestation) according to the method of Hellerstrom et al. (16) and as previously described (17). After 7 days of culture, islets were picked under a stereomicroscope and further processed for RNA extraction. RNA was also extracted from fetal pancreatic mesenchymal cells (fibroblasts) that remained attached to the tissue culture dish once islets had been harvested.
RNA extraction and analysis.
Total RNA from INS-1, NIH 3T3, or islet cells was extracted by the guanidium isothiocyanate method (18). Briefly, pancreases were homogenized in 4 mol/l guanidium thiocyanate solution, submitted to a phenol extraction step, and precipitated twice with 2-propanol. The integrity of the RNA was verified by gel electrophoresis. For Northern blot analysis, 10 μg of RNAs were analyzed by gel electrophoresis in a 1% agarose-formaldehyde gel and transferred onto a Hybond-N nylon membrane (Amersham, Orsay, France). Membranes were cross-linked by exposure to ultraviolet light. Hybridization was conducted according to the established method of Church and Gilbert (19) using probes labeled by random priming. Membranes were then washed three times for 15 min at 65°C in 0.5 × sodium chloride−sodium citrate containing 0.1% SDS. Two probes were used in the present study. To detect Kit mRNA, a 346-bp cDNA fragment encoding to the extracellular domain of KIT was used (10). The probe used to detect NGFI-A mRNA has been previously described (20).
Reverse transcriptase−polymerase chain reaction.
Reverse transcriptase (RT)-polymerase chain reaction (PCR) was performed according to the standard protocol (21). Total RNA (6 μg) was first treated for 30 min at 37°C with RNase-free DNase (Gibco/BRL). cDNA was prepared from total RNA using random hexamers as primers. The reaction was performed in the presence or absence of murine leukemia virus RT (Gibco/BRL). The sets of primers used for the PCR are shown in Table 1.
The cDNAs were quantified by serial dilutions, followed by PCR using cyclophilin as an internal control. The total number of cycles used was 35, and the optimal concentration of MgCl2 was 2 mmol/l. The following PCR profile was used: denaturation at 96°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. The amplification products were separated by electrophoresis on 1.5% agarose gel and revealed with Gelstar (FMC Bioproducts, Rockland, ME).
Induction of early response genes.
Cells (INS-1 and NIH 3T3) were seeded into 100-mm tissue culture plates in their respective growth medium. When the cells reached 50–70% confluence, they were changed to 10 ml of RPMI medium without fetal calf serum. The experiments were initiated 16–20 h after that medium change by the addition of recombinant KITL (final concentration 10 nmol/l; R&D Systems) or an equal volume of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). After a 60-min incubation, RNA was extracted, and Northern blot analysis was performed. When PD98059 (50 μmol/l, in DMSO; Calbiochem) or wortmannin (100 nmol/l in DMSO; Sigma) was used, the cells were pretreated with the inhibitors 1 h before the addition of KITL or with an equal volume of vehicle (DMSO).
Western blot analysis.
INS-1 cells were grown in 100-mm dishes and deprived of serum during the last 24 h. The cells were next washed twice in Krebs-Ringer bicarbonate buffer and cultured during 1 h in the same buffer. KITL (10 nmol/l) or an equal volume of PBS-BSA was added. After 5 or 15 min, the cells were washed twice with cold PBS and lysed in 500 μl of single-detergent lysis buffer containing 50 mmol/l Tris (pH 8.0), 1% nonidet-P40, 150 mmol/l NaCl, 1 mmol/l phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, and 1 mmol/l sodium orthovanadate. Insoluble material was removed by centrifugation at 10,000g at 4°C for 10 min. Equal amounts of proteins were separated by SDS-PAGE in a 10% gel. The proteins were transferred to nitrocellulose membranes (Amersham) that had been preincubated for 1 h in blocking solution (3% BSA, PBS containing 0.1% Tween-20) and then incubated with an antiactive extracellular signal-regulated protein kinase (ERK) antibody (1:20,000; Promega). The membranes were then washed in PBS containing 0.1% Tween-20, and with a 1:2000 dilution of horseradish-peroxidase−conjugated anti-rabbit IgG antibodies. Immunoreactivities were determined using enhanced chemiluminescence plus reaction (Amersham). The membranes were next stripped and rehybridized with an anti-ERK2 antibody (Santa Cruz Biotech) that recognizes both unphosphorylated and phosphorylated forms of ERK.
The 129/Sv KitW-lacZ/+ mice have been described previously (13). In these mice, a lacZ gene containing the SV40 nuclear localization signal was introduced in frame in the Kit gene. 129/Sv KitW-lacZ/+ males were crossed with wild-type females. Pregnant mice at 14.5 or 18.5 days of gestation were killed by cervical dislocation. The embryos were removed from the uterus and the pancreases were dissected.
β-galactosidase staining and immunohistochemistry.
The pancreases were fixed at 4°C in 4% paraformaldehyde in PBS for 2 h, briefly rinsed with PBS, cryoprotected overnight at 4°C in 30% sucrose, and frozen. Consecutive sections (6-μm thick) were cut and collected on gelatinized glass slides. The sections were postfixed in 4% paraformaldehyde, incubated in PBS containing 2 mmol/l MgCl2, and stained at 37°C in X-Gal buffer (phosphate buffer 0.1 mmol/l [pH 7.3], 2 mmol/l MgCl2, 0.02% NP40, 0.01% sodium deoxycholate, 5 mmol/l potassium ferricyanide, and 5 mmol/l potassium ferrocyanide) containing 1 mg/ml X-Gal. After staining for β-galactosidase (β-gal), the sections were rinsed with water, and immunohistochemistry was performed as described below.
For immunohistochemistry, the sections were first incubated for 30 min in PBS containing 3% BSA. Subsequently, the sections were incubated for 2 h at room temperature (or overnight at 4°C) with the primary antibodies. After being washed in PBS containing 0.5% Tween-20 (PBST), the sections were incubated with the appropriate fluorescent secondary antibodies. Finally, the sections were extensively washed in PBST and mounted with a fluorescence protecting medium (Vectashield; Vector Laboratories). The sections were examined and photographed with a Leitz DMRD microscope.
The antisera used in this study were at the following dilutions: guinea pig anti-porcine insulin (Dako), 1:200; mouse anti-porcine glucagon (Sigma), 1:2000; rabbit anti-Pax-6 (1:10, a gift from Dr. S. Saule); rabbit anti−Pdx-1 (1:100, a gift from Dr. P. Serup); and rabbit anti-β-gal (Cappel) 1:500. The fluorescent secondary antibodies used were at the following dilutions: fluorescein anti-guinea pig antibodies (Dako), 1:500; Texas-red anti-mouse antibodies (Jackson), 1:200; and Texas-red anti-rabbit antibodies (Jackson), 1:500.
KIT was expressed in INS-1 cells and was functional.
Northern blot analysis was first performed to determine whether INS-1 cells express mRNA coding for KIT. As shown in Fig. 1, a strong signal was detected in total RNA extracted from INS-1 cells. By contrast, no signal was detected in total RNA extracted from the fibroblastic cell line NIH 3T3.
To test the functionality of the KIT/KITL pathway in INS-1 cells in terms of signal transduction, we first determined whether KITL was able to induce the phosphorylation of ERK-1 and ERK-2 in INS-1 cells. These serine threonine protein kinases play a key role in the transmission of extracellular signals (22) and are phosphorylated upon KITL treatment in responsive cells (23). For that reason, INS-1 cells were treated with KITL or vehicle for 5 or 15 min; ERK phosphorylation was followed by Western blot analysis. As shown in Fig. 2, KITL induced a strong and transient phosphorylation of ERK in INS-1 cells.
Induction of the NGFI-A mRNA steady state level represents a useful indicator of the functional growth factor signaling pathway (24,25,26). To further confirm the functionality of KIT receptors in INS-1 cells, the NGFI-A mRNA steady state level was followed after KITL treatment. As shown in Fig. 3, when INS-1 cells were treated for 60 min with KITL, the NGFI-A mRNA steady state level was increased when compared with INS-1 cells treated for the same time with vehicle alone. This induction of NGFI-A by KITL was blocked when the cells were incubated with the mitogen-activated protein/ERK kinase inhibitor PD098059. By contrast, wortmannin, a potent inhibitor of the phosphatidylinositol (PI) 3-kinase (27), had no effect on the induction of NGFI-A mRNA by KITL.
Kit was expressed in adult and fetal islet cells.
To define whether Kit was expressed in islet cells in vivo, RNA from adult rat islets were hybridized using a probe specific for Kit. As shown in Fig. 4A, a specific signal corresponding to Kit was detected in adult rat islets, indicating that Kit was transcribed by adult islet cells.
The expression of Kit was also studied in fetal islets developed in culture. In this in vitro system, islets that develop in culture can be mechanically separated from the mesenchyme that supports their growth (28). Amplification of cyclophilin was first performed to equalize the amounts of cDNA in the fractions enriched in islets and mesenchyme used in the PCR. Amplification of insulin was next used to determine the level of enrichment in insulin mRNA of the fraction enriched in islets. The islet fraction was enriched in insulin mRNA at least 10-fold (Fig. 4B). PCR to detect Kit mRNA and its ligand was performed next. Kit mRNA was mainly detected in RNA prepared from the fraction enriched in fetal rat islets, whereas its ligand, KITL, was mainly detected in the fraction enriched in mesenchyme from fetal pancreases (Fig. 4B).
Kit was transcribed in a subpopulation of adult β-cells.
To define the cell types expressing Kit in the adult pancreas, we used mice that carry a lacZ gene into the Kit locus. In these mice, the lacZ gene recapitulates the normal expression of the Kit gene and can thus be used to mark Kit-positive cells (13). When triple staining for insulin, glucagon, and β-gal activity was performed, it appeared that β-gal activity was restricted to islets (Fig. 5A−F). All islets were labeled. No β-gal−expressing cells were found in the acinar or duct cells. No β-gal−positive cells were detected in the pancreas of wild-type mice (Figs. 5D–F). Moreover, identical data were observed when β-gal protein was revealed with an anti−β-gal antibody (data not shown). In the islets, β-gal activity was restricted to insulin-expressing cells, with no activity being detected in glucagon-expressing cells. Interestingly, not all β-cells stained positive for β-gal. In fact, in each islet, only a subpopulation of insulin-expressing cells stained positive for β-gal activity. Hence, Kit was specifically expressed in a subpopulation of insulin-expressing cells in the adult pancreas.
Kit exhibited a specific pattern of expression during pancreatic development.
We next studied the spatial and temporal pattern of Kit expression during pancreatic development. Histological analysis indicated that β-gal−positive cells were detected in the pancreas of heterozygous embryos at E14.5 in the epithelium (Fig. 6). Co-staining for insulin, glucagon, and β-gal activity indicated that at this stage, β-gal activity was found in insulin- and glucagon-expressing cells as well as in cells negative for both insulin and glucagon (Figs. 7A–C). No β-gal activity was detected in the pancreas of wild-type embryos (Fig. 7G). At E18.5, β-gal was mainly detected in insulin-expressing cells, and only rarely in glucagon-expressing cells (Fig. 7D−F). This indicated a progressive restriction of Kit expression to insulin-expressing cells during embryogenesis. To determine whether the cells expressing β-gal, but staining negative for endocrine markers, represent precursors for endocrine cells, we determined whether such cells would express Pdx-1 or Pax-6, two transcription factors expressed in endocrine precursor cells (29,30). As shown in Fig. 8, at E14.5, cells that expressed β-gal but stained negative for endocrine markers expressed Pax-6 and Pdx-1.
Kit-expressing cells that stained positive for insulin could be detected in Kit-deficient mice.
We next studied the pancreas of Kit-deficient mice. As shown in Fig. 9, at E18, endocrine cells were present in Kit-deficient mice. Islet cell organization did not differ between wild-type and Kit-deficient mice. Finally, endocrine cells expressing β-gal were present in Kit-deficient mice, clearly indicating that Kit was not necessary for endocrine cell survival.
In the present study, we demonstrated that KIT, the receptor for KITL, is expressed in the insulin-producing cell line INS-1. The receptor is functional in terms of signal transduction in this cell line. We next demonstrated that Kit is also expressed in fetal and adult islets, specifically in a subpopulation of β-cells. Finally, during development, Kit exhibited a specific pattern of expression. Although during embryonic life Kit was expressed in cells located in the immature pancreatic epithelium and in the first insulin- and glucagon-expressing cells, its expression became restricted to β-cells at later stages of development.
We first determined that Kit was expressed in an insulin-producing cell line and in the islets of Langerhans. Although it is well known that Kit is expressed in melanoblasts, primordial germ cells, and hematopoietic progenitors (11), little information has been available concerning the expression of Kit in mature β-cells. Based on PCR experiments using cDNA from β-cell lines as templates, it has been postulated that Kit is expressed in insulin-producing cells (10,31). We confirmed in the present study that Kit is expressed in β-cell lines, such as INS-1 cells, but also in RINm5F cells (data not shown). Moreover, we showed that this receptor is functional in terms of signal transduction, indicating that an insulin-expressing cell can indeed respond to KITL. We have demonstrated that the Kit ligand activates p42 and p44 mitogen-activated protein (MAP) kinases. This activation is followed by the induction of the early responsive gene NGFI-A, a useful indicator of a functional growth factor signaling pathway (3,24,26). This induction of NGFI-A was blocked by treatment with PD98059, an inhibitor of the MAP kinase pathway. On the other hand, wortmannin, a potent inhibitor of PI 3-kinase (27), had no effects. Thus, activation of the MAP kinase, but not the PI 3-kinase, pathway is necessary for KITL-induced early responsive gene expression, such as NGFI-A in INS-1 cells. Finally, in addition to its expression in β-cell lines, Kit was also found in fetal and adult rat islets. To the best of our knowledge, this was the first demonstration of Kit expression in mature β-cells.
In the present study, we used mice that carry a lacZ gene containing the SV40 nuclear localization signal inserted into frame in the Kit gene in order to follow the pattern of expression of Kit during pancreatic development. β-gal activity can thus be used in situ as a sensitive and reliable reporter for the expression of Kit gene transcription. This type of strategy has been recently used with success to describe in detail the pancreatic pattern of expression of specific transcription factors, such as Pdx-1/IDX-1 (32,33). Our data indicate that in the adult pancreas, Kit is specifically expressed in β-cells. β-Gal−positive cells were not found in either the other endocrine cell types or the exocrine tissue. However, not all β-cells express Kit. In fact, although all islets contained insulin-positive cells that stained positive for β-gal, in each islet, ∼10−20% of insulin-positive cells were found positive. This indicated that β-cells are heterogeneous for this specific marker. No specific pattern relating Kit expression and the geographic location of β-cells in the islets was found. Various data from the literature suggest that a functional heterogeneity exists between β-cells (34). For example, it has been shown that β-cells differ in their individual metabolic responsiveness to glucose (35). β-cells have also been found recently to be heterogeneous in terms of insulin gene transcription (36). Finally, morphological observations have indicated that differences between β-cells in terms of the types of granules can be detected in situ (35). Thus Kit represents a new marker for β-cell subpopulations in the adult pancreas. This marker should be useful in efforts to study functional heterogeneity in a pancreatic β-cell population (34).
In this study, we also found that, although during adult life the pancreatic expression of Kit is restricted to β-cells, during pancreatic development at E14, Kit is transcribed both in insulin- and glucagon-expressing cells, which are known to derive from a common precursor (37). Moreover, during embryonic life, Kit is detectable in epithelial cells that stain negative for these endocrine markers but stain positive for transcription factors such as Pax-6 or Pdx-1, which are known to be expressed in endocrine precursor cells (29,30), suggesting that such cells represent endocrine precursor cells. However, the fact that not all Pax-6−expressing hormone-negative cells stained positive for Kit suggests that Kit may be expressed in some, but not all, endocrine precursor cells. It is interesting to note that in the case of the hematopoietic system, Kit (CD117) is routinely used as a marker of stem cells. Moreover, it has been suggested that most pluripotent long-term reconstituting murine stem cells express Kit (38,39,40). Whether this is also the case for pancreatic precursor remains to be demonstrated.
To date, the potential role of Kit and its ligand in the pancreas is unknown. We showed here that Kit is present in embryonic and fetal islets, and that KITL mRNA can be amplified from fetal pancreatic fibroblasts, suggesting that some interactions between these two compartments could be mediated by KITL. However, we were unable to detect an effect of recombinant KITL in an in vitro model of rat embryonic pancreatic epithelium development (41). Moreover, although KITL mRNA could be reproducibly amplified from fetal pancreatic mesenchyme, we were unable to detect a signal by Northern blot analysis or the KITL protein by immunohistochemistry (data not shown), thereby indicating a low level of expression. It thus remains to be determined whether the cells expressing Kit in the pancreas respond to KITL produced locally or to circulating systemic levels of KITL.
Although the present data clearly indicate that Kit is expressed in the endocrine pancreas and follows a specific temporal pattern of expression during development, our results also indicate that endocrine cells are present in the pancreas of Kit-deficient embryos. Thus, either Kit and its ligand play a subtle role during the development of the endocrine pancreas during embryonic/fetal life, or some level of redundancy does exist. It is, however, interesting to note that endocrine cells expressing β-gal are present in Kit-deficient mice, indicating that Kit is not necessary for pancreatic endocrine cell survival. This is at variance with what is found for other cell types, such as melanoblasts or hematopoietic progenitors that fail to survive in Kit-deficient mice (13). Finally, another possibility would be that Kit is implicated in β-cell function during postnatal life. Although INS-1 cells express functional receptors for KITL, we were unable to detect any effect of KITL on insulin secretion. Moreover, because the Kit−/− mice die around birth, the postnatal development and the function of their pancreas cannot be studied.
In conclusion, our data indicate that Kit is expressed by pancreatic endocrine cell subpopulations at specific stages of their development and thus represents a new marker of such specific cells.
|Gene .||Primers .||Size of the amplified fragment .|
|Gene .||Primers .||Size of the amplified fragment .|
This work was supported by grants from the Juvenile Diabetes Foundation International and from the Association pour la Recherche contre le Cancer.
Sophie Mahé is acknowledged for her technical assistance.
Address correspondence and reprint requests to Raphael Scharfmann, PhD, INSERM U457, Hospital R. Debré, 48 Blvd. Sérurier, 75019 Paris, France. E-mail: email@example.com.
Received for publication 31 October 2000 and accepted in revised form 5 June 2001.
BSA, bovine serum albumin; ERK, extracellular signal-regulated protein kinase; β-gal, β-galactosidase; KITL, KIT ligand; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; PBST, PBS containing 0.5% Tween-20; PCR, polymerase chain reaction; PI, phosphatidylinositol; RT, reverse transcriptase; RTK, receptor tyrosine kinase.