(Received for publication, December 29, 1995; and in revised form, February 15, 1996)
From the
The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a >80% reduction of pro-NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro-NT/NN with brain and intestinal phenotype, respectively.
Neuropeptides and peptide hormones are synthesized as part of
larger inactive polypeptide precursors from which they are produced by
cleavage at specific sites, usually pairs of basic residues, by
proprotein convertases (PCs) ()(reviewed in (1, 2, 3) ). The mammalian PCs belong to a
recently identified family of subtilisin-like proteases that were
identified by their homology with the yeast Kex2 protease involved in
the processing of pro-
-mating factor(4, 5) .
These Kex2-related enzymes exhibit different tissue and cellular
distributions (reviewed in Refs. 2 and 3). Thus, furin (6) and
PACE 4 (7) are expressed in most neuroendocrine and
nonendocrine tissues in the body. PC1 (also designated PC3) and PC2 (8, 9, 10, 11) are restricted to
endocrine and neuronal cells. PC4 (12, 13) is
exclusively expressed in germ cells of testes and ovaries. PC5 (also
designated PC6) (14, 15) is widely distributed in
neural, endocrine, and nonendocrine tissues, being abundant in the
periphery, especially in the gut and adrenal. At the cellular level,
furin appears to be confined to the Golgi apparatus while PC1 and PC2
are found in the various compartments of the regulated secretory
pathway including the secretory granules.
Consistent with such
tissue and cellular distributions, furin has been shown to efficiently
process protein precursors that are destined to the constitutive
secretory pathway such as pro--nerve growth factor, proalbumin, or
pro-von Willebrand factor(2, 16) , while PC1 and PC2
have been reported to cleave peptide hormone and neuropeptide
precursors that are routed to the regulated secretory pathway, like
proinsulin(17, 18, 19) ,
proglucagon(20, 21) , or
POMC(22, 23) . Furthermore, a tissue-specific action
of PC1 and PC2 has been shown to be responsible for the differential
processing of POMC in the anterior and intermediate pituitary
lobes(22, 24) . The roles of PACE4, PC4, and PC5 in
proprotein processing are as yet unknown. Even as regards furin, PC1,
and PC2, only a few of the potential physiological substrates for these
enzymes have been identified. The number of proprotein and
hormone/neuropeptide precursors undergoing post-translational cleavage
at basic sequences is considerable, and a major task in the future will
be to identify the enzyme(s) involved in the maturation, often
tissue-specific, of each of these precursors.
Neurotensin (NT) and
neuromedin N(NN) are two structurally related brain and gut regulatory
peptides which are encoded in the same
precursor(25, 26) . Rat pro-NT/NN is depicted in Fig. 1. The four Lys-Arg sequences in the precursor represent
putative processing sites, the cleavage of which could generate various
sets of peptides in addition to NT and NN. Recent evidence indicates
that pro-NT/NN is differentially processed in brain versus intestinal tissues. Thus, in all rat brain regions examined
pro-NT/NN is primarily cleaved at the three most C-terminal dibasic
sequences to generate similar amounts of NT, NN, and a large N-terminal
precursor fragment ending with the residue that precedes
Lys(27, 28) . In the gut, the precursor
is preferentially cleaved at the two most C-terminal pairs of basic
residues, giving rise to comparable amounts of NT and a large
biologically active peptide starting after the signal peptide and
ending with the NN sequence (large NN, Fig. 1) (29, 30, 31) . The first dibasic sequence
(Lys
-Arg
) is poorly cleaved in all the
systems examined so far. At present, nothing is known about the PC(s)
involved in pro-NT/NN processing. Identifying these enzymes will be
necessary for understanding the mechanisms underlying the differential
biosynthesis of NT, NN, and other precursor-derived peptides in
tissues.
Figure 1: Schematic representation of rat pro-NT/NN and the various products detected in the present study. Rat prepro-NT/NN is 169 amino acids long and starts with a 22-residue signal peptide not represented here. The positions of the four Lys-Arg (KR) dibasic sequences are shown.
Neuroendocrine cell lines have proven to be useful models to identify the PCs involved in the maturation of neuropeptide/hormone precursors such as proinsulin(19) , proglucagon(20) , and POMC(22, 24) , and to explain the processing patterns observed for these precursors in the tissues that normally express them. Recently, we reported that the rat medullo-thyroid carcinoma rMTC 6-23 cell line (32) expresses and processes the NT/NN precursor to yield NT, NN, and the large N-terminal fragment in similar amounts(33, 34) . In this respect, rMTC 6-23 cells exhibit the processing pattern observed in brain. We also showed that dexamethasone induces the expression of the NT/NN precursor mRNA in rMTC 6-23 cells and concomitantly increases the cellular content of precursor-derived products without affecting their relative proportions(34) . This is in contrast to rat pheochromocytoma PC12 cells which can be induced to produce large amounts of pro-NT/NN mRNA and protein in response to a combination of nerve growth factor, forskolin, dexamethasone, and lithium, but largely lack the capability to process the precursor at any of its dibasic sequences (35, 36, 37, 38, 39) . Interestingly, PC12 cells have been reported to be devoid of both PC1 and PC2 (2, 3) and transfection of this cell line with PC1 or PC2 resulted in the correct routing and maturation of these enzymes in the regulated secretory pathway (39, 40) and in PC1-mediated cleavage of the NT precursor(39, 41) .
Insight into the mechanism of pro-NT/NN processing could therefore be gained: 1) by identifying the PC(s) present in rMTC 6-23 cells and blocking their action by antisense RNA strategies and 2) by stably transfecting PC12 cells with PC(s) and analyzing pro-NT/NN processing patterns in the transfected cells. Using these approaches, we show here that PC2 is inducible by dexamethasone and plays a key role in pro-NT/NN maturation in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process the NT/NN precursor, PC1 mimicking the processing pattern observed in the gut while PC2 reproduces the pattern observed in the brain and in rMTC 6-23 cells.
Portions of acid extracts from PC12 and
rMTC 6-23 cells were citraconylated, submitted to Arg-directed tryptic
digestion(33, 38) , and assayed for N-terminal
immunoreactive NN (iNN). The value of CTiNN thus obtained provides an
index of the total amount of pro-NT/NN (either processed or
unprocessed) that was synthesized and stored in the cells at the time
of extraction. rMTC 6-23 cell extracts were directly assayed for their
iNT and N-terminal iNN contents. Previous studies in rMTC 6-23 cells
have shown that iNT consists of approximately 95% authentic NT and 5%
large NT, and that N-terminal iNN consists of 100% authentic
NN(33, 34) . PC12 cell extracts were fractionated by
reverse-phase HPLC as described elsewhere(33) . The fractions
were assayed for their content in iNT, C-terminal iNN, N-terminal iNN,
and iK6L. Because of the above described antisera specificity, these
assays measure the amounts of precursor products that are processed at
the Lys-Arg
,
Lys
-Arg
,
Lys
-Arg
, and Lys
-Arg
sequences, respectively. The products detected after HPLC were
NT, NN, large NT, and large NN which eluted with retention times of 40,
43, 68, and 70 min, respectively. NT and large NT were assayed with
antiserum 29G, NN gave equal measurements with antisera NN-Ah and NMR,
and large NN was assayed with antiserum NMN. The results were
normalized for the amount of protein in each extract. The percentages
of cleavage at the Lys
-Arg
,
Lys
-Arg
,
Lys
-Arg
, and Lys
-Arg
sequences were calculated by dividing post-HPLC iNT, C-terminal
iNN, N-terminal iNN, and iK6L, respectively, by CTiNN and by
multiplying these ratio values by 100.
Figure 2: Analysis of PCs content in rMTC 6-23 cells. rMTC 6-23 cells were unstimulated(-) or stimulated (+) by 1 µM dexamethasone for 48 h. AtT-20 cells, GH3 cells, rat testis, and rat ileum were used as positive controls for PC1, PC2, PC4, and PC5 expression, respectively. PC12 cells served as a negative control for PC1 and PC2 expression and as a positive control for furin expression. Total RNA (10 µg) and proteins (50 µg) were analyzed by Northern and Western blotting as described under ``Experimental Procedures.'' A, Northern blot analysis of PC1, PC2, furin, PC4, and PC5 mRNAs. B, Western blot analysis of PC1, PC2, and furin proteins. The C-terminally directed PC2 antiserum 4BF was used for immunoblotting.
Figure 3: Concentration-response for the effect of dexamethasone on PC2 mRNA and protein expression in rMTC 6-23 cells. rMTC 6-23 cells were exposed for 48 h to the indicated concentrations of dexamethasone. Total RNA (10 µg) and proteins (50 µg) were analyzed by Northern and Western blotting as described under ``Experimental Procedures.'' A, Northern blot analysis of PC2 and GAPDH mRNAs. B, Western blot analysis of PC2 using the C-terminally directed PC2 antiserum 4BF. C, quantification of PC2 mRNA and protein by densitometric scanning as described under ``Experimental Procedures.'' The density of the PC2 75- and 66-kDa bands was summed. The data are expressed as the percent of maximal PC2 mRNA (closed squares) and protein (open squares) levels and represent the mean from two to three separate experiments.
Figure 4: Time course for the effect of dexamethasone on PC2 mRNA and protein expression in rMTC 6-23 cells. rMTC 6-23 cells were exposed to 1 µM dexamethasone for the indicated periods of time. Total RNA (10 µg) and proteins (50 µg) were analyzed by Northern and Western blotting as described under ``Experimental Procedures.'' A, Northern blot analysis of PC2 and GAPDH mRNAs. B, Western blot analysis of PC2 using the C-terminally directed PC2 antiserum 4BF. C, quantification of PC2 mRNA and protein by densitometric scanning as described under ``Experimental Procedures.'' The density of the PC2 75- and 66-kDa bands was summed. The data are expressed as the percent of maximal PC2 mRNA (closed squares) and protein (open squares) levels and represent the mean from two to three separate experiments.
Figure 5: Effect of hPC2 antisense mRNA expression on PC2 protein levels and pro-NT/NN conversion in transfected rMTC 6-23 cells. Total RNA (1 µg) and proteins (50 µg) from control (clone 10) and hPC2 antisense cDNA-transfected rMTC 6-23 cells (clones E7, E5, and E14) were submitted to reverse transcriptase-PCR and Western blot analysis as described under ``Experimental Procedures.'' Acid extracts of the cell lines were assayed for their CTiNN, iNT, and iNN contents (see ``Experimental Procedures''). A, ethidium bromide staining of hPC2 (a) and rPC2 (s) cDNAs obtained by reverse transcriptase-PCR. B, Western blot analysis of PC2 using the C-terminally directed PC2 antiserum 4BF. C, quantification of PC2 protein levels (closed bars) and pro-NT/NN processing (open bars). PC2 levels were determined by densitometric scanning as described under ``Experimental Procedures.'' The density of the PC2 75- and 66-kDa bands was summed. The data are expressed as the percent of PC2 protein levels in the control (clone 10) and represent the mean from two separate experiments. Processing efficiency was determined by calculating the ratio (in %) of iNT and iNN over CTiNN. The two values thus obtained for each clone were similar and only the iNT/CTiNN ratio values are shown here. CTiNN contents were (in pmol/mg of protein) 6.4 ± 0.5, 4.4 ± 0.3, 18.7 ± 1.8, and 7.3 ± 0.3 in extracts from clones 10, E7, E5, and E14, respectively (mean ± S.E. from quadruplicate determinations in two separate experiments).
Figure 6:
Northern blot analysis of PC1, pro-NT/NN,
and GAPDH mRNAs and Western blotting of PC1 in wild type and
PC1-transfected PC12 cells. PC12 cells were stimulated with nerve
growth factor, dexamethasone, forskolin, and Li for 48
h. Total RNA (10 µg) and proteins (50 µg) were analyzed by
Northern and Western blotting as described under ``Experimental
Procedures.'' A, Northern blot analysis of PC1,
pro-NT/NN, and GAPDH mRNAs. B, Western blot analysis of
PC1.
Figure 8: Percentages of cleavage of the dibasic sequences in pro-NT/NN by PC1- and PC2-transfected PC12 cells. The values represent the mean ± S.E. from the determinations obtained in the six individual PC12/PC1 and PC12/PC2 transfectants (see Table 1and Table 2).
PC2-transfected PC12
cells expressed varying levels of the 2.8-kilobase PC2 mRNA whereas
wild type PC12 cells were devoid of PC2 mRNA (Fig. 7A).
Western blot analysis with a N-terminally-directed PC2 antiserum which
preferentially detects the 66-kDa PC2 protein confirmed these data (Fig. 7B). The levels of pro-NT/NN mRNA expression were
variable in the selected PC12/PC2 transfectants (Fig. 7A). They were mirrored by the amounts of CTiNN
measured in these clones (Table 2). Pro-NT/NN processing analysis
showed that the PC2 transfectants produced principally NN and NT (Table 2). Large NN was in general not detectable except in the
two clones (E2.11 and L2.2) that had the highest concentrations of
CTiNN. In these clones, large NN was 5-10 times less abundant
than NN. Large NT was detected only in the highest CTiNN-producing
clone (L2.2). As with the PC12/PC1 cells, no iK6L could be detected in
the PC2 transfectants (not shown), indicating that PC2 did not cleave
the Lys-Arg
dibasic site in pro-NT/NN. The
percentages of cleavage of the Lys-Arg sequences in pro-NT/NN (Table 2, Fig. 8) revealed an order of preference for PC2
that was Lys
-Arg
=
Lys
-Arg
Lys
-Arg
. Note that this order markedly
differs from that found for PC1.
Figure 7:
Northern blot analysis of PC2, pro-NT/NN,
and GAPDH mRNAs and Western blotting of PC2 in wild type and
PC2-transfected PC12 cells. PC12 cells were stimulated with nerve
growth factor, dexamethasone, forskolin, and Li for 48
h. Total RNA (10 µg) and proteins (50 µg) were analyzed by
Northern and Western blotting as described under ``Experimental
Procedures.'' A, Northern blot analysis of PC2,
pro-NT/NN, and GAPDH mRNAs. B, Western blot analysis of PC2
using the N-terminally directed PC2 antiserum
7BF.
One of the goals of the present study was to identify the
PC(s) responsible for pro-NT/NN processing in the rMTC 6-23 cell line.
Among the convertases whose presence was tested in rMTC 6-23 cells,
only furin and PC2 were detected. This suggests that furin or PC2 could
be involved in pro-NT/NN processing. Two pieces of evidence stand
against furin as being a pro-NT/NN convertase. First, as recalled in
the Introduction, furin, a ubiquitous enzyme, appears to be principally
involved in the processing of precursor proteins that, unlike
pro-NT/NN, are routed to the constitutive secretory pathway. Second,
PC12 cells which do express furin (present study and (2) and (3) ) are virtually unable to process pro-NT/NN into mature
products(37, 38, 39) . This leaves the
neuroendocrine cell-specific convertase, PC2, as the most likely
pro-NT/NN convertase candidate in rMTC 6-23 cells. This hypothesis was
directly tested by stably transfecting antisense PC2 mRNA in rMTC 6-23
cells and assessing the consequences of antisense expression on PC2
protein levels and pro-NT/NN processing. PC antisense strategies have
been successfully used by others for demonstrating the role of PC2 in
proglucagon processing in TC1-6 cells (20) and of
PC1 in POMC processing in AtT-20 cells(23) . Our data show that
rMTC 6-23 clones which expressed PC2 antisense mRNA exhibited a massive
reduction of PC2 protein levels (up to 90%) which was paralleled by a
marked inhibition of pro-NT/NN processing (>80%). These observations
strongly argue in favor of PC2 being the major endogenous pro-NT/NN
convertase in rMTC 6-23 cells.
Previous studies have shown that pro-NT/NN mRNA and protein expression was stimulated by dexamethasone in rMTC 6-23 cells(34) . We show here that the glucocorticoid also increased PC2 mRNA and protein levels in these cells. The concentration-response and time dependence for the effect of dexamethasone on PC2 expression were similar to those previously reported for pro-NT/NN expression(34) . Coregulation of pro-NT/NN and PC2 by dexamethasone might have physiological relevance. Thus, glucocorticoids have been shown to up-regulate pro-NT/NN mRNA and NT levels in hypothalamic neurons both in vitro and in vivo(46, 47) . The induction of pro-NT/NN will put an increased demand on its maturation. One way to cope with this would be to concomitantly increase the synthesis of relevant convertase(s), as observed here in the case of rMTC 6-23 cells. Most brain regions process pro-NT/NN with a pattern similar to that described in rMTC 6-23 cells(27, 28, 34) . In addition, PC2 is the most abundant convertase found in brain(3) . It would be interesting to see if PC2 is expressed in a dexamethasone sensitive manner in the hypothalamic neurons that produce pro-NT/NN. Hormonal coregulation of PC(s) and precursor substrate(s) expression may be a general phenomenon (23, 48) . In particular, it has been reported that dexamethasone coregulated POMC and PC2 levels in AtT-20 cells, although in this case the glucocorticoid effect was inhibitory(48) .
Another goal of the present work was to compare PC1- and
PC2-mediated pro-NT/NN processing patterns. A major finding of this
study is that PC1 and PC2 stably transfected into PC12 cells were both
able to process the NT/NN precursor but with different patterns of
peptide production. Thus, the major products obtained with PC1 were NT
and large NN. NN was also produced but in 4-fold lower concentrations
than large NN. With PC2, the major products observed were NT and NN,
large NN being barely detectable. It is interesting that the pattern of
processing observed with PC1 resembles that found in the gut (27, 29, 30, 31) while PC2
reproduces the processing pattern described in the brain and in the
rMTC 6-23 cell line(28, 33, 34) . We have
obtained preliminary immunocytochemical data showing that PC1
colocalizes with pro-NT/NN in the endocrine N cells of the rat ileum. ()Thus, the coexpression profile of PC1 and PC2 with
pro-NT/NN in systems that produce pro-NT/NN-derived products is
consistent with the pattern of pro-NT/NN processing by these enzymes as
determined here in transfected PC12 cells.
Differential processing
by PC1 and PC2 has been reported for other prohormone precursors such
as POMC and proinsulin. In the case of POMC, PC1 appears to be
responsible for the formation of large products including ACTH and
-lipotropin in the corticotrophs of the anterior pituitary,
whereas PC2 processes these products further to generate smaller
peptides such as the melanotropins, corticotropin-like intermediate
lobe peptide, and
-endorphin in the intermediate lobe of the
pituitary(22, 23, 24) . In proinsulin, the
insulin B and A chains are separated by a connecting peptide (C
peptide) which is linked to both chains by dibasic sequences. Several
lines of evidence indicate that in the insulin secretory
-granules
PC1 preferentially cleaves the B chain-C peptide junction, while PC2
cleaves the C peptide-A chain
junction(17, 18, 49, 50) . Most
recently, proglucagon was also shown to be differentially processed by
PC1 and PC2, PC1 reproducing the processing pattern observed in the
endocrine L cell of the gut and PC2 generating the pattern found in the
pancreatic
cell(51) . In general, it appears that PC1
cleaves multipeptide-producing precursors at a limited number of
dibasic sites to generate large biologically active peptides, whereas
PC2 processes additional dibasic sites to liberate the smaller active
peptides. Such a pattern of action for PC1 and PC2 is consistent with
the present observation that PC1 cleaves the Lys-Arg sequence that
precedes NN much less efficiently than the two Lys-Arg sequences that
flank NT in contrast to PC2 which processes the three Lys-Arg dibasic
sites with a similar efficiency.
The analysis of a number of PC12 transfectants that expressed varying amounts of PCs allows us to evaluate the incidence of PC1 and PC2 expression level on pro-NT/NN processing pattern and efficiency. Although there was some variability in the extent of dibasic site cleavages among the PC1- and PC2-transfected cells, the order of site preference for each endoprotease was similar within either series of transfectants. Thus, the level of convertase expression does not seem to greatly influence the qualitative pattern of pro-NT/NN processing by either PC1 or PC2. Similar conclusions were reached regarding the processing of POMC in AtT-20 cells that overexpressed PC1 or PC2(24, 52) . With respect to processing efficiency, somewhat higher pro-NT/NN conversion ratios were obtained in PC12/PC1 as compared to PC12/PC2 transfectants. This was also the case in another study(39) . It is, however, difficult to compare the efficiency of PC1 and PC2 in the present work because their concentration relative to one another is unknown. Our data with rMTC 6-23 cells show that PC2 can quite efficiently process pro-NT/NN (>95%). Recent studies have revealed that PC2 activation and enzymatic activity in the cell is under the control of another neuroendocrine tissue-specific cellular protein designated 7B2(53, 54, 55) . It is therefore possible that the efficiency of PC2 may vary from one cell line to another depending on the cellular levels of 7B2 expression.
It would be interesting to know if the dibasic sites in pro-NT/NN are cleaved by PC1 and PC2 in a precise temporal order and if this order differs for both enzymes. This could provide some clues as to the pro-NT/NN sequence elements that direct PC1 and PC2 substrate specificity. The rMTC 6-23 cell line and the stable PC12/PC1 and PC12/PC2 transfectants characterized here should provide useful models to address this issue.