Role of laminin-1, collagen IV, and an autocrine factor(s) in
regulated secretion by lacrimal acinar cells
Lanlin
Chen,
J. Douglas
Glass,
Staci C.
Walton, and
Gordon W.
Laurie
Department of Cell Biology, University of Virginia, Charlottesville,
Virginia 22908
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ABSTRACT |
Adhesion to novel basement membrane component BM180 in the
presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell
Physiol. 39): C1743-C1750,
1996]. The identity of the active laminin-1 site and
the possibility that other promoters of coupling are present in the
acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the
1 and
1 chains, but not
1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the
1-integrin subunit, and several
growth factors. Increasing cell number in serum-free medium revealed an
unknown, serum-maskable, secretion-enhancing activity with a remarkable
specificity for regulated secretion. Stimulus-secretion coupling,
therefore, appears to be modulated by several extracellular factors
whose relative contributions remain to be determined.
coupling; tear; exocytosis; signaling; integrin
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INTRODUCTION |
FROM ITS EARLIEST FORMATIVE stages as a cordlike
ectodermal invagination of conjunctival origin, the lacrimal gland (20, 33) has some form of basement membrane investment. For many years,
basement membrane was viewed simply as a supportive structure. Indeed,
enzymatic dissolution of basement membrane is a prerequisite for acinar
cell dispersion (9). Molecular studies now reveal this thin, resilient
extracellular membrane to be a complex, adhesive gel of laminins,
collagen IV, perlecan, growth factors, and other components both known
and unknown (28).
Particular attention has been paid to mechanisms and consequences of
cellular adhesion to basement membrane, an interaction linked in part
to differentiation of epithelial and other cell types (8).
Differentiation of mammary epithelial cells, as manifested by the
quantity and nature of constitutive casein secretion, is basement
membrane dependent (25). Could the same be true of the multistep
regulated secretory pathway in excitable cells, such as lacrimal acinar
cells? Extrapolation from descriptive studies in pancreas (4) suggest
that development of regulated secretion is a complex process. Cell
polarization coincides with the appearance of protein synthetic and
signal transduction machinery, surface agonist receptors, and
expression of secretory proteins and, as a consequence, the capacity to
release nascent secretory proteins by the regulated secretory pathway.
Previously, we searched for differentiation factors that may be
residents of lacrimal acinar basement membranes (14). Advantage was
taken of a novel basement membrane substrate (referred to as BMS; Ref.
17). Adhesion to BMS was sufficient to reverse the characteristic
propensity of isolated lacrimal acinar cells to lose secretagogue
responsiveness in culture. Combined use of fractionated BMS and
anti-BMS monoclonal antibodies led to the identification of BM180, a
novel lacrimal and parotid gland basement membrane protein with
cell adhesion activity that appeared to act in the
presence of laminin-1 to promote stimulus-secretion coupling (14). Here
we explore which laminin-1 sites may be active and whether other
extracellular molecules have a modulatory role in regulated secretion
by lacrimal acinar cells.
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METHODS |
Preparation of substrate.
A 10 mM EDTA extract of Engelbreth-Holm-Swarm (EHS) tumor
matrix (BMS) was prepared as previously described (17). BMS contains numerous components including laminin-1, BM180 (14), and collagen IV.
Purified laminin-1 was kindly provided by Dr. Roy Ogle (University of
Virginia). BMS was diluted with DMEM and added at 0.044-0.06, 0.177, 0.354, 0.531, or 0.708 mg/cm2 to 96- or 48-well culture
plates kept chilled on ice. Laminin-1 and also collagen I and Matrigel
(both purchased from Collaborative Research, Bedford, MA) were
similarly coated at 0.531 mg/cm2.
For ELISA examination of basement membrane synthesis by collagen I-adherent cells, a 0.06 mg/cm2
collagen coating was used. Concentrations of 0.044-0.06
mg/cm2 gave rise to a monolayer.
Concentrations >0.044-0.06
mg/cm2 formed a gel when incubated
for 1 h at 37°C.
Antibodies and synthetic peptides.
Antibody and peptide reagents utilized are summarized in Table
1. All antibodies, except those directed
against entactin, were purified on protein A Sepharose (Pharmacia Fine
Chemicals, Piscataway, NJ) or G protein agarose (Pierce, Rockford, IL)
or, alternatively, were purchased in purified form. Rabbit antibody concentration was determined using the extinction coefficient for
rabbit IgG (13.5 [A1%1cm280]).
Chain-specific anti-laminin-1 antibodies were provided by Dr. Y. Yamada
(National Institute of Dental Research, Bethesda, MD). Chain
specificity was independently verified (17). Anti-entactin antisera
(6), including a negative control anti-glutathione S-transferase antiserum,
were supplied by Dr. A. Chung (University of Pittsburgh, Pittsburgh,
PA). A polyclonal anti-rat
1-integrin antiserum (2) was
provided by Dr. T. Borg (University of South Carolina, Columbia, SC).
ELISA detection of BM180 was performed with monoclonal antibody (MAb)
3E12 (14).
Isolation of lacrimal acinar cells.
Lacrimal acinar cells were isolated from 4- to 6-wk-old male
Sprague-Dawley rats, as previously described (14). Briefly, an
intracardiac wash of the vasculature with DMEM was followed by mincing
and incubation in alternating solutions of EDTA and enzyme (9, 23).
Released single acinar cells were collected by passage through
sequential 160-µm and 25-µm Nitex filters followed by
centrifugation on a discontinuous 10%, 30%, 60% Percoll gradient. Acinar cells sediment at the interface between 30 and 60%. Cell viability was 77-95%, as assessed by trypan blue exclusion. Cells were plated at 0.5-2.8 × 105
cells/cm2 on BMS, laminin-1,
collagen I, or Matrigel in serum-containing medium (MOM; Ref. 9). Cells
were also plated at 0.3-3.5 × 105
cells/cm2 on BMS in serum-free MOM
(SFMOM; Ref. 9) medium. MOM consisted of high-glucose DMEM containing
dexamethasone (1 ng/ml), putrescine (1 mM), epidermal growth factor
(EGF; 50 ng/ml), L-ascorbic acid (25 µg/ml), insulin (6 µg/ml), transferrin (6 µg/ml), selenous acid (6 ng/ml), reduced glutathione (10 µg/ml), HEPES (15 mM), gentamicin (50 µg/ml), and heat-inactivated fetal calf serum (10%). SFMOM was identical to MOM, except for the omission of calf serum and
addition of basic fibroblast growth factor (bFGF; 100 ng/ml). In
inhibition studies, gelled BMS was incubated with DMEM-diluted anti-laminin-1, anti-entactin or anti-growth factor antibodies (10-100 µg/100 µl) for 1-2 h at 37°C and washed two
times with DMEM before addition of freshly isolated cells. In other
experiments, freshly isolated cells were preincubated with
anti-
1-integrin antibodies
(10-100 µg/100 µl) or synthetic peptide (100 µM) for 1 h at
37°C. Cells in the presence of antibody or peptide were then plated
on BMS.
Secretion assay.
The tear protein peroxidase was used as a marker of constitutive and
regulated secretion. Cultures were gently washed twice overnight
(18-24 h), and fresh medium was added. Constitutively secreted
peroxidase was then collected for 100 min. Peroxidase secreted by the
regulated secretory pathway was collected in the same manner following
replacement with fresh medium to which had been added carbachol
(10
4 M) and vasoactive
intestinal peptide (VIP;
10
8 M). Cultures were
terminated by digestion of BMS with dispase (40 U/ml; Collaborative
Research) in the presence of 0.1% trypsin and 1.1 mM EDTA (GIBCO BRL,
Grand Island, NY). Released cells were collected and retained for
determination of total cellular peroxidase and DNA (14). Quantitation
of secreted peroxidase was determined using the method of Herzog et al.
(10). All secretion values were normalized to cellular DNA.
ELISA.
Presence of laminin-1 and BM180 in overnight collagen-adherent cultures
of mouse lacrimal acinar cells (2 × 105
cells/cm2) were detected by
ELISA. Isolation of mouse lacrimal cells was similar to that used for
rats, with the omission of the Percoll gradient and Nitex filtering.
Cultures, or collagen-coated (0.06 mg/cm2) wells without cells,
were PBS washed, fixed with 4% formaldehyde, and then PBS washed and
blocked with PBS containing 1% BSA and 0.01% Tween 20. Subsequent
steps, including incubation with antibodies against intact mouse
laminin-1 or with anti-BM180 MAb 3E12 (14), detection, and analysis,
were all carried out using a standard ELISA format, as previously
described (14). Mouse lacrimal acinar cells were used to avoid
background staining between the anti-rat secondary antibody (required
to detect 3E12) and rat cells.
Statistical analysis.
All values are expressed as means ± SE. Student's
t-test was used where noted to assess
statistical significance.
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RESULTS |
Lacrimal acinar cells freshly released from their native basement
membranes adhere to BMS, a 10 mM EDTA extract of mouse EHS tumor
basement membrane (Fig. 1), and after 24 h
in culture remain capable of secreting tear proteins in a physiological
manner (Fig. 2; Ref. 14). In
contrast, cells on plastic are poorly adherent and generally lose much
of their capacity to respond to carbachol and VIP (Fig.
2A). Regulated secretion remains
constant at 40-50 mU tear peroxidase/µg cellular DNA,
irrespective of the amount of underlying BMS (Fig.
2A; 41 ± 1 mU/µg,
n = 62, for 0.531 mg/cm2), suggesting that key
components are saturating at the lowest levels applied, even in the
absence of the gelled configuration (< 0.177 mg/cm2) common to native
basement membranes. Acinar cells on gelled collagen I were equally
responsive (Fig. 2B). This result is
a manifestation of the commonly observed propensity of epithelial cells
to elaborate native basement membranes soon after adhesion to
collagen I (Fig. 3; Ref. 25).
Indeed, synthesis of both laminin-1 and BM180 could be readily detected
(Fig. 3) by ELISA. Lower secretory levels were observed for cells
cultured on gelled Matrigel (Fig. 2B) or purified laminin-1 (Fig.
2B; Ref. 14). Both constitutive and
regulated tear secretion were serum enhanced but not serum dependent
(constitutive secretion 4.4 ± 0.8 and 2 ± 0.7 mU/µg; regulated secretion 41 ± 1 and 11.2 ± 1.7 mU/µg, for
serum-containing and serum-free media respectively;
n = 40-62). Carbachol-stimulated secretion was dose dependent and atropine inhibitable (Table
2).

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Fig. 1.
Appearance of rat lacrimal acinar cells on basement membrane substrate
(BMS). Freshly isolated cells just after plating
(A and
B) or 24 h later
(C and
D). Cells cultured for 24 h are
adherent and display numerous secretory granules (small raised bumps)
concentrated in apical cytoplasm. Secretory granules contain a
number of tear proteins, including peroxidase. Cells were cultured in
serum-containing medium (MOM) on BMS (0.531 mg/cm2). Similar culture
conditions were used in experiments represented in Figs.
2-7, except as noted. Bars, 100 µm (for
A and
C) and 20 µm (for
B and
D).
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Fig. 2.
Regulated secretory pathway is particularly active in BMS- and collagen
I-adherent lacrimal acinar cells. A:
adhesion to BMS enhances capacity for regulated peroxidase secretion in
a dose-independent manner between 0.044 and 0.708 mg/cm2. Plastic-adherent cells (0 mg/cm2) display lower secretion,
an activity attributable to presence of serum. At concentrations
>0.044 mg/cm2, BMS is in form of
a gel. Values represent 7 (0-0.354 and 0.708 mg/cm2) or 62 (0.531 mg/cm2) independent
measurements. B: use of gelled
collagen I (Coll; 0.531 mg/cm2)
or Matrigel (Mg; 0.531 mg/cm2)
as adhesion substrate is equally (collagen I;
P = 0.8), or less (Matrigel;
P < 0.001) effective. Gelled
laminin-1 (Ln; 0.531 mg/cm2)-adherent cells display
less capacity for regulated secretion
(P < 0.001). Occasional batches (not
shown) of possibly BM180- and/or collagen IV-contaminated
laminin-1 are equally active. Values represent 7 (collagen I), 10 (laminin-1), or 19 (Matrigel) independent measurements. Here and in
Figs. 4-6, except as noted, regulated peroxidase secretion was
assessed after stimulation with
10 4 M carbachol and
10 8 M vasoactive intestinal
peptide; cumulative secretion over 100 min was normalized to µg of
cellular DNA.
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Fig. 3.
Elaboration of laminin-1 and BM180 by collagen I-adherent lacrimal
acinar cells (2 × 105
cells/cm2). Overnight cultures
of mouse lacrimal acinar cells (solid bars), or cell-free collagen
I-coated wells incubated overnight with serum-containing medium (open
bars), were formaldehyde fixed, blocked, and then immunostained with
antibodies against intact mouse laminin-1 (Ab LN) or mouse BM180 (Ab
BM180) using an ELISA approach. Ctrl, result when primary antibody was
omitted. Wells were coated with 0.06 mg/cm2 of collagen I. Use of rat
lacrimal cells gave rise to background staining with anti-rat secondary
antibody required to detect Ab BM180. This problem did not exist for
detection of rabbit Ab LN, for which an identical level of basement
membrane synthesis could be detected. OD, optical density.
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Table 2.
Dose response effect of carbachol with or without VIP and atropine
on peroxidase secretion by BMS-adherent lacrimal acinar cells
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Anti-laminin-1 and anti-collagen IV antibodies suppress regulated
secretion.
Previous studies suggested that BM180 and laminin-1 together played key
roles in promoting stimulus-secretion coupling in BMS-adherent lacrimal
acinar cells (14). To determine which laminin-1 site(s) was involved
and whether other basement membrane-associated molecules contributed,
an antibody blocking approach was used (Fig.
4; Tables 3
and 4). Antibodies against intact laminin-1 partially suppressed regulated secretion by 26% (Fig.
4A), an effect that was reproduced
with anti-
1 (35%) and anti-
1 (33%) but not with two different
anti-
1, laminin chain-specific antibodies (Fig.
4B). Anti-collagen IV antibodies
tested in parallel were also partially inhibitory (37%; Fig.
4A). Constitutive secretion was
unaffected (Fig. 4A,
inset). These values are similar to
the level of inhibition (30-40%) commonly observed in the
presence of anti-BM180 MAb 3E12 (14). In contrast, domain-specific
anti-entactin antibodies (Table 3) and several different anti-growth
factor antibodies (Table 4) had no effect.

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Fig. 4.
Anti-laminin-1 and anti-collagen IV antibodies partially inhibit
BMS-enhanced regulated secretion. A:
preincubation of BMS with antibodies against intact laminin-1 (Ab LN)
or collagen (Ab IV) suppresses capacity for regulated secretion (solid
symbols) in a dose-dependent manner. Values are expressed as % of
no-antibody control and are means ± SE of 5 independent
measurements; t-test comparing BMS vs.
BMS + Ab LN or BMS + Ab IV at 50 µg/well revealed a
P < 0.001. Inset: there was little or no effect
of Ab LN and Ab IV on constitutive secretion (Unstim; open symbols);
t-test with same format as above
indicated no significant difference (P = 0.24-0.27). Constitutive secretion values were 116 ± 16 and
114 ± 18% for Ab LN and Ab IV, respectively, at 100 µg/well.
Values are means ± SE of 5 independent measurements.
B: association of laminin-1 activity
with 2 distinct domains. Preincubation of BMS with antibodies against
1 chain YIGSR site (Ref. 7; amino acids 925-933; Ab 1) or
carboxy-terminal 1 chain site (amino acids 1420-1439; Ab 1)
partially inhibits regulated secretion. Not inhibitory were
neutralizing antibodies against carboxy-terminal SN-peptide site (Ref.
17; 1 chain amino acids 2179-2198; Ab 1[SN]) or
IKVAV site (Ref. 27; 1 chain amino acids 2097-2108; Ab
1[IK]). Values are means ± SE of 5 independent
measurements; t-test comparing BMS vs.
BMS + Ab 1 or BMS + Ab 1 revealed a
P < 0.003;
t-test comparing BMS vs. BMS + Ab
1[SN] or BMS + Ab 1[IK] showed no
significant difference (P = 0.95 and
0.21, respectively).
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Lack of effect of
anti-
1-integrin and soluble
RGD peptide on regulated secretion.
Although information is lacking on lacrimal acinar cells, cellular
adhesion to laminin-1 and collagen IV is most commonly mediated by
1-integrins. Four notable
exceptions include
6
4,
V
3,
-dystroglycan, and the putative 67/32-kDa laminin-1 receptor (19).
The BM180 receptor(s) is unknown. To examine whether blocking cell
surface
1-integrins would in
turn affect stimulus-secretion coupling, acinar cells were cultured
overnight in the presence of
anti-
1-integrin antibody (Fig.
5). Regulated and constitutive secretion,
assessed 24 h later, were unaffected (Fig.
5A). An alternative strategy is to
interfere with RGD-dependent adhesion mechanisms [i.e.,
V
3-laminin-1
(13),
5
1-fibronectin
(11), and
IIb
3-fibrinogen
(12)] by plating cells in the presence of soluble GRGDS synthetic
peptide. Use of GRGDS peptide had no effect. Curiously, the control
peptide GRGESP was slightly inhibitory (Fig.
5B).

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Fig. 5.
Anti- 1-integrin antibody and
RGD peptide have no apparent effect on either regulated (solid bars) or
constitutive (open bars) secretory pathways.
A: preincubation of
anti- 1-integrin antibody with
acinar cells, followed by 24-h culture on BMS in presence of antibody,
does not interfere with secretory capacity. Similar results were
obtained in serum-free cultures (not shown). Values represent 3 (50 µg) or 6 (10, 100 µg) independent measurements.
B: incubation with RGD peptide (100 µM) has no effect on regulated secretion, whereas some slight
suppression appears to occur with control RGE peptide
(P = 0.037). Values are means ± SE
of 4 independent measurements.
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Acinar cells produce a secretion-enhancing factor(s).
In addition to tear proteins, lacrimal acinar cells may produce
insoluble (i.e., basement membrane) and soluble materials that could
help establish a suitable microenvironment for regulated secretory
function. To examine this possibility, freshly isolated cells were
plated at increasing density on BMS, and secretion was assessed after
normalization to cellular DNA (Fig. 6).
Normalized regulated secretion increased with plating number in
serum-free cultures, whereas constitutive secretion remained constant
(Fig. 6A). Addition of serum (Fig.
6B) masked this effect. Cellular aggregation in serum-free cultures was minimal (Fig.
7), suggesting that reformation of native
acinar cell-like structures could not serve as a mechanism for enhanced
secretion. These results are interpreted as tentatively revealing the
presence of an autocrine secretion-enhancing factor(s) that
specifically acts on the regulated secretory pathway to augment
stimulus-secretion coupling.

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Fig. 6.
Presence of a putative autocrine enhancing activity (or activities) is
revealed by culturing BMS-adherent acinar cells at increasing density
without serum. A: in serum-free
medium (SFMOM) capacity for regulated (Stim; solid symbols), but not
constitutive (Unstim; open symbols), secretion is cell number
dependent. Similar results were obtained whether cells were adherent to
0.044 (squares) or 0.531 (circles)
mg/cm2 BMS. Values were normalized
to µg cellular DNA and are means ± SE of 3 (1.1, 1.8, and 2.1 × 105
cells/cm2), 6 (2.8 × 105
cells/cm2), or 12 (1.4 × 105
cells/cm2) independent
measurements. B: inclusion of serum in
medium (MOM) masked or eliminated this effect. Values are means ± SE of 2 (1.1 and 2.1 × 105
cells/cm2) or 3 (all others)
independent measurements.
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Fig. 7.
Appearance of overnight serum-free cultures plated at increasing
density on BMS. A: 0.35 × 105
cells/cm2.
B: 0.7 × 105
cells/cm2.
C: 1.4 × 105
cells/cm2.
D: 2.1 × 105
cells/cm2.
E: 2.8 × 105
cells/cm2.
F: 3.5 × 105
cells/cm2. Cells were adherent to
0.06 mg/cm2 BMS. Cells remain, for
the most part, dispersed. In contrast, serum-containing cultures tend
to display density-dependent aggregation (for example, Fig.
1C), a phenomenon that is not linked
to enhanced secretion (Fig. 6B).
Bar, 200 µm.
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DISCUSSION |
Development of the regulated secretory pathway is an intricate example
of cellular differentiation possibly activated by adhesive signaling
between the exocrine acinar cell and component(s) of the acinar cell
basement membrane. Use of antibodies and serum-free or serum-containing
lacrimal acinar cell cultures has here resulted in the identification
of basement membrane collagen IV, two sites in laminin-1, and an
unidentified autocrine factor(s) as likely playing contributory roles
in the maintenance of the regulated secretory pathway in vitro.
Advantage was taken of the observation that gelled BMS supported
stimulus-secretion coupling by overnight cultures of lacrimal acinar
cells (14). Dissection of BMS by gel filtration previously identified a
novel lower-molecular-weight activity defined by a monoclonal antibody
as BM180, whose function appeared to be dependent on laminin-1 (14).
The nature of laminin-1 active site(s) and the possible presence of
accessory activators of stimulus-secretion coupling were investigated
by plating lacrimal acinar cells overnight on BMS in the presence of
anti-laminin
1 and
1 chain or anti-collagen IV antibodies. Each
reduced regulated secretion by 33-40% but left secretion by the
constitutive pathway untouched. Not inhibitory were a diverse panel of
antibodies with specificities for two laminin
1 chain sites; three
entactin globules and a bridging domain; the growth factors bFGF, tumor
necrosis factor-
, EGF, insulin-like growth factor (IGF) II, and
transforming growth factor (TGF)-
; and
1-integrins.
These results are in keeping with the concept that stimulus-secretion
coupling is dependent on a highly selective group of basement membrane
molecules that, through receptor ligation, may upregulate one or
several elements in the coupling pathway(s). Noteworthy was the
association of apparent coupling activity with laminin
1 and
1
chains, rather than the strongly adhesive
1 chain G domain through
which most cellular attachment to laminin-1 occurs (19, 26). The
1
chain epitope, located near the laminin-1 cross region (amino acids
929-933), curiously includes the YIGSR sequence that was
previously thought to be a major cell adhesion site via its apparent
interaction with a 32/67-kDa nonintegrin receptor (7). More recent
studies attribute cell adhesion activity of the cross region to a
cryptic RGDS site in the
1 chain, which serves as ligand for the
V
3
integrin receptor (13). The inability of soluble RGDS (and of an
anti-
1-integrin antibody) to
inhibit regulated secretion underlines the apparent complexity of this region and offers an opportunity to reexplore the possible functional significance of the YIGSR site or adjacent sites that might have been
partially blocked by anti-YIGSR peptide antibody. How the
1 chain
epitope (amino acids 1420-1439), with differing location (carboxy-terminal E8 region) and sequence, contributes to the role of
laminin-1 in regulated secretion and whether it and/or the
1
chain are capable of synergy with BM180 remains to be determined. When
the lack of
1-integrin or RGD
involvement and the apparent requirement for cellular contact with
BM180 and two uncommon sites in laminin-1 are taken together, it
appears that developmental activation of stimulus-secretion coupling
may be the main responsibility of supplemental, rather than primary,
adhesion mechanisms. Such an arrangement could subtly modulate acinar
cell function in the absence of detachment-associated apoptosis.
A striking observation was the augmentation of normalized regulated,
but not constitutive, secretion as lacrimal acinar cell plating density
was increased under serum-free conditions. Microscopic examination of
cultures suggested that enhancement was not due to the formation of
higher order cellular aggregates, such as acini, the appearance of
which (29) may depend on the presence of contaminating cellular
aggregates at the time of plating. Curiously, serum seemed to promote
density-dependent aggregation, yet secretion remained constant. What is
the identity of this previously undescribed secretion-enhancing
activity, which is presumed to be of lacrimal acinar cell origin
(although contribution from a contaminating mesenchymal or ductal cell
cannot be excluded)? From its earliest stages of development, the
lacrimal gland is influenced by a complex and dynamic array of soluble
and matrix signals. Thought to be destined mainly for the corneal
surface are a number of lacrimal gland-derived growth factors expressed
by acinar cells [EGF, TGF-
, and TGF-
(30, 32)],
ductal cells [EGF (22)], or periacinar connective tissue
cells [hepatocyte growth factor (HGF) (15)]. bFGF and
receptor mRNA for bFGF and HGF (15) have also been detected but with
unknown cellular origin. In addition, both the growth factor-rich serum
transudate common to all tissues and BM180 in the lacrimal acinar cell
basement membrane are potential or demonstrated (BM180) modulators of
tear protein secretion. Lacrimal EGF expression is first
detected 2 wk after birth, in keeping with its apparent role in eyelid
opening. In nonocular tissues, TGF-
stimulates epithelial,
endothelial, and fibroblast proliferation, and HGF is involved in
aggregation morphogenesis (1). bFGF could have multiple influences,
including epithelial differentiation and promotion of blood vessel
growth (31).
Many of these factors tentatively appear to play little or no role in
the development of stimulus-secretion coupling by lacrimal acinar
cells. Although exogenous bFGF, but not EGF or IGF-I, has been reported
to stimulate regulated amylase secretion by cultured pancreatic acini
using an atropine-insensitive pathway (3), inclusion of bFGF or EGF in
our serum-free lacrimal acinar cell cultures is insufficient to support
regulated secretion at low plating densities. TGF-
and
platelet-derived growth factor have been respectively detected at 8.5 ng/ml and 64 pg/ml in BMS (18), among the few growth factors surveyed.
The compositionally similar Matrigel contains, in addition, bFGF (1 ng/ml) and IGF-I (7 ng/ml) (16). Yet, increasing BMS 12-fold or
preincubating BMS with neutralizing anti-TGF-
, -EGF, -bFGF, or
-IGF-II antibodies had no effect on regulated secretion. Lack of a BMS
dose response also argues against the candidacy of BM180, laminin-1, or
collagen IV, although one might suggest that the cellular surface of
gelled vs. coated BMS may not differ in terms of molar levels of
available matrix-bound components. Other candidate growth factors
remain that could possibly support regulated secretion. Because serum masked the effect, it may be an alternative source of the same active
factor(s). Experiments testing whether the activity (or activities) is
soluble (via use of conditioned media) or, alternatively, deposited
onto the BMS coat are needed. When all the data are taken
together, it is apparent that the regulated secretory pathway is
modulated by multiple environmental influences. At the level of the
acinar cell-basement membrane interface, there exists BM180, laminin-1,
and collagen IV, which appear to act in a
1-integrin-independent manner.
Also acting on the cells is a previously undescribed
secretion-enhancing activity (or activities), presumably derived from
lacrimal acinar cells either as a soluble factor or as a matrix factor,
which could possibly synergize with basement membrane to promote
lacrimal acinar cell differentiation and regulated secretory function.
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ACKNOWLEDGEMENTS |
We gratefully acknowledge Drs. Albert Chung, Tom Borg, Roy Ogle,
and Yoshi Yamada for protein and antibodies.
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FOOTNOTES |
This work was supported by National Eye Institute Grant EY-09747 (to G. W. Laurie). J. D. Glass was supported in part by an institutional grant
from the Juvenile Diabetes Foundation.
Present addresses: L. Chen, Div. of Nephrology, Dept. of Internal
Medicine, University of Virginia, Charlottesville, VA 22908; J. D. Glass, Dept. of Anesthesiology, University of Virginia, Charlottesville, VA 22908.
Address for reprint requests: G. W. Laurie, Department of Cell Biology,
Box 439, Health Sciences Center, University of Virginia,
Charlottesville, VA 22908.
Received 5 February 1997; accepted in final form 15 April 1998.
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REFERENCES |
1.
Brinkmann, V.,
H. Foroutan,
M. Sachs,
K. M. Weidner,
and
W. Birchmeier.
Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells.
J. Cell Biol.
131:
1573-1586,
1995[Abstract].
2.
Carver, W.,
I. Molano,
T. A. Reaves,
T. K. Borg,
and
L. Terracio.
Role of the alpha 1 beta 1 integrin complex in collagen gel contraction in vitro by fibroblasts.
J. Cell. Physiol.
165:
425-437,
1995[Medline].
3.
Chandrasekar, B.,
and
M. Korc.
Basic fibroblast growth factor is a calcium-mobilizing secretagogue in rat pancreatic acini.
Biochem. Biophys. Res. Commun.
177:
166-170,
1991[Medline].
4.
Chang, A.,
and
J. D. Jamieson.
Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin.
J. Cell Biol.
103:
2353-2356,
1986[Abstract].
5.
Chen, L.,
V. Shick,
M. L. Matter,
S. M. Laurie,
R. C. Ogle,
and
G. W. Laurie.
Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L494-L503,
1997[Abstract/Free Full Text].
6.
Dong, L.-J.,
J.-C. Hsieh,
and
A. E. Chung.
Two distinct cell attachment sites in entactin are revealed by amino acid substitutions and deletion of the RGD sequence in the cysteine-rich epidermal growth factor repeat 2.
J. Biol. Chem.
270:
15838-15843,
1995[Abstract/Free Full Text].
7.
Graf, J.,
Y. Iwamoto,
M. Sasaki,
G. R. Martin,
H. K. Kleinman,
F. A. Robey,
and
Y. Yamada.
Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding.
Cell
48:
989-996,
1987[Medline].
8.
Gumbiner, B. M.
Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell
84:
345-357,
1996[Medline].
9.
Hann, L. E.,
R. S. Keller,
and
D. A. Sullivan.
Influence of culture conditions on the androgen control of secretory component production by acinar cells from the rat lacrimal gland.
Invest. Ophthalmol. Vis. Sci.
32:
2610-2621,
1991[Abstract].
10.
Herzog, V.,
H. Sies,
and
F. Miller.
Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation.
J. Cell Biol.
70:
692-706,
1976[Abstract].
11.
Hynes, R. O.
Integrins: versatility, modulation and signalling in cell adhesion.
Cell
69:
11-25,
1992[Medline].
12.
Kieffer, N.,
and
D. R. Phillips.
Platelet membrane glycoproteins: functions in cellular interaction.
Annu. Rev. Cell Biol.
6:
329-357,
1990.
13.
Kramer, R. H.,
Y.-F. Cheng,
and
R. Clyman.
Human microvascular endothelial cells use
1 and
3 integrin receptor complexes to attach to laminin.
J. Cell Biol.
111:
1233-1243,
1990[Abstract].
14.
Laurie, G. W.,
J. D. Glass,
R. A. Ogle,
C. M. Stone,
J. R. Sluss,
and
L. Chen.
"BM180": a novel basement membrane protein with a role in stimulus-secretion coupling by lacrimal acinar cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1743-C1750,
1996[Abstract/Free Full Text].
15.
Li, Q.,
J. Weng,
R. R. Mohan,
G. L. Bennett,
R. Schwall,
Z.-F. Wang,
K. Tabor,
J. Kim,
S. Hargrave,
K. H. Cuevas,
and
S. E. Wilson.
Hepatocyte growth factor and hepatocyte growth factor receptor in the lacrimal gland, tears, and cornea.
Invest. Ophthalmol. Vis. Sci.
37:
727-739,
1996[Abstract].
16.
Mannuzza, F. J.
Removal of soluble growth factors from matrigel basement membrane matrix and the demonstration and quantitation of insoluble, matrix-bound TGF-beta (Abstract).
Mol. Biol. Cell
3:
226a,
1992.
17.
Matter, M. L.,
and
G. W. Laurie.
A novel laminin E8 cell adhesion site required for lung alveolar formation in vitro.
J. Cell Biol.
124:
1083-1090,
1994[Abstract].
18.
Matter, M. L.,
and
G. W. Laurie.
A putative sub-10-kDa basement membrane activity required for lung alveolar formation in vitro.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L489-L494,
1996[Abstract/Free Full Text].
19.
Mercurio, A. M.
Laminin receptors achieving specificity through cooperation.
Trends Biochem. Sci.
5:
419-423,
1995.
20.
Mircheff, A. K.,
D. W. Warren,
and
R. L. Wood.
Hormonal support of lacrimal function, primary lacrimal deficiency, autoimmunity, and peripheral tolerance in the lacrimal gland.
Ocul. Immunol. Inflamm.
4:
145-172,
1996.
21.
Nomizu, M.,
W. H. Kim,
A. Yamamura,
S.-Y. Song Utani,
A. Otaka,
P. R. Roller,
H. K. Kleinman,
and
Y. Yamada.
Identification of cell binding sites in the laminin
1 chain carboxyl-terminal globular domain by systematic screening of synthetic peptides.
J. Biol. Chem.
270:
20583-20590,
1995[Abstract/Free Full Text].
22.
Obata, H.,
H. Horiuchi,
Y. Dobashi,
T. Oka,
M. Sawa,
and
R. Machinami.
Immunohistochemical localization of epidermal growth factor in human main and accessory lacrimal glands.
Jpn. J. Ophthalmol.
37:
113-121,
1993[Medline].
23.
Oliver, C.,
J. F. Waters,
C. L. Tolbert,
and
H. K. Kleinman.
Growth of exocrine acinar cells on a reconstituted basement membrane gel.
In Vitro Cell. Dev. Biol.
23:
465-473,
1987[Medline].
24.
Sephel, G. C.,
K. I. Tashiro,
M. Sasaki,
D. Greatorex,
G. R. Martin,
Y. Yamada,
and
H. K. Kleinman.
Laminin A chain synthetic peptide which supports neurite outgrowth.
Biochem. Biophys. Res. Commun.
162:
821-829,
1989[Medline].
25.
Streuli, C. H.,
and
M. J. Bissell.
Expression of extracellular matrix components is regulated by substratum.
J. Cell Biol.
110:
1405-1415,
1990[Abstract].
26.
Sung, U.,
J. J. O'Rear,
and
P. D. Yurchenco.
Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein.
J. Cell Biol.
123:
1255-1268,
1993[Abstract].
27.
Tashiro, K.,
G. C. Sephel,
B. Weeks,
M. Sasaki,
G. R. Martin,
H. K. Kleinman,
and
Y. Yamada.
A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth,
J. Biol. Chem.
264:
16174-16182,
1989[Abstract/Free Full Text].
28.
Timpl, R.,
and
J. C. Brown.
Supramolecular assembly of basement membranes.
Bioessays
18:
123-132,
1996[Medline].
29.
Vanaken, H.,
I. Vercaeren,
F. Claessens,
R. De Vos,
C. Dewolf-Peeters,
J. P. Vaerman,
W. Heyns,
W. Rombauts,
and
B. Peeters.
Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.
Exp. Cell Res.
238:
377-388,
1998[Medline].
30.
Van Setten, G. B.,
S. Macauley,
M. Humphreys-Beher,
N. Chegini,
and
G. Schultz.
Detection of transforming growth factor-
mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-
in human tears.
Invest. Ophthalmol. Vis. Sci.
37:
166-173,
1996[Abstract].
31.
Vlodavsky, I.,
J. Folkman,
R. Sullivan,
R. Fridman,
R. Ishai-Michaeli,
J. Sasse,
and
M. Klagsbrun.
Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.
Proc. Natl. Acad. Sci. USA
84:
2292-2296,
1987[Abstract].
32.
Yoshino, K.,
R. Garg,
D. Monroy,
Z. Ji,
and
S. C. Pflugfelder.
Production and secretion of transforming growth factor beta (TGF-
) by the human lacrimal gland.
Curr. Eye Res.
15:
615-624,
1996[Medline].
33.
Zoukhri, D.,
and
D. A. Dartt.
Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium.
Am. J. Physiol.
268 (Cell Physiol. 37):
C713-C720,
1995[Abstract/Free Full Text].
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