(Received for publication, August 16, 1995; and in revised form, January 24, 1996)
From the
Prohormone convertases PC2 and PC3, yeast Kex2-family
endoproteases specific to the regulated secretory pathway, cleave
proinsulin to insulin in the secretory granules of pancreatic
cells. The well-differentiated
cell line MIN6 expresses PC2 and
PC3 and another regulated secretory pathway-specific protein
chromogranin A. Furin, another yeast Kex2 endoprotease, exists in the trans-Golgi networks of many cell types. The
cell line
RINm5F (a cell line that is less differentiated than the MIN6 cell
line) does not express the regulated pathway-specific proteins, but
strongly expresses furin. We suspected that furin expression may cause
the decrement of regulated secretory pathway-specific proteins. To test
this hypothesis, we expressed a furin cDNA with a metallothionein
promoter in MIN6 cells. With Zn
stimulation of furin
expression, the messages of PC2, PC3, and chromogranin A decreased, and
the processing of proinsulin to mature insulin became less efficient.
The furin-expressing MIN6 cells exhibited less insulin content and
weakened insulin secretion in response to a high glucose concentration.
The conditioned medium from furin-expressing MIN6 cells also exerted a
decrease of PC2 and PC3 expression in unaltered MIN6 cells. Thus,
proteins cleaved by furin inside the cells or by truncated furin shed
into the culture medium appear to cause decreased PC2 and PC3
expression, insulin content, and glucose-responsive insulin secretion
in MIN6 cells.
Pancreatic cells possess well-differentiated
characteristics, such as the formation of insulin secretory granules,
the processing of proinsulin to mature insulin, and the
glucose-stimulated secretion of insulin.
cells synthesize insulin
as proinsulin, a precursor peptide, at the rough endoplasmic reticulum,
from which transport vesicles carry the proinsulin to the trans-Golgi networks. At the trans-Golgi networks,
proinsulin is sorted to a secretory pathway that is well regulated by
an extracellular stimulus, such as glucose(1) . Through this
pathway, proinsulin (B chain-Arg-Arg-C peptide-Lys-Arg-A chain) is
packaged into immature granules and processed to mature insulin (B
chain-S-S-A chain), which is then stored in dense-core
granules(2) . When this secretory system is stimulated, insulin
molecules stored in the dense-core granules are secreted into the
extracellular space. These granules contain at least three distinct
groups of processing enzymes: the prohormone convertases PC2 and PC3
(also called PC1), carboxypeptidase H, and amidation
enzymes(3, 4, 5) . Processing of proinsulin
into mature insulin requires cleavage by PC2 and PC3 at the paired
basic residues and their subsequent removal by carboxypeptidase
H(3, 4, 6) .
In addition to the regulated
secretory pathway, cells are equipped with a secretory pathway
that is not regulated by an extracellular stimulus. Along this
constitutive secretory pathway, proteins are transported from the trans-Golgi networks to plasma membranes by small transport
vesicles(1, 7) . Furin, from the family of yeast Kex2
endoproteases to which PC2 and PC3 also belong(8, 9) ,
is located at the trans-Golgi network(10) . While PC2
and PC3 are uniquely expressed in the regulatory pathway of
neuroendocrine cells, furin is thought to be present in the
constitutive pathway of virtually all cell types(11) . Furin is
specific for the unique cleavage sequence
-Arg
-X
-(Lys/Arg
)-Arg
(RX(K/R)R)(12, 13) . This
furin-specific RX(K/R)R motif is found in a number of growth
factor precursors(12) , including nerve growth factor,
platelet-derived growth factor, transforming growth factor
,
activin A, the adhesion molecule cadherin, metalloproteinases such as
stromelysin 3(14) , and matrix metalloproteinase
X1(15) , and, in some growth factor proreceptors (12) including those for insulin receptor, insulin-like growth
factor 1 receptor, and hepatocyte growth factor receptor (oncoprotein MET). Several reports indicate that furin is responsible for
the formation of bioactive proteins such as transforming growth factor
(16) , stromelysin 3(14) , and insulin
receptor(17) . We previously found that when mutant proinsulin
with furin cleavage sites was expressed in the mouse fibroblast-like
cell line NIH3T3, containing high levels of furin, and the Chinese
hamster ovary-derived cell line CHO which expressed low levels of
furin, NIH3T3 cells produced more bioactive insulin than did CHO
cells(18) . Thus, while furin is localized in virtually all
cell types(11) , increased expression of furin may generate
more bioactive proteins from their precursors, and the increased
protein levels may induce a change in cell growth as well as
differentiation of cell functions. Furin expression in mature
cells may induce notable changes in their highly differentiated
characteristics, such as the formation of insulin secretory granules,
the processing of proinsulin to mature insulin, and glucose-stimulated
secretion of insulin.
To investigate this possibility, we chose one
well-differentiated cell line, MIN6(19, 20) ,
and two less-differentiated cell lines, HIT-T15 (21) and
RINm5F(22) . We compared the expression of the prohormone
convertases PC2 and PC3, the regulatory pathway marker protein
chromogranin A(23) , and the constitutive pathway enzyme furin
among the three
cell lines. Since furin expression appears to be
correlated with an undifferentiated state of
cell function, we
prepared a furin-expressing MIN6 cell line, MIN6-mf, under the control
of a metallothionein promoter. The present study investigates the
decrement of the differentiated characteristics of
cells using
the MIN6-mf cell line.
The media from the rat pancreatic islets, and the three
cell lines MIN6, HIT-T15, and rat insulin I gene-expressing RINm5F were
applied to a 1.0 120-cm column of Sephadex G-50 superfine gel
(Pharmacia Biotech Inc.) equilibrated with 100 mM acetic acid,
as described previously(18) . The proportion of proinsulin that
was converted to insulin was quantified by measuring the areas of
proinsulin and insulin fractions on the gel chromatogram, and the
area
/area
+
area
was calculated.
Immunofluorescence was studied
using MIN6 and RINm5F cells cultured on poly-L-lysine-coated
glass coverslips for 2 to 3 days and fixed with 4% paraformaldehyde in
a 0.1 M phosphate buffer at a pH of 7.4 for 1 h at room
temperature. These cells were then treated with 50 mM NHCl in phosphate-buffered saline (PBS) for 20 min to
quench any free aldehyde and made permeable by treatment with PBS
containing 0.1% saponin and 0.4% bovine serum albumin (BSA) for 30 min.
The specimens were incubated with 1% BSA-PBS for 30 min, followed by
another 12-h incubation at 4 °C with anti-furin serum diluted at
1:4000. After the cells were washed with PBS, they were incubated for 1
h with indocarbocyanine-conjugated affinity-purified donkey anti-rabbit
IgG (Jackson Immunoresearch, West Grove, PA), diluted with 1% BSA-PBS,
and mounted with PermaFluor (Immuron, Pittsburgh, PA). The specimens
were examined through a Olympus BX50 microscope equipped with a BX-FLA
incident illuminator.
Figure 1: Northern blot analysis of regulatory pathway prohormone convertases PC2, PC3, and the regulatory pathway protein chromogranin A (CgA), and the constitutive pathway enzyme furin. 18 S(-) and 28 S (-*) represent the corresponding sizes of ribosomal RNAs. Note that the bands of the three regulatory pathway protein mRNAs were clearly visible, but a furin mRNA band was not found in the MIN6 cells. The furin was observed strongly in RINm5F cells, which did not contain any of the three regulatory pathway protein messages.
Figure 2: Immunocytochemical staining of furin in RINm5F and MIN6 cells. Staining of furin was intense in the perinuclear region of RINm5F cells (A), but faint in MIN6 cells (B).
Figure 3:
Expression of furin, PC2, and PC3 in MIN6
cell lines with different passage numbers. MIN6 cell lines at 16, 25,
and 35 passages were used for RNA isolation. Expression of furin, PC2,
and PC3 messages were assessed on Northern blots. -Actin message
was used to measure blotting efficiency.
We next compared insulin content and glucose-responsive insulin secretion between the MIN6 cells with the passage numbers 16 and 35. Insulin content was almost 2-fold higher in the cells at passage 16 than in the cells at passage 35 (Table 3). Insulin secretion in response to 1 mM and 5 mM glucose was similar in MIN6 cells at 16 and 35 passages. However, when the glucose concentration was raised form 5 mM to 25 mM, the cells at passage 16 showed nearly a 2-fold increase in insulin secretion, while the cells at passage 35 showed an increase of only 1.2-fold (Fig. 4). Thus, PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion decreased with increasing passage number, whereas furin expression increased.
Figure 4: Glucose-responsive insulin secretion in MIN6 cell lines at passages 16 and 35. Glucose-responsive insulin secretion from MIN6 cells was assessed at 1, 5, and 25 mM glucose concentrations. Insulin secretion stimulated by 1 mM glucose was expressed as 1, then that at 5 and 25 mM glucose was plotted as fold increase. The cells at passage 35 showed less insulin secretion than those at passage 16 in response to 25 mM glucose. Bars represent the mean ± S.D.
Figure 5:
Expression of furin in
Zn-stimulated MIN6-mf cells. A, MIN6-mf
cells were cultured for 0, 24, 48, and 72 h in the medium containing
100 µM Zn
. Isolated RNA (10 µg) was
electrophoresed on an agarose gel prior to Northern blot analysis.
-Actin message was used to measure blotting efficiency. B, MIN6-cells with the empty expression vector pMEP4 (MIN6-0
cells) were prepared and cultured for 0, 24, 48, and 72 h in the medium
containing 100 µM Zn
. RNA isolation,
electrophoresis, and Northern blot analysis were carried out as in A. A blotted membrane was probed for the expression of furin,
PC2, and PC3.
Upon immunocytochemical staining of furin,
stained granular dots were amassed in the perinuclear region in the
Zn-stimulated cells, unlike in the unstimulated cells (Fig. 6, A and B). As mentioned above, furin
staining was visible even in unstimulated cells because these cells
were at passage 26 and had began to express furin as shown in Fig. 5, A and B.
Figure 6:
Immunocytochemical staining of furin in
MIN6-mf cells cultured in the presence or absence of
Zn. Immunocytochemical staining of furin was
performed on cells cultured for 48 h in the absence of 100 µM Zn
(A) and in the presence of
Zn
(B). The Zn
-stimulated
cells showed a strong accumulation of stained granular dots in the
perinuclear region (B). Unstimulated cells showed fewer
granular dots scattered in the cytoplasm (A).
In contrast with the
increase in furin expression, the expression of PC2 and PC3 decreased
extensively in Zn-stimulated MIN6-mf cells cultured
for 48 and 72 h (Fig. 7). This decrease of PC2 and PC3 messages
by Zn
was not observed in the empty vector-introduced
MIN6-0 cells (Fig. 5B). MIN6-mf cells cultured
for 72 h also showed a decrease in chromogranin A message. Thus, it
appears that increased furin induction decreased the expression of the
regulated pathway proteins PC2, PC3, and chromogranin A. As expected
from the decrement of PC2 and PC3, Zn
-stimulated
MIN6-mf cells produced a higher proportion of proinsulin to the total
IRI than did unstimulated MIN6-mf cells (54% versus 13%) (Fig. 8). The Zn
-stimulated MIN6-mf cells
(high furin induction) exhibited less insulin content than unstimulated
MIN6-mf (low furin induction) at 72 h after the Zn
addition (Table 4). This is consistent with the decrease in
insulin content as unaltered MIN6 cells progress from passage 16 (low
furin induction) to passage 35 (moderate furin induction) as shown in Table 3.
Figure 7:
Expression of PC2, PC3, and chromogranin A
mRNAs in Zn-stimulated MIN6-mf cells. The same RNA
isolated for Northern blot analysis was assessed for PC2, PC3, and
chromogranin A (CgA) expression. PC2 and PC3 messages were
decreased in the MIN6-mf cells cultured for 48 and 72 h in the presence
of 100 µM Zn
. Cells cultured for 72 h
also showed a marked decrease in chromogranin A
message.
Figure 8:
Gel
filtration profiles of IRI in the culture medium of MIN6-mf cells with
and without Zn stimulation. MIN6-mf cells were
cultured for 72 h in the presence or absence of 100 µM Zn
. Culture medium was collected every 24 h. IRI
in the culture medium collected between 48 and 72 h was separated with
a Sephadex G-50 superfine gel column. The proinsulin peak of MIN6-mf
cells stimulated by Zn
was larger than that of
unstimulated MIN6-mf cells. Molecular size was calibrated with blue
dextran (V
), potassium ferricyanide (V
), and synthetic human insulin. Similar
elution profiles were obtained in three other chromatography
tests.
With the decrement of regulated pathway-specific
proteins, glucose-responsive insulin secretion was diminished. The MIN6
cells at passage 16 secreted a 2.0-fold higher level of insulin in a 25
mM glucose medium than in a 1 mM glucose medium (Fig. 4). This glucose-dependent increase became only 1.6-fold
in MIN6-mf cells without Zn stimulation, probably due
to the increase in cell line passage (passage 26) (Fig. 9). The
glucose-induced secretion was further reduced when the cells were
stimulated with 100 µM Zn
for 48 h.
Zn
-stimulated MIN6-mf cells exhibited only a 1.2-fold
increase in insulin secretion when the glucose concentration was raised
from 5 mM to 25 mM (Fig. 9). However, the
increase from 1 mM to 5 mM glucose resulted in
similar increases in furin expression (2.1- to 2.3-fold) in all three
cell conditions, MIN6 cells at passage 16 and MIN6-mf cells with and
without Zn
stimulation. Thus, the greatest change in
glucose-responsive insulin secretion was a marked decrease observed in
Zn
-stimulated MIN6-mf cells when glucose
concentrations were shifted from 5 mM to 25 mM.
Figure 9:
Glucose-responsive insulin secretion in
MIN6-mf cell lines with or without stimulation of Zn.
Glucose-responsive insulin secretion in MIN6-mf cells was assessed at
1, 5, and 25 mM glucose concentrations. MIN6-mf cells were
cultured for 48 h in the presence or absence of 100 µM Zn
, then processed for the insulin secretion
assay. Insulin secretion at 1 mM glucose was expressed as 1,
then that at 5 and 25 mM glucose was plotted as fold increase.
MIN6-mf cells cultured in the presence of 100 µM Zn
showed less insulin secretion than those
cells cultured in the absence of Zn
. Bars represent the mean ± S.D.
Figure 10:
Effect of conditioned media from
Zn-stimulated and unstimulated MIN6-mf cells on the
expression of PC2 and PC3 in unaltered MIN6 cells. Conditioned medium A
was derived from unstimulated MIN6-mf cells cultured for 48 h;
conditioned medium B was derived from Zn
-stimulated
MIN6-mf cells cultured for 48 h. MIN6 cells were cultured for 48 h with
either conditioned medium A or B. RNA was isolated from MIN6 cells
cultured in each medium. Northern blot analysis was performed to
examine PC2 and PC3 expression. Both PC2 and PC3 expressions were lower
in the MIN6 cells cultured with the conditioned medium B than in those
cultured with the conditioned medium A. This result was reproduced even
with the dialyzed Zn
-free media A and
B.
The present results demonstrate that when furin is induced in
the pancreatic cell line MIN6, expression of the regulatory
pathway-specific prohormone convertases PC2 and PC3 and the secretory
granule-specific protein chromogranin A, insulin content, and
glucose-stimulated insulin secretion are decreased. MIN6 cells
exhibited typical characteristics of mature
cells, including the
presence of the three regulatory pathway-specific proteins PC2, PC3,
and chromogranin A, and a conversion ratio of proinsulin to insulin
similar to that found in mature islet
cells. The cells also
retain an insulin secretion capacity that can be regulated by a
physiological range of glucose
concentrations(19, 20) . A previous study observed
that pancreatic
cells purified by a fluorescence-activated
cell-sorting system exhibited only 1.5- to 2.0-fold insulin secretion
when the glucose concentration was raised from 3 mM to 20
mM, although the addition of cAMP-generating secretagogues
together with glucose strongly facilitates the insulin
secretion(35) . Thus, the glucose-responsive insulin-secreting
capacity of MIN6 cells is well-maintained, although it is considerably
lower than that of pancreatic
cells exhibited in the
islet(36) . In contrast, RINm5F cells illustrated
characteristics opposite to those of the well-differentiated
cells. These characteristics included a low level of insulin, virtually
no regulated secretory pathway protein messages, no glucose-responsive
insulin secretion(36, 37) , and an especially high
level of furin expression. The presence of furin in RINm5F suggests
that this enzyme may be expressed in the less differentiated state of
cells. Indeed, rat fetal and infant
cells showed strong
furin immunostaining (data not shown), but adult
cells stained
only faintly(38) .
Since furin appeared to be expressed in
less-differentiated cells, we examined the furin expression in
MIN6 cells of different passage numbers. For studies such as these,
MIN6 is the
cell line of choice because the cells retain their
glucose-responsive insulin-secreting
capacity(19, 20) . In many well-differentiated
cell lines, this glucose-responsive insulin secretion is known to be
gradually diminished with the increase of cell line
passages(39, 40) , as seen in MIN6. With increasing
MIN6 cell line passages and the resulting decline in secretion
capacity, PC2 and PC3 messages also decreased and insulin content was
diminished. In contrast, furin expression increased with increasing
cell passages.
Proinsulin is converted to mature insulin through the
regulated secretory pathway(1) . On the other hand, furin is
thought to cleave precursor proteins that are secreted through a
constitutive secretory pathway. Thus, furin probably has a minor role
in processing proinsulin. Furin cleaves the consensus processing site
-Arg-X
-(Lys/Arg
)-Arg
(12, 13) . For cleavage to occur properly, more basic
amino acids are required ahead of the general cleavage site of most
propeptide hormones,
-(Lys/Arg
)-Arg
. If furin is
highly expressed in
cells, the bioactive proteins cleaved from
their precursors would increase in concentration. To examine whether
furin is a primary cause for the decline of PC2 andPC3 expression and
for the diminished glucose-responsive insulin secretion, we developed a
new cell line MIN6-mf with a furin expression system that was regulated
by Zn
. With an increase in furin expression, all
three regulatory pathway-specific proteins (PC2, PC3, and chromogranin
A) were diminished. The MIN6-mf cells stimulated with Zn
generated a larger proportion of proinsulin to total IRI than did
MIN6-mf cells without Zn
stimulation. Two reasons may
be considered for the increase in proinsulin proportion. First, the
decrease of prohormone convertases PC2 and PC3 may have increased the
proportion of unprocessed proinsulin. Second, the decrease of regulated
pathway proteins may have eventually driven proinsulin to a
constitutive pathway. Since furin is known to recycle between the trans-Golgi network and plasma
membrane(33, 41) , the furin recycling may increase a
protein flow of the constitutive pathway, which results in an augmented
transport of proinsulin molecules outside the cells. In a mouse
pituitary tumor-derived cell line AtT20, which carries both regulatory
and constitutive pathways, a part of human proinsulin expressed in this
cell line was sorted to a constitutive pathway where proinsulin is
processed to an intermediate form des-31,32-split
proinsulin(42) . In the MIN6-mf cell line, it remains to be
investigated whether proinsulin is processed to this intermediate form.
Insulin content and glucose-stimulated insulin secretion were also
diminished in Zn-stimulated MIN6-mf cells. Proinsulin
may have escaped by way of a rapid secretion of newly formed immature
granules or through the constitutive pathway to the culture medium,
which may result in the decrease of insulin content in MIN6 cells. The
resulting diminished insulin content may lead to the decrease of
insulin secretion in response to glucose. In addition to this
possibility, we need to assess the impairment of the glucose-sensing
system, namely the decreased expression of type 2 glucose transporter
and glucokinase in furin-expressing MIN6-mf cells.
Furin generates
secretory peptides such as platelet-derived growth factor, transforming
growth factor , and activin A as well as membrane-bound proteins
such as cadherin and insulin
receptor(12, 14, 15, 16, 17) .
To examine if extracellular peptides cleaved by furin cause the loss of
differentiation in MIN6 cells, we prepared the conditioned medium from
the Zn
-stimulated and unstimulated MIN6-mf cells. The
conditioned medium from the Zn
-stimulated cells
induced the decrement of PC2 and PC3 expression in the genetically
unaltered MIN6 cells. Thus, we can say at least that some extracellular
peptides cleaved by furin must have exerted the loss of differentiation
in
cells, although we cannot say whether furin cleaved precursor
peptides intracellularly or extracellularly.
Activation of the
constitutive secretory pathway appears to be correlated with furin
expression. We do not know what factors are involved in the activation
of the constitutive pathway, but once furin is induced, furin-cleaved
bioactive proteins including a number of growth factors may lead
well-differentiated cells to an undifferentiated state. Since the
decline of
cell functions are often observed in insulinomas (43, 44) and non-insulin-dependent diabetes
mellitus(45) , understanding the physiological switching from a
regulated secretory pathway to a constitutive pathway may aid in
understanding the onset of such disorders.