and processing of mast cell secretory granule components Frida Henningsson*, Sonja Hergeth*, Robert Cortelius, Magnus A˚ brink and Gunnar Pejler Swedish University of Agricultural Scienc
Trang 1and processing of mast cell secretory granule components Frida Henningsson*, Sonja Hergeth*, Robert Cortelius, Magnus A˚ brink and Gunnar Pejler
Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Center, Uppsala, Sweden
Mast cells (MCs) are characterized by their large
con-tent of electron-dense secretory granules, and these
granules are released following MC activation, a
pro-cess that can be accomplished by various mechanisms,
including antigen-mediated crosslinking of
surface-associated IgE and exposure to neuropeptides,
anaphy-latoxins or calcium ionophores [1,2] The MC granules
contain a broad array of bioactive compounds, with
the exact composition being dependent on the
partic-ular species and subclass of MC [1,3] Histamine is a
major constituent of all types of MC, and it is now
well recognized that MC granules can contain a
num-ber of different cytokines, such as tumor necrosis
factor-a, interleukin (IL)-4, IL-5, IL-13, transforming
growth factor-b and vascular endothelial growth factor
[2] Moreover, the MC granules contain b-hexosamini-dase and a number of MC-specific neutral proteases: chymases, tryptases and carboxypeptidase A (CPA) [4,5] In addition, MC granules contain large amounts
of highly sulfated and thereby negatively charged pro-teoglycans (PGs) of the serglycin (SG) type, and it is these PGs that give the typical metachromatic staining
of MCs with cationic dyes [6] In MCs, SG PGs can accommodate either (or both) chondroitin sulfate or heparin side chains, depending on MC subclass [7] The processes involved in MC degranulation, in par-ticular the signal transduction pathways, have been the subject of intense investigation [8,9] In contrast, strik-ingly little is known regarding the actual formation of
MC secretory granules For example, the factors that
Keywords
mast cells; proteases; proteoglycans;
serglycin; sorting
Correspondence
G Pejler, Swedish University of Agricultural
Sciences, Department of Molecular
Biosciences, The Biomedical Center,
Box 575, 751 23 Uppsala, Sweden
Fax: +46 18 550762
Tel: +46 18 4714090
E-mail: Gunnar.Pejler@bmc.uu.se
*These authors contributed equally to this
work
(Received 28 June 2006, revised 15 August
2006, accepted 4 September 2006)
doi:10.1111/j.1742-4658.2006.05489.x
In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases However, the mechanisms behind these phenomena are not known In this study, we addressed these issues by studying the functionality and morphol-ogy of secretory granules as well as the fate of the secretory granule prote-ases in bone marrow-derived mast cells from serglycin+⁄ +and serglycin–⁄ – mice We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defect-ive in serglycin–⁄ – mast cell granules The low levels of mast cell proteases present in serglycin–⁄ – cells had a granular location, as judged by immu-nohistochemistry, and were released following exposure to calcium iono-phore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degrada-tion, exocytosis or defective intracellular processing In contrast, b-hexosa-minidase storage and release was not dependent on serglycin Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive path-way of secretion, but rather that serglycin may be involved in the retention
of the proteases after their entry into secretory vesicles
Abbreviations
BMMC, bone marrow-derived mast cell; CPA, carboxypeptidase A; MC, mast cell; mMCP, mouse mast cell protease; PG, proteoglycan;
SG, serglycin; TEM, transmission electron microscopy.
Trang 2determine the sorting of granular components are
lar-gely undefined, and the mechanisms that lead to the
assembly of the electron-dense, metachromatically
staining granules seen in mature MCs have been
poorly investigated In a recent study, we generated a
mouse strain in which the SG gene was targeted [10]
We found that, in the absence of SG, mature
metach-romatically staining granules were not observed
Fur-thermore, we noted that storage, but not mRNA
expression, of the various MC proteases was
dramatic-ally defective in SG–⁄ – MCs However, the underlying
mechanisms behind these observations were not
defined The aim of this study was therefore to provide
insights into this issue by determining the fate of
SG-dependent proteases in cells with an inactivated SG
gene Our results are compatible with a model of
secre-tory granule maturation in which SG PG is not
involved in the transport of compounds into secretory
vesicles, but is essential for retention of certain
constit-uents after their entry into the granules
Results
This study was undertaken to resolve the mechanism
behind the previously observed severe granule defects in
SG–⁄ – MCs [10] Given the dramatic effects of the SG
knockout on granular staining properties and storage of
proteases, it was first important to determine whether
the lack of SG affected the actual assembly of granules
and whether secretory granules were also functional in
the absence of SG To this end, bone marrow cells
were recovered from SG+⁄ +and SG–⁄ –mice and were
in vitrodifferentiated into mature bone marrow-derived
MCs (BMMCs) by culturing in medium containing IL-3
[11,12] Cells were recovered at various stages of cellular
differentiation, and their morphology was examined
after staining with May–Gru¨nwald ⁄ Giemsa At day 0,
as expected, no cells with an MC-like appearance were
observed Starting from day 5, cells containing ‘empty’
(May–Gru¨nwald ⁄ Giemsa-negative) vesicles were
observed Such vesicles were seen both in SG+⁄ +and
SG–⁄ – cells, indicating that their formation was not
dependent on SG When the cells were cultured further,
the number of May–Gru¨nwald ⁄ Giemsa-negative
vesi-cles gradually decreased in both SG+⁄ +and SG–⁄ –cells
This was accompanied by the appearance, from about
day 12, of May–Gru¨nwald ⁄ Giemsa-positive granular
structures in SG+⁄ + cells A gradual increase in
May–Gru¨nwald ⁄ Giemsa staining was seen over time in
SG+⁄ +cells In contrast, May–Gru¨nwald ⁄
Giemsa-pos-itive vesicles were not seen in SG–⁄ –cells at any stage of
differentiation (not shown) These results are in
agree-ment with those of a previous study [10]
One potential explanation for the lack of May– Gru¨nwald ⁄ Giemsa-negative vesicles in SG–⁄ – cells at later stages of differentiation could be that immature granules are generated in the absence of SG, but that the lack of SG causes their disruption Alternatively, secretory granules could be present at late stages of maturation also in SG– ⁄ – cells, but not be visible by conventional microscopy To provide further insights into this issue, we examined the cells at the ultrastruc-tural level by transmission electron microscopy (TEM) The TEM analysis indeed revealed the existence of secretory granule-like organelles in SG–⁄ – cells, and these organelles were found in approximately equal numbers as in SG+⁄ + cells (Fig 1; upper panels) However, the morphology of the granules was differ-ent; whereas dense core formation was seen in SG+⁄ + granules, the contents of the SG–⁄ – granule were of more amorphous character, without defined electron-dense cores (Fig 1; lower panels)
To address whether the secretory granules were functional, we measured the ability of the MCs to release b-hexosaminidase, a granule component, upon exposure to calcium ionophore A23187 As shown in Fig 2A, equal amounts of b-hexosaminidase were released by SG+⁄ +and SG–⁄ –cells after calcium iono-phore stimulation, and the kinetics of release were sim-ilar Furthermore, the levels of b-hexosaminidase in conditioned medium from nonstimulated cells were similar in cultures of both genotypes (Fig 2A), indica-ting that the lack of SG PG did not result in increased spontaneous release of b-hexosaminidase These find-ings indicate that the general ability of MCs to degran-ulate is not dependent on SG Experiments were also undertaken to investigate whether the level of stored b-hexosaminidase is influenced by SG Although b-hexosaminidase activity was already detected at day
0, the intracellular content of this enzyme increased markedly after 6 days of culture, and reached a plat-eau from about day 12 (Fig 2B) Both the kinetics of accumulation and the level of maximal storage were virtually identical in SG+⁄ + and SG–⁄ – cells, indica-ting that the storage of b-hexosaminidase is independ-ent of SG
The results above indicate that functional MC secre-tory granules are formed independently of SG PG Hence, the defective storage of MC proteases in the absence of SG [10] is not due to defects in the forma-tion or funcforma-tionality of granular compartments In order to understand the mechanism by which SG PG promotes storage of these compounds, the strategy in the next set of experiments was to follow the fates
of the SG-dependent proteases when SG was absent
To address these issues, we examined the expression,
Trang 3cellular storage, processing and secretion of SG and of
various MC proteases at different stages of MC
differ-entiation, as described below
SG core protein transcript was already clearly
detectable at day 0, but the level of transcript appeared
to increase after5 days of culture (Fig 3A) In
con-trast, no detectable mRNA for mouse MC protease 5
(mMCP-5; a chymase), the tryptase mMCP-6 or CPA
was detected at day 0 Starting from day 5, however,
mMCP-6 and CPA transcripts were clearly detected,
and they appeared to increase further after 12 days of
culture The onset of mMCP-5 mRNA expression was
somewhat delayed, with clearly detectable transcript
being seen from day 12 mMCP-5, mMCP-6 and CPA
transcripts were detected in both SG+⁄ + and SG–⁄ –
cells, and the kinetics as regards onset of mRNA
expression were similar in both genotypes, indicating
that the knockout of SG does not affect cellular
differ-entiation into MCs, as judged by the transcription of
the MC protease genes
Further experiments were carried out to examine
how the mRNA expression profiles of SG+⁄ + and
SG–⁄ –cells were reflected at the protein level (Fig 3B) Immunoblot analysis of SG+⁄ + cell extracts showed that neither of the MC proteases were present at day
0 mMCP-5 protein was detected starting from day 12, i.e at the same time as when gene transcription was first seen mMCP-5 protein accumulated over time, with a plateau of maximal storage seen after 26 days
of culture mMCP-6 storage showed similar kinetics as for mMCP-5 In contrast, CPA protein was detected
as early as after 5 days of culture, and a maximal plat-eau of storage was already seen at day 12 Both pro-CPA and mature pro-CPA were detected in SG+⁄ +cells
A dramatically different pattern was seen in SG–⁄ – cells mMCP-5 protein was not detected at any time point, and mMCP-6 protein, although being detect-able, was present at markedly lower levels than in
SG+⁄ + cells Notably, however, mMCP-6 accumula-tion in SG–⁄ – cells showed similar kinetics as in
SG+⁄ + cells In contrast, the total amounts of CPA antigen (pro-CPA + mature CPA) were approximately equal in SG– ⁄ – and SG+ ⁄ + cells An interesting observation was that only the pro-form of CPA was
Fig 1 Transmission electron micrographs The upper panels show representative mature (5 weeks of culture) bone marrow-derived mast cells (BMMCs) from serglycin (SG)+⁄ +and SG–⁄ –mice (original magnification 5000·) The lower panels show enlarged granules (original magnification · 40 000).
Trang 4detected in SG–⁄ – cells, indicating that pro-CPA
processing into mature protease is strongly dependent
on SG A plausible explanation for this finding is that
the protease(s) that are responsible for the pro-CPA
processing is dependent on SG In agreement with
such a notion, we showed recently that pro-CPA
processing was defective in cathepsin E–⁄ – MCs and
that the storage of cathepsin E in MCs is dependent
on heparin PG [13] Thus, a likely explanation for the
defective pro-CPA processing in SG–⁄ –MCs is that the
lack of SG also results in defective cathepsin E storage
and that this, in turn, results in defective pro-CPA
processing, leading to an accumulation of pro-CPA
Next, we investigated the possibility that the
pro-teases were constitutively secreted in the absence of
SG PG Conditioned media were collected at different stages of MC differentiation, and were analyzed for the presence of secreted MC proteases As shown in Fig 3C, mMCP-5 protein was present at low levels
in conditioned medium from SG+⁄ + cells after prolonged culture, but was absent in medium from
SG– ⁄ – cells In contrast, mMCP-6 protein was clearly detected, starting from about day 14, in conditioned medium from both SG+⁄ +and SG–⁄ – cells CPA, pre-dominantly in its mature form, was secreted into the medium by SG+⁄ + cells, starting at about day 5 In contrast, only the pro-form of CPA was secreted by
SG–⁄ –cells The total level of secreted CPA (pro-CPA and mature CPA) was somewhat higher in medium from SG–⁄ –than in that from SG+⁄ +MCs, in partic-ular at early time points (Fig 3C) Note that, at early time points, pro-CPA dominated over mature protease, both intracellularly (Fig 3B; day 5) and in conditioned medium from SG+⁄ + cells (Fig 3C; days 6 and 12), indicating that efficient processing of pro-CPA is dependent on the degree of MC maturation In accord-ance with this notion, only the mature form of CPA is detected in fully mature connective tissue-type MCs recovered in vivo [13], and only the pro-form of CPA
is detected in poorly differentiated transformed cell lines of MC origin (M Grujic & G Pejler, unpub-lished results)
The results above indicate that mMCP-6 and pro-CPA are secreted by SG–⁄ – MCs A possible explan-ation for these findings would be that the absence of
SG causes missorting of mMCP-6 and pro-CPA into the constitutive rather than into the regulated pathway
of secretion If indeed this were the case, mMCP-6 and pro-CPA would not be present in the secretory gran-ules, and exposure of SG–⁄ – cells to MC-degranulating agents would not cause increased release of mMCP-6 and pro-CPA If, on the other hand, mMCP-6 and pro-CPA are in fact located in secretory granules also
in SG–⁄ –cells, MC degranulation would be expected to induce their release In order to address these possibil-ities, SG+⁄ +and SG–⁄ –MCs were exposed to the cal-cium ionophore A23187, a compound that is regularly used as an MC secretagogue [14] As shown in Fig 4A, exposure of SG+⁄ + cells to A23187 resulted
in clearly detectable mMCP-6 and CPA in conditioned medium Strikingly, calcium ionophore stimulation also resulted in the release of mMCP-6 and pro-CPA
by SG–⁄ – cells (Fig 4A) The implication of these find-ings is that mMCP-6 and pro-CPA are sorted into releasable secretory vesicles despite the absence of SG
To obtain further evidence for this, SG–⁄ – cells were stained for mMCP-6 antigen, before and after expo-sure to calcium ionophore In resting SG–⁄ – cells,
0
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+/+ non-stim +/+ A23187 -/- non-stim -/- A23187
A
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Days of culture
+/+
B
Fig 2 b-Hexosaminidase content and release (A) Conditioned
media from mature (5 weeks of culture) serglycin (SG)+⁄ + (filled
symbols) and SG – ⁄ – (open symbols) cells were analyzed for
b-hex-osaminidase activity, without stimulation (squares) or after addition
of A23187 (circles) (B) SG+⁄ + (filled squares) and SG–⁄ – (open
squares) cells taken at different stages (days 0–33) of
differenti-ation in interleukin (IL)-3-containing medium were analyzed for total
intracellular b-hexosaminidase activity Results are expressed as
percentages, where the b-hexosaminidase content in SG + ⁄ + cells
at day 33 is set as 100% Results are expressed as means of
tripli-cate determinations ± SD.
Trang 5mMCP-6 was found in granule-like compartments
close to the plasma membrane (Fig 4C), indeed
sup-porting the idea that mMCP-6 is transported into
secretory granules even in the absence of SG PG
Fur-thermore, after exposure to A23187, SG–⁄ – cells
showed signs of degranulation and it was also evident
that the released granules stained positively for
mMCP-6 (Fig 4C) Preimmune serum gave only
dif-fuse overall staining of SG–⁄ –cells and a total absence
of granular staining of either unstimulated or
A23187-stimulated cells (Fig 4C)
Next, we investigated the possibility that the MC proteases are subjected to degradation by lysosomal proteases when SG PG is absent For this purpose, cells were incubated with NH4Cl in order to raise the
pH of acidic intracellular compartments, including lysosomes and secretory granules, and thereby inacti-vate lysosomal proteases Incubation of SG–⁄ – MCs with NH4Cl did not affect the level of intracellular mMCP-6, indicating that degradation by lysosomal mechanisms is not a primary fate of mMCP-6 when
SG is absent (Fig 5) In contrast, NH4Cl caused an
SG
mMCP-6 mMCP-5
CPA HPRT
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mMCP-6
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Fig 3 mRNA expression and protein analysis (A) Total RNA was prepared from serglycin (SG)+⁄ +and SG–⁄ –bone marrow-derived cells after different durations (days 0–33) of culture in medium containing interleukin (IL)-3 The RNA was used for analysis of the expression of mouse mast cell protease (mMCP)-5, mMCP-6, carboxypeptidase A (CPA) and SG by RT-PCR Expression of hypoxanthine–guanine phopho-ribosyltransferase was used as housekeeping control (B) Cell extracts were prepared from cells taken at various stages (days 0–33) of differ-entiation and were subjected to immunoblot analysis using antisera towards mMCP-5, mMCP-6 and CPA (C) Secretion of MC proteases from SG + ⁄ + and SG – ⁄ – cells Conditioned media were recovered from SG + ⁄ + and SG – ⁄ – cells at various stages (days 0–33) of differentiation
in IL-3-containing medium The media were concentrated and subjected to immunoblot analysis for mMCP-5, mMCP-6 and CPA The results shown are representative of three independent experiments.
Trang 6accumulation of mMCP-5 protein in SG–⁄ – cells,
whereas mMCP-5 levels were not affected in SG+⁄ +
MCs (Fig 5; upper panel) This indicates that
mMCP-5, in the absence of SG, is degraded by proteases with
low pH optima, possibly in lysosomal compartments
Interestingly, several ‘lysosomal’ proteases, e.g
cathep-sin C, cathepcathep-sin D and cathepcathep-sin E, have been found
not only in lysosomes but also in MC secretory
gran-ules [13,15,16] Thus, the degradation of mMCP-5 does
not necessarily have to involve transport to lysosomes,
but could in fact occur within the secretory granule
compartment NH4Cl did not cause any noticeable
accumulation of pro-CPA or mature CPA in either
SG+ ⁄ + or SG– ⁄ – MCs However, the NH4Cl
treat-ment resulted in the accumulation of an intermediate
form of CPA, of somewhat lower molecular weight than pro-CPA (Fig 5; lower panel) Most likely, this compound represents an intermediate in processing These findings indicate that the processing of pro-CPA occurs in (at least) two steps, and that the processing
of the intermediate form of CPA to mature protease is dependent on a (lysosomal?) protease with an acidic
pH optimum Control experiments showed that NH4Cl did not affect cellular viability (not shown)
Degradation by the proteasome pathway could con-stitute an alternative degradative pathway in the absence of SG However, incubation of cells with lac-tacystin, an inhibitor of proteasome function, did not cause any accumulation of MC proteases in SG– ⁄ –
MCs (not shown)
C
Fig 4 Protease release after mast cell (MC) degranulation Serglycin (SG)+⁄ +and SG–⁄ – MCs (after 5 weeks of culture) were treated with calcium ionophore A23187 (A) Medium fractions from SG+⁄ +and SG–⁄ –cells were subjected to immunoblot analysis using anti-sera towards carboxypeptidase A (CPA) and mouse mast cell protease (mMCP)-6 Note the increase in extracellular mMCP-6 and CPA antigen, in both SG + ⁄ + and SG – ⁄ – cul-tures, after stimulation with A23187 (B) Cell fractions from SG+⁄ +and SG–⁄ –cells were analyzed for mMCP-6 and CPA by immuno-blotting (C) Cytospin slides were prepared from nontreated and A23187-treated SG–⁄ – cells and were immunohistochemically stained for the presence of mMCP-6 anti-gen Note the granular staining for mMCP-6, both before and after A23187 stimulation The results shown are representative of three independent experiments.
Trang 7As shown in Fig 3A, SG core protein mRNA was
already expressed at day 0 However, maximal MC
protease accumulation was not obtained until about
day 26 (Fig 3B), a finding that may appear
contradict-ory, considering the strong dependence of the MC
pro-teases on SG for storage This indicates that the levels
of stored proteases are not directly related to the
amount of SG core protein mRNA being expressed
One potential explanation could be that the amount of
actual sulfated PGs is not directly correlated with the
level of SG mRNA, and it was therefore of interest to
also follow the levels of sulfated PGs during the course
of MC differentiation To this end, SG+⁄ + MCs at
different stages of differentiation were biosynthetically
labeled with 35SO42– 35S-labeled PGs were recovered
both from conditioned medium (secreted PGs) and
from cell extracts, and were quantified As shown in
Fig 6A, the levels of secreted PGs were similar at all
time points tested In contrast, the levels of
intracellu-lar PGs increased markedly over time Notably, the
level of intracellular PGs did not reach a plateau, as
observed for the proteases (Fig 3B) Rather, the level
of cell-associated PGs showed a continuous increase
over time (Fig 6A) Notably, the latter is in good
agreement with the relatively late appearance of May–
Gru¨nwald ⁄ Giemsa-positive granules as compared to
the onset of SG mRNA expression (see above)
Next, the possibility that the different abilities of MCs to store proteases at different stages of differenti-ation could be due to differences in the electrostatic charge of the PGs was addressed For this purpose,
MC PGs recovered at different time points during the course of MC development were examined by anion exchange chromatography At early time points (day 10), there was a distinction between two separate PG populations, one with low anionic charge density [coeluting with standard chondroitin sulphate A (CS-A)] and one population with a markedly higher charge (coeluting with standard pig mucosal heparin) (Fig 6B) Similar elution profiles were seen for PGs recovered from the cell layer and from conditioned medium In contrast, only highly charged PGs were seen at day 23 (Fig 6B) and day 34 (not shown), again with similar charge densities being displayed by cell-associated and extracellular PGs Together, these results indicate that the MC maturation process is associated with both increased total synthesis of sulfated PGs and increased charge density of the synthesized PGs
Discussion
Although the knockout of both SG [10] and N-de-acetylase⁄ N-sulfotransferase-2 [17,18], the latter being
an enzyme involved in the sulfation of heparin chains attached to the SG core protein, has provided strong evidence for a crucial role of PGs in mediating the storage of secretory granule compounds in MCs, the mechanism behind these observations has not been established One potential mechanism would be that
SG is important for the formation of the secretory granule However, we show here that SG–⁄ –MCs also displayed clearly discernible secretory vesicle-like struc-tures By conventional microscopy, such vesicles were May–Gru¨nwald ⁄ Giemsa-negative and, interestingly, May–Gru¨nwald ⁄ Giemsa-negative vesicles were also seen in SG+⁄ + cells at early stages of differentiation Most likely, these structures represent immature secre-tory granules in which the PG content is too low to stain with May–Gru¨nwald ⁄ Giemsa In accordance with this, it was observed that the intracellular content of highly sulfated PGs was relatively low at the corres-ponding (early) time point At later stages of differenti-ation, in contrast, SG+⁄ + MCs showed staining with May–Gru¨nwald ⁄ Giemsa, and this correlated well with
a marked increase in the recovery of highly sulfated intracellular PGs
The presence of secretory vesicle-like structures in
SG–⁄ – cells was also supported at the ultrastructural level TEM analysis showed the presence of highly elec-tron-dense granules in SG+⁄ + cells, but also showed
Fig 5 Inhibition of lysosomal proteases Serglycin (SG)+⁄ + and
SG – ⁄ – cells (after 5 weeks of culture) were incubated for 6 h with
20 m M NH4Cl, or for 20 h with 5 m M NH4Cl Cell extracts were
subsequently subjected to immunoblot analysis using antisera
towards carboxypeptidase A (CPA) and mouse mast cell protease
(mMCP)-6 The arrow indicates a ‘semiprocessed’ form of CPA.
The results shown are representative of three independent
experi-ments.
Trang 8an abundance of granule-like organelles in SG–⁄ –cells.
Importantly, however, the granule matrix in SG–⁄ –cells
was more amorphous than in SG+⁄ +cells, and showed
less defined dense core formation Our results also
pro-vide epro-vidence that SG–⁄ – cells retain the full capability
to undergo stimulus-induced degranulation, as
deter-mined by the ability to release b-hexosaminidase in
response to calcium ionophore Together, our data thus
indicate that SG PG is not necessary for the formation
of MC secretory granules, and nor is SG involved in
mechanisms of degranulation
Another possible explanation for the storage defects
seen in SG–⁄ –MCs would be that SG PG is needed for
correct intracellular sorting of the MC proteases into
the secretory granules, the alternative fate being secre-tion by the constitutive pathway If this was the case,
it would be expected that SG-binding compounds such
as the MC proteases would be preferentially released into the extracellular space by SG–⁄ – MCs Such mis-sorting would result in excessive accumulation of granule compounds in conditioned medium from
SG–⁄ –cells We here provide evidence that CPA, in its pro-form, is secreted at higher levels by SG–⁄ – cells than by their SG+⁄ + counterparts, indeed indicating that the lack of SG PG causes increased constitutive release However, rerouting into the constitutive path-way of secretion does not seem to be a general effect
on all MC proteases when SG PG is lacking, as shown
0.0 0.5 1.0 1.5 2.0 2.5
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Fig 6 Analysis of sulfated proteoglycans Serglycin (SG) + ⁄ + bone marrow cells were cultured for different times (10, 23 34 days)
in medium containing interleukin (IL)-3 and were biosynthetically labeled with 35 SO42– (A) Total recovery of 35 S-labeled proteogly-cans per 1 · 10 6
cells into cell (filled bars) and medium (hatched bars) fractions (B) Charge density of sulfated proteoglycans.
35 S-labeled proteoglycans isolated from cell and medium fractions, both derived from
SG + ⁄ + cells, were mixed with internal stand-ards of heparin (hep) and chondroitin sulfate (CS) and were subjected to anion exchange chromatography on a DEAE–Sephacel col-umn The column was eluted with a linear gradient of LiCl Fractions were analyzed for radioactivity (filled symbols) and for uronic acid in order to detect the internal standards (open symbols; A 530 ) The results shown are representative of two independent experi-ments.
Trang 9by the fact that mMCP-6 and mMCP-5 were found at
similar or higher levels in medium from SG+⁄ + cells
than in medium from their SG–⁄ – counterparts
More-over, our data provide evidence that the SG-dependent
MC proteases are in fact present in functional
secre-tory granules even in the absence of SG PG This
indi-cates that transport of MC proteases into the secretory
granule compartments can occur independently of
SG PG Hence, SG does not appear to function as
general sorting vehicle for MC proteases
From the data presented here and previously [10], it is
clear that knockout of the SG gene results in a major
reduction of mMCP-5, mMCP-6 and CPA storage
However, the blockade is not complete, indicating that
the proteases can actually be stored to some extent in
MC granules even in the absence of SG PGs to which
they can bind In turn, this may be explained by low
lev-els of granular storage in the absence of any partner PG
An alternative explanation could be that there are low
levels of PGs other than SG in the MC granule, and that
such PG species can provide some compensation for the
lack of SG in terms of promoting MC protease storage
However, the presence of non-SG PG species within
MC granules remains to be demonstrated
So, how does the lack of SG cause such a dramatic
reduction of stored MC proteases? Although general
mechanisms involved in the intracellular sorting of
granule components are still relatively poorly defined,
two major hypotheses have emerged: ‘sorting by entry’
and ‘sorting by retention’ [19] In the sorting by entry
hypothesis (e.g the mannose 6-phosphate system [20]),
each secretory granule compound has a unique sorting
signal that interacts with a cognate receptor on the
luminal side of specific regions in the trans-Golgi
network, leading to budding from the trans-Golgi
network of vesicles containing only molecules with the
corresponding specific sorting signals In the sorting by
retention hypothesis, certain compounds entering the
immature granules may carry sorting motifs that
inter-act with the limiting membrane, but luminal proteins
that are not associated with the trans-Golgi network
membrane may also be included in the budding vesicle
According to this hypothesis, the contents of the
imma-ture granule are subsequently refined, both by
conden-sation of selected compounds and by removal of others
by vesicular transport, e.g to lysosomes for destruction,
or to the extracellular space by ‘constitutive-like’ or
‘piecemeal’ exocytosis [19,21] This process will
eventu-ally result in secretory granule maturation Although
we cannot at this stage with certainty define the role of
SG PG in this process, our results are clearly
compat-ible with a model in which SG organizes secretory
gran-ule maturation according to the sorting by retention
hypothesis In support of this, all of the MC proteases that have been shown to be SG-dependent for storage, i.e mMCP-4, mMCP-5, mMCP-6 and CPA (this study and [10]), display high affinity for sulfated glycosami-noglycans [22–25] It is therefore possible that
mMCP-4, mMCP-5, mMCP-6 and CPA are transported into immature granules independently of SG, but that their retention within the granules is dependent on their tight electrostatic interaction with SG PG However, our data indicate that interaction with SG PG is not an absolute prerequisite for retention of all granule com-pounds within the granule, as shown by the lack of SG dependence for the storage of b-hexosaminidase The sorting for retention model of granule matur-ation implies that compounds not selected for retent-ion are expelled from the maturing granule by vesicular transport In line with this model, our results suggest that mMCP-5 is targeted to degradation if not retained by SG We also see a marked secretion of pro-CPA by SG–⁄ – cells, possibly as a consequence
of defective retention However, there is also secretion
of CPA, albeit in its mature form, from SG+ ⁄ + cells mMCP-6 is also secreted by SG–⁄ – cells, but in con-trast to pro-CPA and mature CPA, mMCP-6 secretion was somewhat higher in SG+⁄ + cells than in their
SG–⁄ – counterparts However, the level of mMCP-6 protein in the conditioned medium from SG–⁄ – cells was considerably higher than the intracellular level, indicating that secretion rather than storage is the dominating pathway for mMCP-6 in the absence of
SG One possible explanation for these findings is that there is continuous low-level release of secretory gran-ule compounds in normal MCs, a process often referred to as ‘piecemeal’ degranulation [21] In the absence of SG as a retention vehicle, this slow release may constitute the dominating pathway
In summary, this study has provided the first insights into the mechanism by which SG PG regulates
MC secretory granule homeostasis
Experimental procedures
Cell culture
experi-ments were approved by the local ethical committee BMMCs were obtained by culturing femural and tibial bone marrow cells in DMEM (SVA, Uppsala, Sweden), supplemented with 10% heat-inactivated fetal bovine serum
2 mm l-glutamine (SVA) and 50% WEHI-3B conditioned medium (which contains IL-3) for 3 weeks The cells were
Trang 10kept at a concentration of 500 000 cellsÆmL)1, and the
med-ium was changed every third day
Staining
Three hundred thousand cells were collected on cytospin
slides (700 r.p.m., 5 min) and stained with
3 min, and then stained with May–Gru¨nwald (Merck,
Sol-lentuna, Sweden) for 15 min After being washed with
water, the slides were stained with 5% Giemsa (Merck) in
water for 10 min
TEM
Cells were fixed for 6 h in 2% glutaraldehyde in a 0.1 m
sodium cacodylate buffer supplemented with 0.1 m sucrose,
and this was followed by 1.5 h of postfixation in 1%
osmium tetroxide dissolved in the same cacodylate buffer
After dehydration in ethanol, the cells were embedded in
the epoxy resin Agar 100 (Agar Scientific, Stansted, UK)
Ultrathin sections were placed on copper grids covered with
a film of polyvinyl formal plastic (Formvar; Agar Scientific)
and contrasted with uranyl acetate and lead citrate
Elec-tron micrographs were taken with a Hitachi elecElec-tron
micro-scope (Hitachi Ltd, Tokyo, Japan)
RT-PCR
Total RNA was isolated using the NucleoSpin RNA II kit
(Macherey Nagel, Du¨ren, Germany) Total RNA was used
for first-strand cDNA synthesis using SuperScriptII for
RT-PCR using primers specific for the MC proteases and
SG Hypoxanthine–guanine phosphoribosyltransferase was
used as a positive control for the RT-PCR The PCR
prim-ers used were as specified elsewhere [10]
Immunoblotting
main cultures, centrifuged at 300 g, (Megafuge 1.0R; Heraeus;
equipped with a swing out rotor), resuspended in 1 mL of
serum-free medium (as described above), and further cultured
overnight The cells were thereafter pelleted by centrifugation
at 300 g (10 min, Megafuge 1.0R; Heraeus; equipped with a
swing out rotor), and both the pellet and the medium fraction
analysis, the recovered media were concentrated 50 times
using Amicon Ultra-4 centrifugal filter device (Millipore,
sample buffer containing 5% b-mercaptoethanol Cell pellets
buffer containing 5% b-mercaptoethanol Immunoblotting
was carried out as previously described [10]
Proteoglycan isolation and analysis
) from days 9, 22 and 33 of culture were biosynthetically labeled overnight with 0.32 mCi of
Cells were pelleted by centrifugation for 10 min at 300 g (Megafuge 1.0R; Heraeus; equipped with a swing out
until further analysis For isolation of cell fraction glycos-aminoglycans, cell pellets were solubilized in 500 lL of
Then, the solubilisates were diluted with 9.5 mL of
contain-ing 0.4 mL of DEAE–Sephacel, equilibrated with 50 mm
media were loaded directly onto the columns After
(pH 5.5) Four fractions of 1100 lL each were collected and analyzed for radioactivity by scintillation counting Fractions containing radioactive material were pooled and
anion exchange chromatography on a 5 mL column of DEAE–Sephacel connected to an FPLC system The col-umn was eluted with a gradient of increasing concentra-tions of LiCl, from 0.05 m to 2 m in 50 mm sodium acetate
internal standard, 200 lL of a mixture of unlabeled heparin
Sweden) was added to each sample before anion exchange chromatography analysis Internal standards were detected
by the carbazole method: 25 lL of each fraction was mixed
10 lL of carbazole reagent (0.125% carbazole in 96% eth-anol) The samples were boiled for 10 min and cooled, and the absorbance at 530 nm was measured
Inhibition of proteasome and lysosome function
) were cultured in 5 lm lactacystin (Affiniti Research Products, Exeter, UK) After incubation overnight, cells were pelleted, solubilized and subjected to immunoblotting for mMCP-5, mMCP6 and CPA Lyso-somal function was inhibited by incubation of cells with 5
solubi-lized and subjected to immunoblotting
Degranulation
cells were incubated for 120 min in the presence of 2 lm of the calcium