1 Department of Biological Sciences, Purdue University, West Lafayette, IN
47907-1392, USA
2 Departments of Paediatrics and Physiology and Pharmacology, Child Health
Research Institute, University of Western Ontario, London, Ontario N6C 2V5,
Canada
* Author for correspondence (e-mail: sfk{at}bilbo.bio.purdue.edu)
Accepted 23 April 2003
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Summary |
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Key words: Pancreas, Exocytosis, bHLH, Transcription, Cell communication
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Introduction |
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Gap junctions are trans-membrane protein structures that allow passage of
small solutes between adjacent cells
(Kumar and Gilula, 1996). Each
gap junction consists of a plaque of connexons, which are composed of
hexameric complexes of connexin proteins. Nineteen different connexin genes,
each with a specific tissue distribution, exist in the mouse
(Willecke et al., 2002
). The
exocrine pancreas expresses connexin32 (Cx32; also referred
to as Gjb1) and connexin26 (Cx26; also referred to
as Gjb2), which is typical of most serous exocrine cells, including
cells of the salivary glands, seminal vesicles and lacrimal glands
(Hsieh et al., 1991
;
Meda et al., 1993
). The
existence of many different connexins with unique tissue specificities
highlights the ability of gap junctions to provide a selective permeability
between cells. Specific connexin expression patterns, coupled with various
combinations of hexameric junctions and states of protein phosphorylation,
contribute to a highly regulated process of intercellular communication
(Goodenough et al., 1996
;
Lampe and Lau, 2000
).
Stimulated acinar cells utilize gap junctions to communicate with adjacent
cells in a common acinus to carefully regulate and coordinate the secretion of
their protein products. Loss of connexin expression leads to increased levels
of basal and stimulated exocytosis in acinar cells
(Chanson et al., 1998
),
confirming the importance of gap junctions in the exocytosis pathway.
Although the expression and function of gap junctions are well
characterized, the molecular regulatory circuits that control the spatial and
temporal expression patterns of the connexin genes are not known. Elucidating
these regulatory factors is of significant interest since much of the function
associated with a specific gap junction complex is dependent on the types and
amounts of connexins it contains (Hill et
al., 2002; Niessen et al.,
2000
; Niessen and Willecke,
2000
). Expression of the connexin gene family is probably
regulated by key transcription factors that exhibit similar patterns of
spatial expression. As previously mentioned, the Cx32 and
Cx26 genes are co-expressed in cells known to exhibit regulated
exocytosis (Meda et al.,
1993
). One transcription factor that shares a similar, but not
identical, expression pattern is the basic helix-loop-helix (bHLH) protein
Mist1 (Pin et al., 2000
).
Members of the bHLH family are instrumental in the development of numerous
organ systems, and gene ablation studies in mice have revealed an essential
role for bHLH proteins in the development of different cell types within the
pancreas (Jenny et al., 2002
;
Kageyama et al., 2000
;
Krapp et al., 1998
). Mice
deficient in BETA2/NeuroD or neurogenin3 fail to develop
insulin-producing ß-cells, develop diabetes and die within a few days of
birth (Jenny et al., 2002
;
Naya et al., 1997
). Similarly,
targeted disruption of the exocrine-specific factor PTF1-p48 results in a
complete lack of exocrine cells within the pancreas, causing early postnatal
death (Kawaguchi et al., 2002
;
Krapp et al., 1998
).
Deletion of the Mist1 gene (Mist1KO), while
not lethal, causes disorganization in acinus formation and numerous defects in
pathways controlling regulated exocytosis
(Pin et al., 2001). These
include altered expression of genes involved in calcium mobilization
(IP3 Receptor Type 3) and secretagogue
signaling (CCK A receptor). In an effort to establish if there is a
relationship between gap junction communication and the
Mist1KO phenotype, we examined the expression and function
of connexin molecules in Mist1KO acinar cells. In this
report, we show that Mist1KO acini are deficient in gap
junction-mediated intercellular communication, mainly due to the loss of
Cx32 expression. At all time points examined, Cx32 mRNA and
protein levels are greatly reduced in the Mist1KO exocrine
pancreas, as well as in all exocrine organs that normally express the
Mist1 gene. Although Mist1KO acinar cells
continue to express the Cx26 gene, Cx26 protein does not accumulate
in gap junction plaques, suggesting that Cx32 is required for stable
incorporation of Cx26 into the membrane. Additional studies using pancreatic
acinar cell lines, transgenic mice and Mist1 expression plasmids have
confirmed that Mist1 transcriptionally regulates expression of the
Cx32 gene. We conclude that Mist1 functions as a positive regulator
of Cx32 gene expression and, in the absence of Mist1, acinar cell gap
junctions and intercellular communication pathways become disrupted.
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Materials and Methods |
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Acini isolation
Pancreatic acini were obtained following standard collagenase protocols
(Ohnishi et al., 1997).
Briefly, animals were euthanized and the pancreas was removed and placed into
KRB (0.1 M NaCl, 1 mM MgCl2, 5 mM KCl, 0.5 mM
Na2HPO4, 33 mM NaHCO3, 0.5 mM
CaCl2, 4 mg/ml glucose, 15 mg/ml glutamine and MEM amino acids)
buffer containing 0.1 mg/ml trypsin inhibitor. Pancreatic tissue was injected
with a collagenase solution (100 U/ml in KRB plus 2.5 mg/ml BSA), gassed with
O2 and incubated at 37°C at 110 rpm for 10 minutes. Tissue
fragments were transferred to a fresh collagenase solution, gassed and
incubated for an additional 40 minutes at 37°C with shaking. Acini were
separated by manual pipetting through decreasing pipette orifices, and passed
through a 150 µm nylon net filter. The filtrate was centrifuged at 500 rpm
for 3 minutes and incubated at 37°C until ready to use. Cells were plated
on glass coverslips in Waymouth's medium containing 1% penicillin and
streptomycin (Pen/Strep) and 0.5% fetal bovine serum (FBS).
Dye coupling and electrophysiology
Glass microelectrodes were prepared from filamented microcapillary tubing
(World Precision Instruments, Kwik-Fill 1B100F-3) using a Narishige vertical
microelectrode puller. The glass microelectrodes were backfilled with 10 mM
6-carboxyfluorescein and individual acinar cells were injected using
hyperpolarizing pulses of current (50 nA; 100 ms pulse per second) for 1
minute. Transfer of dye was monitored for at least three minutes and images
were recorded using a digital camera.
Experiments to detect electrical coupling between acinar cells was
performed as previously described (Mao et
al., 2000). Briefly, glass microelectrodes were backfilled with 1
M KCl, single cells were injected using hyperpolarizing pulses of current (50
nA; 100 ms) and the transfer of current to adjacent cells was recorded. The
recording electrode was inserted into either an adjacent cell or into a cell
that was positioned
200 µm away from the current-injecting electrode.
Cell membrane potentials were recorded and plotted as current (mV)
vs. time.
Gene constructs
The mouse Cx32 P1 promoter spanning 680 to +20
(Hennemann et al., 1992;
Neuhaus et al., 1995
) was
cloned from genomic DNA using PFU DNA polymerase and promoter-specific primers
containing flanking restriction sites. The 700 bp PCR product was digested
with BamHI and BglII and cloned into the pGL2-Basic
luciferase reporter plasmid from Clontech. The entire promoter region was
verified by DNA sequencing. All other DNA constructs used in this report have
been described previously (Krapp et al.,
1998
; Lemercier et al.,
1998
).
Cell culture and DNA transfections
Cells from the pancreatic exocrine cell line AR42J were propagated in
growth medium consisting of 40% F12K, 25% hgDMEM, 25% F12, 10% FBS, 1%
Pen/Strep. Cells (1.6x106) were transfected by
electroporation (350 mV, 960 µF; 0.4 cm gap) using a BioRad GenePulser and
10 µg reporter (Cx32p-Luc), 5 µg of the appropriate
transcription factor expression plasmid (pcDNA3, pcDNA3-Mist1,
pcDNA3-Mist1mut basic, pcDNA3-Mist1mut helix I,
pcDNA3-E12, pcDNA3-E47, pcDNA3-HEB, pcDNA3-PTF1-p48, pcDNA3-NeuroD,
pcDNA3-MyoD, pcDNA3-Mash1) and 5 µg pRL-Null Renilla luciferase control
vector per experimental group. Cells were harvested 48 hours later by scraping
in Promega Passive Lysis Buffer and luminescence values were determined using
the Promega Dual Luciferase Reporter Assay System. A minimum of three
independent transfections were performed for all gene constructs.
RNA expression analysis
For RT-PCR, 2 µg of total RNA was reverse transcribed using the
Superscript II reverse transcription system (Gibco BRL). cDNA reactions were
then amplified using gene-specific primers for Mist1
(5'-GCGCGTACGGCCTCGAAT-3', 5'-CAAGCCCTAGAGAAGATG-3'),
Cx32 (5'-GCAACCAGGTGTGGCAGTGC-3',
5'-CGGAGGCTGCGAGCATAAAGAC-3'), and ß-actin
(5'-ATTGTTACCAACTGGGACG-3', 5'-TCTCCTGCTCGAAGTCTAG). Target
sequences were amplified for 35 cycles using 95°C/40 seconds, 55°C/40
seconds and 72°C/55 seconds conditions. All primer pairs amplified regions
crossing intron borders.
RNA isolation and hybridization blots
Total organ RNA was isolated using the QIAGEN RNeasy isolation system
following the manufacturer's recommended protocol. Mice were euthanized and
perfused with 12 ml of cold PBS and then tissues were removed and disrupted
using a Tissue-Tearor mechanical homogenizer. Samples were resuspended in
RNase-free water and stored at 80°C. For RNA blot analysis, 20 Mg
of total RNA was precipitated in ethanol, resuspended in 20 µl of RNA
loading buffer and heated at 65°C for 15 minutes until completely
dissolved. RNA was loaded on a 1.2% formaldehyde agarose gel and
electrophoresed in 1x MOPS buffer (0.5 M Mops, 0.01 M EDTA). After
separation, RNA was transferred by capillary action to Hybond+
membranes in 10x SSC. Hybridizations were performed using Clontech's
ExpressHyb buffer according to the manufacturer's recommendations.
Isotopically labeled probes were synthesized by standard procedures using the
Ambion Decaprime kit.
Immunohistochemistry
Frozen and paraffin-embedded pancreatic sections were processed for
immunohistochemistry by standard procedures. Briefly, tissues were removed and
frozen without fixation in OCT and 5 µm sections were cut at
20°C using a Zeiss cryostat. Tissue sections were fixed in 4%
paraformaldehyde, 0.1% Triton X-100 in PBS and then blocked for 1 hour with
the Mouse on Mouse (MOM) reagent (Vector). For paraffin immunohistochemistry,
pancreatic tissue was fixed in 10% formalin, embedded in paraffin and
sectioned at 5 µm. Sections were rehydrated, treated with 100 µg/ml
proteinase K and post-fixed in 10% formalin for 10 minutes. Samples were
washed with PBS, permeabilized with 0.1% Triton X-100 and blocked using 5% BSA
and 0.1% Triton X-100. Primary antibodies were then added to frozen or
paraffin-embedded sections for 1 hour at room temperature. Primary antibodies
included mouse myc 9E10 (1:200, Developmental Studies Hybridoma Bank), mouse
connexin32 (1:50, Developmental Studies Hybridoma Bank or 1:100, Chemicon),
rabbit connexin32 (1:200, Zymed Laboratories), rabbit connexin26 (1:200, Zymed
Laboratories), mouse ß-galactosidase (1:100, Developmental Studies
Hybridoma Bank), rabbit amylase (1:250, Sigma), rabbit occludin (1:100, Zymed)
and rabbit ß-catenin (1:1000, Sigma). Following primary antibody
addition, sections were incubated with biotinylated or Texas Red-conjugated
secondary antibodies (1:200 Vector) for 10 minutes at room temperature
followed by incubation with Oregon Green-conjugated tertiary antibodies
(Molecular probes) for 5 minutes. Coverslips were mounted with Fluorosave
reagent and examined using an Olympus fluorescence microscope. Images were
captured using a QImager MicroImager II digital camera and Empix Northern
Eclipse software.
Protein immunoblot assays
Tissue protein extraction, protein electrophoresis and immunoblotting were
performed as described previously (Pin et
al., 2000). For immunoblot analysis, 50 µg of whole cell
protein extracts were electrophoresed on acrylamide gels, transferred to PVDF
membranes and incubated with primary antibodies against Mist1, Cx32,
ß-gal and ß-actin. Following secondary antibody incubation the
immunoblots were developed using an ECL kit (Pierce) as per manufacturer's
instructions.
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Results |
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The progressive deterioration of tissue architecture that is observed in
Mist1KO mice resembles chronic pancreatic injury and is
associated with changes in a number of gene products
(Pin et al., 2001). In many
cases, the timing of the observed molecular changes suggests that they are
secondary events occurring as a consequence of cell morphology, and not as a
consequence of direct regulation by Mist1. In order to investigate whether the
loss of Cx32 protein represents an early or late event associated with
deletion of the Mist1 gene, we performed a time course analysis of
Cx32 expression in WT and Mist1KO pancreatic tissue
(Fig. 2). Tissue samples taken
between postnatal day 1 (PN1) and adulthood revealed strong Cx32
immunofluorescence in WT pancreas, whereas virtually no Cx32-containing gap
junctions were detected in Mist1KO sections, regardless of
age (Fig. 2A-F).
|
Several mechanisms regulate protein accumulation, including changes in
protein stability or mRNA transcription. To establish if the loss of Cx32
protein in Mist1KO acini was due to a corresponding loss
of Cx32 transcripts, RNA blot analyses were performed on total
pancreas RNA isolated from WT and Mist1KO mice. As shown
in Fig. 2G, Cx32 mRNA
levels in all Mist1KO samples were reduced by 75-85%
compared to WT controls. This was particularly evident in the samples taken
from 3-week-old mice, at which time the expression of Cx32 peaks in
WT pancreas possibly because of the maturation of the regulated exocytosis
process (Jamieson et al.,
1988). The difference in Cx32 mRNA levels was consistent
across all time points examined, with the first indication of a significant
decrease detected at PN1 (Fig.
2G). These results indicate that in Mist1KO
acini, Cx32 gene expression is never activated to levels in WT acinar
cells, and that the decrease in Cx32 protein most probably reflects changes in
mRNA transcription or mRNA stability.
Mist1KO pancreatic acinar cells are defective in
intercellular communication
Given the significant reduction in Cx32 gene expression, we set
out to examine whether Mist1KO cells remain functionally
coupled. For these initial experiments, individual acini were isolated from WT
and Mist1KO mice and stained for Cx32 protein. As observed
previously in the pancreatic sections, Cx32 was detected in the membranes of
WT acini while Mist1KO acini were devoid of Cx32 staining
(Fig. 3A,B). WT and
Mist1KO acini then were tested for the ability to transfer
6-carboxyfluorescein, a small molecular mass dye, into adjacent cells. As
expected, dye transfer rapidly occurred in acinar cells isolated from control
mice (Fig. 3C,D). In all
instances (n=15 acini from 4 WT mice) three or more adjacent cells
exhibited dye transfer. In contrast, acini isolated from
Mist1KO mice were highly defective in dye transfer. No
transfer of 6-carboxyfluorescein was detected in any of the neighboring acinar
cells of Mist1KO mice (n=12 acini from 4 mice),
even after extended incubation times (Fig.
3E-H).
|
We next characterized WT and Mist1KO cells with respect
to electrical coupling. Hyperpolarizing pulses of current (50 nA; 100 msec)
were injected into one cell within an isolated acinus and the spread of
current to neighboring cells was monitored. As shown in
Fig. 3I, electrical coupling
between adjacent cells from WT acini was readily detected. Electrical coupling
was also evident at interelectrode distances of 200 µm, suggesting
that widespread coupling of WT acinar cells occurred within a single acinus
(data not shown). In contrast, Mist1KO acini showed a
complete absence of electrical coupling. In all cases, no transfer of the
hyperpolarizing current was ever detected, even when multiple adjacent cells
were tested (Fig. 3J). The
complete loss of cell-cell communication in Mist1KO acinar
cells is surprising since acini from Cx32KO mice maintain
some coupling properties (Chanson et al.,
1998
). These findings suggest that other factors that are
instrumental in establishing communication networks may also be affected in
Mist1KO mice.
Connexin26 mRNA levels are retained, but protein abundance
is altered, in Mist1KO mice
The complete loss of dye transfer and electrical coupling in
Mist1KO acinar cells suggested that all gap junctional
proteins were affected in these cells. Formation of normal pancreas gap
junctions requires the co-expression of Cx32 and a second connexin protein,
Cx26 (Meda, 1996;
Zhang and Nicholson, 1994
).
Cx26 gene expression occurs in all acinar cells of the pancreas and
the protein often cooperates with Cx32 to form heteromeric channels
(Meda et al., 1993
;
Stauffer, 1995
). In fact, Cx26
and Cx32 are often co-localized in the same cells
(Zhang and Nicholson, 1994
)
and our immunohistochemical analysis confirmed that both proteins were
localized to the same gap junction plaques in WT pancreatic acini
(Fig. 4A-C). To determine if
Mist1 regulates the expression of both genes in a similar fashion, we
characterized the expression and localization of Cx26 in
Mist1KO acinar tissue. As shown in
Fig. 4D, RNA blot analysis
revealed comparable Cx26 transcript levels in both WT and
Mist1KO acinar cells. Surprisingly, no difference in
Cx26 mRNA levels could be detected between normal and
Mist1KO mice at any time point examined (PN1 to adult)
(data not shown), suggesting that transcriptional regulation of the
Cx32 and Cx26 genes remains under separate control. To
confirm if this expression pattern was also observed at the protein level,
Cx26 antibodies were used to detect Cx26 in Mist1KO
pancreatic acini. Immunoblot analysis of 3-week old pancreatic tissue revealed
equivalent levels of Cx26 protein in WT and Mist1KO
samples (Fig. 4E).
Surprisingly, Cx26 protein was not detected in pancreatic acinar cells
obtained from older (10-weeks old and adult) Mist1KO mice,
despite the continued presence of Cx26 transcripts
(Fig. 4D). Examination of
additional young animals (PN1 to 6-weeks old) by immunofluorescence confirmed
the presence of Cx26 protein within pancreatic acinar cells, but instead of
the expected gap junction staining pattern observed in WT and Mist1
heterozygous (Mist1Het) samples
(Fig. 4F,H), Mist1KO acinar cells exhibited a diffuse cytoplasmic
staining (Fig. 4G,I). As
predicted from the immunoblot analysis
(Fig. 4E), this intracellular
staining pattern persisted until 6 weeks of age, after which time the Cx26
protein was no longer detected in Mist1KO cells
(Fig. 4K). The significant
mislocalization and reduction in Cx26 protein most likely contributes to the
defects in cell communication observed in Mist1KO acini.
These results suggest that the loss of Cx32, coupled with the overall
disorganization of the Mist1KO acinar cells, affects the
assembly, trafficking, and/or stability of Cx26-containing gap junctions to
the plasma membrane, resulting in the eventual loss of unassembled Cx26
protein.
|
Mist1 functions as a positive regulator of Cx32
transcriptional activity
Our studies have shown that Cx32 gene expression, but not
Cx26, is altered in the Mist1KO pancreas. The
specific loss of RNA expression for only one of the connexins strongly
suggests that Mist1 exerts its control directly on Cx32 gene
transcription. Indeed, analysis of Mist1 and Cx32 gene
expression profiles revealed a striking overlap in tissue distribution, with
co-expression observed in all secretory exocrine cells, including the
pancreas, submandibular gland, parotid gland and seminal vesicles
(Fig. 5A). The exception to
this pattern of common expression was in liver hepatocytes
(Fig. 5A) and neuronal
supporting cells (data not shown), both of which do not undergo regulated
exocytosis and only express Cx32 but not Mist1
(Fig. 5A)
(Evert et al., 2002;
Meda et al., 1993
;
Nicholson et al., 2001
;
Pin et al., 2000
). Analysis of
various tissues in Mist1KO animals also revealed a
striking correlation between Mist1 and Cx32 gene expression.
Tissues that normally express Mist1 mRNA and protein, including the
lacrimal and submandibular glands, exhibited greatly reduced levels of
Cx32 transcripts (Fig.
5B) and an almost complete absence of Cx32-containing gap junction
complexes (Fig. 5C-H). In
contrast, expression of Cx32 transcripts and formation of
Cx32-containing gap junction plaques remained unchanged in tissues that do not
normally express Mist1, such as liver hepatocytes
(Fig. 5B,I,J). These results
support the hypothesis that Mist1 functions as a tissue-specific regulator of
Cx32 gene expression in secretory exocrine cells.
|
In order to examine whether Mist1 directly controls expression of the
Cx32 gene, we cloned a portion of the mouse Cx32 P1 promoter
(Neuhaus et al., 1995)
spanning 680 to +20 into a luciferase reporter plasmid
(Cx32p-Luc) and tested it for expression in the pancreatic exocrine
cell line AR42J. Introduction of Cx32p-Luc into AR42J cells resulted
in very low basal luciferase activity (Fig.
6A). Co-transfection of cells with the Cx32p-Luc reporter
and an expression plasmid encoding Mist1 generated a 15- to 20-fold
increase in Cx32p-Luc expression
(Fig. 6A). Similar results also
were obtained with the exocrine cell line ARIP (data not shown).
Interestingly, no other bHLH transcription factor tested in this assay was
able to activate expression of the Cx32p-Luc gene. These included
both ubiquitously expressed Class A bHLH factors (E12, E47, HEB) and tissue
restricted Class B bHLH factors (PTF1-p48, NeuroD, MyoD, Mash1)
(Fig. 6B). The ability of Mist1
to activate expression of the Cx32p-Luc gene was dependent on its DNA
binding and dimerization properties. Mutant Mist1 proteins that were defective
in DNA binding (Mist1mut basic) or protein dimerization
(Mist1mut helix 1) (Lemercier
et al., 1998
), did not activate Cx32p-Luc expression
(Fig. 6C). From these studies
we conclude that an active Mist1 protein is required to generate full
Cx32 gene expression in secretory acinar cells.
|
To more rigorously test the importance of active Mist1 protein for
Cx32 gene transcription, we took advantage of the fact that the
Mist1mut basic protein functions as a dominant-negative factor to
repress endogenous Mist1 activity
(Lemercier et al., 1998). We
reasoned that expression of Mist1mut basic in pancreatic acinar
cells would inhibit the endogenous Mist1 protein and down-regulate the
expression of Mist1 target genes. Transgenic mice expressing a myc-tagged
Mist1mut basic protein under the transcriptional control of the
pancreas-specific elastase 1 promoter
(elastasep-Mist1mut basic-myc)
(Heller et al., 2001
;
Kruse et al., 1995
), were
generated and examined for Mist1mut basic expression as well as for
the presence of Cx32-containing gap junction plaques. As shown in
Fig. 7, pancreatic acinar cells
expressing the Mist1mut basic protein showed a significant decrease
in Cx32 expression, whereas adjacent acinar cells that did not express the
Mist1mut basic transgene exhibited normal expression and
localization of Cx32. In addition, the Mist1mut basic-expressing
cells no longer produced Cx26-containing gap junction complexes and instead
showed the diffuse cytoplasmic Cx26 staining that was evident in
Mist1KO samples (data not shown). These results
demonstrate that loss of Mist1 activity, through either deletion of the
Mist1 gene or through inhibition of endogenous Mist1 protein
activity, leads to the loss of Cx32 expression. We conclude that
Mist1 functions as a positive regulator of Cx32 gene expression in
exocrine acinar cells and, in the absence of Mist1, gap junctions and
intercellular communication pathways become disrupted.
|
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Discussion |
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Each connexin protein is distinct and the expression pattern for each
displays a tissue and cell-type specificity
(Plum et al., 2000). The
regulated expression of the connexin genes results in the selective passage of
molecules because of differing permeability characteristics of the channels.
For instance, Ins(1,4,5,)P3 travels through Cx32 homomeric
channels more readily than channels composed of Cx43 or Cx26
(Niessen et al., 2000
). In
many cases, connexin gene expression is also altered as cells respond to
changes in local environmental conditions
(Petrich et al., 2002
). These
observations suggest that connexin genes are regulated by tissue-specific
transcription factors that are responsive to intracellular signaling pathways.
Unfortunately, molecular details of the transcriptional control of individual
connexin genes remain largely unknown.
In an effort to identify the importance of gap junctions in acinar cell
morphology and function, we examined connexin expression in mice lacking the
bHLH transcription factor Mist1. Mist1KO pancreatic acinar
cells are highly disorganized (Pin et al.,
2001), suggesting that intercellular junctions could be
compromised. Indeed, this study has revealed that Mist1KO
acinar cells exhibit a specific loss of connexin expression and gap junctional
communication. This defect is primarily caused by reduced expression of the
Cx32 gene, which leads to severe depletion of gap junction formation
and gap junction-mediated cellular communication.
While it could be argued that the loss of Cx32 is a consequence of cellular
disorganization and not directly of a loss of Mist1 activity, several pieces
of evidence support a model of direct transcriptional regulation. First, one
would predict that a simple disruption of the gap junctions by cellular
disorganization would lead to decreases in both connexin genes expressed in
pancreatic acini. Cx32 and Cx26 proteins are co-expressed in many of the same
cell types and readily form heteromeric gap junctions
(Meda et al., 1993;
Stauffer, 1995
). However, in
Mist1KO acinar cells, Cx26 gene transcription is
maintained while Cx32 gene transcription is greatly reduced, implying
that the transcriptional control of these two genes is distinct. Secondly,
analysis of the Cx32 gene reveals two separate promoters, P1 and P2
(Neuhaus et al., 1995
). The P1
promoter directs Cx32 gene expression in liver, salivary glands and
pancreatic epithelial cells, while the downstream P2 promoter is responsible
for Cx32 expression in cells of the spinal cord and brain
(Neuhaus et al., 1995
).
Studies from the Ruch laboratory (Koffler
et al., 2002
; Piechocki et
al., 2000
) have shown that liver-specific expression of the
Cx32 gene relies on the winged-helix transcription factor HNF-1,
which is also present in the pancreas. However, adult pancreatic HNF-1
expression is primarily confined to cells of the endocrine compartment where
Cx32 is not expressed (Edlund,
2002
; Nammo et al.,
2002
). Other HNF family members (HNF-6, HNF-3ß) are present
in the exocrine pancreas (Rausa et al.,
1997
), but they do not activate Cx32 gene expression in
transfection assays (unpublished data). Instead, our data suggest that the P1
promoter is activated in acinar cells by Mist1. Sequence analysis of the P1
promoter reveals 5 E-box regulatory sites that potentially bind Mist1, and a
mutant form of Mist1, which does not bind DNA, fails to activate Cx32
expression. The observed repression of the Cx32 gene in pancreatic
acinar cells expressing a dominant-negative Mist1 protein confirms that
endogenous Mist1 activity is essential for acinar cell Cx32
transcription. As predicted, the reduction in Cx32 gene expression in
Mist1KO mice is observed only in cell types that normally
express Mist1, such as the exocrine cells from the pancreas, salivary
glands, lacrimal gland and seminal vesicle. From these studies we conclude
that Mist1 functions as a direct positive regulator of Cx32 gene
expression and, in its absence, acinar cell gap junctions and intercellular
communication pathways become disrupted.
Although the DNA binding and dimerization domains of Mist1 are required to achieve Cx32 gene transcription, the precise mechanism(s) by which Mist1 activates Cx32 gene expression is not known. In this regard, it will be essential to determine if Mist1 functions as a homodimer or as a heterodimer with another bHLH protein expressed in acinar cells. Preliminary data suggest that Mist1 homodimers are the preferred protein complex in the pancreas (T. Tran, unpublished data). However, Mist1 homodimers alone are not sufficient to activate the endogenous Cx32 gene in non-acinar cell types (unpublished data), suggesting that acinar cells contain co-factors that modify the Mist1 protein, or that serve as accessory transcription factors, to allow full Cx32 gene expression. Indeed, regulation of the Cx32 promoter is probably complex, since in the absence of Mist1 the Cx32p-Luc reporter gene remains active at a low basal level. This expression is not surprising given that the 680 bp 5' flanking sequence contains protein binding sites for many general transcription factors including Sp1, NF1, Stat5 and SRF (data not shown). Future studies will focus on determining if Mist1 interacts with these other transcription factors that are bound to the Cx32 promoter to influence acinar-specific gene expression. Identification of the specific Cx32 promoter elements and additional acinar cell transcription factors will be essential to establish their role in activating Cx32 gene expression.
These studies have also provided new insight into the differential
regulation of acinar-specific connexin proteins. While Cx32 appears
to be regulated at the transcriptional level, this is not the case for
Cx26. Despite normal levels of Cx26 mRNA, very few
Cx26-containing gap junctions are detected in Mist1KO
acinar cells. Instead, Cx26 protein accumulates within the cytoplasm and does
not associate with the cellular membrane. This observation supports the
hypothesis that Cx26 prefers to form gap junctions with Cx32
(Zhang and Nicholson, 1994).
Similar events have also been reported for Cx32KO mice,
where significant reductions in Cx26 gap junctions are observed, resulting in
decreased dye transfer (Chanson et al.,
1998
). Interestingly, lacrimal glands from
Cx32KO animals exhibit transient diffuse patterns of Cx26
protein accumulation that is similar to our observations with
Mist1KO mice (Walcott
et al., 2002
). However, the complete phenotypes associated with
Mist1KO and Cx32KO mice are quite
distinct. Cx32KO acinar cells continue to display some
electrical coupling (Chanson et al.,
1998
) and the disorganization observed in
Mist1KO mice is not evident in Cx32KO
pancreatic tissue (data not shown). The alterations in cell morphology in
Mist1KO acini probably contribute to the loss of cell
communication and indicate that the loss of Cx32 protein is not solely
responsible for the phenotypic abnormalities observed in
Mist1KO animals. It remains possible that several key
defects in regulated exocytosis, including altered CCK AR and
Ins(1,4,5)P3 receptor expression
(Pin et al., 2001
), may
contribute to the inefficient dye transfer and loss of electrophysiological
coupling found in this current study. The disorganization of
Mist1KO acinar cells is evident early in embryogenesis,
prior to significant gap junction organization (C. Johnson, personal
communnication). Indeed, results from our laboratory support the idea that
Mist1KO pancreatic acinar cells do not reach a fully
differentiated state and are developmentally blocked as immature exocrine
cells. Thus, although Mist1 clearly has a role in controlling Cx32
gene expression, additional Mist1 target genes are probably important in
establishing and maintaining normal acinar cell polarity and function.
Identification of these genes will be crucial to fully understanding the role
of Mist1 in these complex cellular processes.
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Acknowledgments |
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