Reproductive Endocrine Unit Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114
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ABSTRACT |
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INTRODUCTION |
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A particular neuropeptide-encoding gene may be expressed throughout the organism in a variety of tissues and cell types with different embryonic origins (1). Many of them are expressed in the central nervous system as well as in peripheral tissues, including the gastrointestinal tract and specific endocrine organs. However, whether cell-specific expression of a neuropeptide gene in ontogenically different tissues is regulated by similar or identical mechanisms remains unclear.
Somatostatin is a neuropeptide hormone whose expression is restricted to cells in the peripheral and central nervous systems, as well as to parafollicular cells of the thyroid gland, D cells of the digestive tract, and D cells of the pancreatic islets of Langerhans (2). Among these tissues, the endocrine pancreas has provided an informative model with which to study transcriptional mechanisms of control of cell-specific somatostatin gene expression, due, in no small part, to the availability of a number of pancreatic cell lines that recapitulate phenotypic features of pancreatic islet cells. Using such cell lines, previous studies by several investigators have shown that pancreatic D cell-specific expression of the somatostatin gene is the consequence of the binding of a number of nuclear proteins to well defined DNA cis-regulatory elements located in its promoter region. These elements include a cAMP-response element (CRE) located in relative proximity to the TATA box (3, 4, 5, 6), several tissue-specific enhancers that provide binding sites for homeodomain transcription factors (7, 8, 9), and several silencer elements (10).
In the central nervous system, somatostatin was first discovered in the hypothalamus and was found subsequently to be widely distributed in other areas, including hippocampus, cerebral cortex, and basal forebrain, where it appears to serve as an inhibitory neurotransmitter released from small interneurons (11). In the rat, the first somatostatin-positive cells in the forebrain appear at embryonic day 14 (E14) (12, 13). In some cells, this expression is transient, whereas in others it is maintained in the adult brain, and thus it has been proposed that in addition to its role as a neurotransmitter, somatostatin may have trophic effects on target cells during brain development (14, 15, 16).
The transcriptional mechanisms that control cell-specific somatostatin gene expression in the central nervous system are unknown. In the present study, we report on the establishment of conditionally immortalized somatostatin producing cell lines derived from rat embryonic brain. By using transient transfection assays, DNA mutagenesis, and DNA-protein binding assays, we show that expression of the somatostatin gene in neural cells is regulated by the strong positive activity of the CRE, which is under the influence of at least three negative acting upstream elements. A functional analysis of the somatostatin gene promoter in neural cells indicated that these negative-acting elements correspond to previously described pancreatic enhancers that bind homeodomain transcription factors.
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RESULTS |
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Positive- and Negative-Acting Promoter Elements Regulate Neural
Cell Expression of the Somatostatin Gene
To obtain information about the activity of the somatostatin gene
promoter region in neural cells, we carried out transient transfection
assays in hippocampal RH1.C4 cells and in cortical RC2.E10 cells.
Initially, we used the plasmid SMS900, a chloramphenicol
acetyltransferase (CAT) reporter plasmid that contains a fragment of
the somatostatin gene spanning nucleotides -900 to +54 (4, 5), and
compared its activity to that of a Rous sarcoma virus enhancer-driven
CAT reporter (RSVCAT). Figure 3B shows
that the level of expression of SMS900 CAT in RH1.C4 cells was similar
to that determined in pancreatic RIN-1027-B2 cells (5), whereas SMS900
CAT expression in RC2.E10 cells was lower.
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Requirement of the CRE for Promoter Activity in RC2.E10 Cells
The above experiments suggest that the CRE plays a prominent role
as a DNA cis-regulatory element mediating somatostatin gene
expression in neural cells. Indeed, it appears that somatostatin gene
transcription is potently activated by this element, and that its
activity is down-regulated by upstream negative modulatory elements. To
examine this notion in detail, we chose to focus our attention on
cortex-derived RC2.E10 cells, because the CAT activity generated by
SMS65 in these cells was significantly higher than in
hippocampus-derived RH1.C4 cells, indicating that the CRE is more
potent in the former than in the latter cell type. In addition, our
5'-deletion analysis suggested that in RC2.E10 cells relatively potent
repressor elements may be needed to counteract the activity generated
by the CRE.
We introduced an internal four-base deletion in SMS900 that
disrupts the CRE motif (from TGACGTCA to TG-CA), and determined the
CAT activity generated by the resulting plasmid, SMS900CRE, after
transient transfection into RC2.E10 cells. These experiments indicated
that the integrity of the CRE is required for somatostatin promoter
function in neural cells, because disruption of the CRE resulted in
reduced CAT activities indistinguishable from background levels (Fig. 3A
). In addition, these results lend additional support to the
hypothesis that the resulting transcriptional activity imparted by the
full-length promoter is the result of the activity of repressor
elements that counteract the transcriptional effect of positive
regulatory elements that require the presence of an intact CRE.
DNA Elements Containing "TAAT" Motifs in the Somatostatin
Promoter Act as Negative Modulators of Transcription in Neural
Cells
To search for additional promoter elements located upstream from
the CRE that regulate somatostatin gene expression in neural cells, we
studied promoter elements that have been shown previously to regulate
somatostatin gene expression via binding of homeodomain transcription
factors in pancreatic cells.
The SMS-UE (nucleotides -114 to -78) contains a so-called domain B (UE-B) with a core TAAT motif that binds homeodomain transcription factors in pancreatic cells (5, 7, 8, 20). Two other homeodomain-binding regulatory elements in the somatostatin gene promoter, SMS-TAAT1 and SMS-TAAT2, located at positions -449 to -445 and -295 to -292, respectively, have been described (8, 20). Inspection of the DNA sequence of the somatostatin promoter revealed the existence of a previously unidentified TAAT motif located between nucleotides -368 and -365, which we named SMS-TAAT3. To determine whether these elements regulate the expression of the somatostatin gene in neural cells, we carried out transient transfections in RC2.E10 cells and tested CAT activities of plasmids in which the TAAT motif of each one of them had been altered by site-directed mutagenesis. Reduced binding of nuclear proteins from RC2.E10 cells to these mutated sequences was confirmed by electrophoretic mobility shift assay (EMSA) (data not shown).
Disruption of each one of the aforementioned TAAT motifs
independently resulted in increases in CAT activity relative to the
wild-type SMS900 (Fig. 4A). The highest
increase (
4-fold) was found with plasmid SMS900T1M (SMS-TAAT1
mutant), followed by both SMS900T2M (SMS-TAAT2 mutant) and SMS900UEBM
(SMS-UE-B mutant). Disruption of SMS-TAAT3 (plasmid SMS900T3M) only
resulted in a modest (<2-fold) increase in CAT activity. Mutations of
more than one of these motifs simultaneously, in different
combinations, did not result in further increases in CAT activity (data
not shown).
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Placing either SMS-TAAT1 or SMS-TAAT2 immediately upstream from the CRE
resulted in a marked decrease in SMS65 CAT activity (Fig. 4B). The
decrease observed with TAAT1-SMS65 was more pronounced than that
observed with TAAT2-SMS65, resulting in levels of CAT expression barely
above background levels. In contrast, SMS-TAAT3 did not significantly
reduce levels of SMS65 CAT expression. Thus, SMS-TAAT1 and SMS-TAAT2,
but not SMS-TAAT3, appear to be relatively strong repressors of
CRE-dependent transcription. In an analogous manner, our
5'-deletion experiments indicated that SMS-UE-B can also repress
CRE-mediated transcription (compare the activities of SMS120 and SMS65
in Fig. 3C
).
Binding of Nuclear Proteins to TAAT-Containing Elements
The activities of SMS-TAAT1, SMS-TAAT2, and SMS-UE-B as DNA
cis-acting elements that control somatostatin gene
expression have been previously studied in pancreatic cells, where they
appear to act as positive regulatory elements. Because our studies
indicate that those elements act as transcriptional repressors in
neural cells, it was of interest to determine whether the complement of
nuclear proteins that bind to them is different in neural and in
pancreatic cells. For that purpose, we carried out EMSA with synthetic
oligonucleotides corresponding to SMS-TAAT1, SMS-TAAT2, SMS-TAAT3, or
SMS-UE-B, using nuclear extracts from cortical RC2.E10 cells or from
pancreatic RIN-1027-B2 cells. When the SMS-TAAT1 probe was used, three
distinct DNA-protein complexes were found with nuclear extracts from
RC2.E10 cells (Fig. 5). Specificity of
these complexes was determined by competition with unlabeled SMS-TAAT1
oligonucleotide added in excess to the binding reaction. Addition of an
oligonucleotide of unrelated sequence failed to compete. A similar
pattern was found when SMS-TAAT2 or SMS-TAAT3 probes were used, with
the exception that an additional complex with relatively fast
electrophoretic mobility (complex 4) was found with SMS-TAAT3 (Fig. 5
).
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When the UE-B probe was used, three closely migrating bands with
relatively slow electrophoretic mobilities were detected with nuclear
extracts of RC2.E10 cells, with the fastest one exhibiting the
strongest intensity (Fig. 5). In contrast, only two complexes were
detected when RIN-1027-B2 nuclear extracts were used, and in this case
the strongest intensity was exhibited by the band with slower
electrophoretic mobility.
To investigate the presence of proteins that recognize somatostatin
TAAT sites in the developing rat brain, we prepared nuclear extracts
from the forebrain of E17 fetuses removed from timed-pregnant rats and
assessed DNA-protein binding by EMSA. We found that the pattern of
bands generated by SMS-TAAT1 and SMS-TAAT2, and SMS-UE-B in extracts of
E17 forebrains shows similarities with those found with nuclear
extracts of RC2.E10 cells. For SMS-TAAT1 and SMS-TAAT2, we detected
complexes 1, 2, and 3, which were found to comigrate with the
corresponding ones observed with nuclear extracts of RC2.E10 cells
(Fig. 6A). As shown in Fig. 6A
, specificity of binding of these complexes to DNA was confirmed by
competition with unlabeled oligonucleotides. When we used the SMS-UE-B
probe, three closely migrating complexes were found in embryonic
forebrain extracts, with similar electrophoretic mobilities to those
observed with RC2.E10 nuclear extracts (Fig. 6B
). Specificity of
complexes from embryonic forebrain bound to SMS-UE-B was confirmed by
competition experiments with nonlabeled oligonucleotides (not shown).
Thus, these experiments support the notion that in the developing brain
in vivo, somatostatin gene-regulatory elements are
recognized by similar proteins to those found in cortex-derived RC2.E10
cells.
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Stimulation of Somatostatin Gene Transcription by cAMP
Previous studies carried out in pancreatic cells have provided
evidence in support of synergistic interactions between CRE-binding
proteins and proteins that bind to SMS-UE or TAAT elements (5, 21).
However, those studies were carried out in pancreatic cell lines in
which somatostatin gene transcription is not stimulated by cAMP, likely
due to a defect in the phosphorylation of nuclear factor CRE-binding
protein (CREB) (6). Therefore, we investigated whether
regulatory TAAT elements participate in the modulation of the
transcriptional responses induced by cAMP in neural cells. To this end,
we transfected RC2.E10 cells with either SMS900 or with similar
plasmids, in which each one of the TAAT elements had been individually
mutated, and treated them with the cAMP analog 8-Br-cAMP (1
mM). Treatment of RC2.E10 cells with 8-Br-cAMP for 16
h resulted in an 8-fold increase in the CAT activity generated by
SMS900 (Fig. 7). That response was
abolished by deletion of four bases in the CRE (Fig. 7
). The magnitude
of the response of SMS900 to 8-Br-cAMP stimulation was blunted by
mutations in SMS-TAAT1 (4.9-fold), SMS-TAAT2 (3.4-fold), or SMS-UE-B
(5.1-fold) but not significantly altered by mutations in SMS-TAAT3
(7.7-fold).
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Treatment of RC2.E10 cells with 8-Br-cAMP resulted in a 2.8-fold
increase in the CAT activity generated by SMS65 (Fig. 7). Placing
either TAAT3 or SMS-UE-B upstream from the CRE did not significantly
alter the strength of the response to 8-Br-cAMP (2.3-fold and 2.7-fold,
respectively) (Fig. 7
). However, the presence of SMS-TAAT2 resulted in
a significant increase in the fold stimulation elicited by 8-Br-cAMP
(4.6-fold, P < 0.05, Students t test, as
compared with SMS65). In contrast, SMS-TAAT1 inhibited
cAMP-dependent stimulation of SMS65 (Fig. 7
). Thus, these results
are consistent with the notion that, whereas TAAT1 represses both basal
and cAMP-stimulated transcription, TAAT2 represses basal transcription
but appears to facilitate cAMP-stimulated transcription.
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DISCUSSION |
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A progressive elevation in somatostatin CAT activity was observed, especially in cortical RC2.E10 cells, when the size of the promoter truncations generated by 5'-deletions increased, yielding levels of expression significantly higher than those observed with the full-length promoter. This was particularly evident as the proximity of the 5'-end of the remaining promoter fragment approached position -65, immediately upstream of the CRE. Thus, it appears that the stepwise removal of upstream negative modulatory elements results in the increasing manifestation of the activity of a strong positive regulatory element that would otherwise remain relatively repressed to maintain appropriate levels of expression. Our data identified the CRE as the predicted strong positive regulator, not only because of its location downstream to nucleotide -65, but also because its removal by a 5'-deletion to position -42 or by an internal 4-base deletion in the full-length promoter results in levels of CAT activity indistinguishable from background. Thus, the CRE appears to play a pivotal role in the regulation of basal somatostatin gene expression in neural cells. Studies in pancreatic and thyroid cells indicate that the somatostatin CRE recognizes a complex array of transcription factors generating between three and seven complexes by EMSA (4, 10, 22). We have observed a similar degree of complexity using nuclear extracts of RC2.E10 cells (our unpublished results), although the identity of the proteins that mediate basal CRE-dependent transcription in neural cells remains to be determined.
No evidence of regulatory elements has previously been reported in studies with pancreatic cells in a distal region of the promoter spanning nucleotides -750 to -900. We observed that the decrease in CAT activity after the deletion of this region was more pronounced in hippocampal RH1.C4 cells (13% of SMS900) than in cortical RC2.E10 cells (40% of SMS900). Somatostatin message levels appeared higher in RH1.C4 than in RC2.E10 cells, and our studies comparing the activity of SMS900 to that of RSVCAT suggest that the somatostatin gene is more efficiently expressed in RH1.C4 than in RC2.E10 cells. Therefore, these results suggest the existence of positive elements between -900 and -750 that act more efficiently in hippocampal than in cerebrocortical cells and thus may play a role in the control of region-specific expression within the central nervous system.
The presence of a neuron-restricted silencer element (NRSE) (23, 24, 25, 26, 27, 28, 29) in the promoters of certain neural and pancreatic genes suggests a possible mechanism for their expression both in neurons and in pancreatic islet cells (30, 31, 32). Although the somatostatin gene promoter does not contain an NRSE-like sequence, our studies indicated the presence of several regions of the somatostatin promoter that contain transcriptional repressor elements. In hippocampal RH1.C4 cells, 5'-deletion experiments allowed us to map the location of these repressor elements within positions -750 to -550, -345 to -250, and -120 to -65. In pancreatic cells, distal negative control elements have only been documented between nucleotides -425 and -345, but not upstream from that position (5). Therefore, repressor elements located between nucleotides -750 and -550 may represent neural-specific elements important for the modulation of adequate levels of expression of the somatostatin gene in the nervous system.
Notably, deletion of a region containing PS1 and PS2, two previously identified proximal silencer elements that repress transcription in pancreatic cells, located between nucleotides -250 and -120 (10), did not result in an increase of activity in hippocampal cells, suggesting that these elements are not active in these cells or that this region contains additional unidentified positive elements that offset the activity of PS1 and PS2. However, in cerebrocortical RC2.E10 cells, PS1 and PS2 are likely to be active, because a deletion from nucleotides -250 to -120 resulted in a significant increase in CAT activity. Thus, it appears that the region spanning nucleotides -345 to -65 contains transcriptional repressor elements, including SMS-TAAT2 (between -345 and -250), PS1 and PS2 (between -250 and -120), and SMS-UE-B (between -120 and -65), which seem to be required for down-regulation of the strong transcriptional activity elicited by the CRE in RC2.E10 cells.
SMS-TAAT1 was found to act as a strong repressor of CRE-dependent transcription in RC2.E10 cells. However, our initial 5'-deletion experiments did not allow us to predict that SMS-TAAT1 would act as a repressor, because removal of the fragment spanning -550 to -425 (SMS-TAAT1 is located between -449 and -445) resulted in decreased rather than increased transcriptional activity. This apparent discrepancy could be explained by the existence of two positive regulatory elements in close proximity to SMS-TAAT1 that can provide binding sites for GATA-type transcription factors (33).
The negative regulatory elements characterized in this study, SMS-TAAT1, SMS-TAAT2, and SMS-UE-B, have been previously identified in pancreatic cells as enhancers that bind homeodomain-type transcription factors (8, 9, 20, 21), and it is likely that in neural cells these elements also bind homeodomain proteins. A number of homeodomain-encoding genes, including Isl-1, Pax-6, Pbx, and Brn-4, have been found to be expressed in both endocrine pancreas, where some of them regulate somatostatin gene transcription, and brain (20, 21, 34, 35, 36, 37, 38). Our EMSA data suggest that a protein with a homeodomain that resembles that of IDX-1 protein is present in neural cells and binds to SMS-UE-B. Although IDX-1 gene expression has been considered to be restricted to stomach, duodenum, and pancreas, it is possible that a gene encoding a protein similar to IDX-1 is expressed in the central nervous system (39). SMS-TAAT1 and SMS-TAAT2 do not appear to bind an IDX-1-like protein. However, a homeodomain protein related to Orthodenticle and Pax may bind to these elements (our unpublished observations).
It remains to be determined whether homeodomain transcription factors that recognize TAAT elements in neural cells act as transrepressor proteins or whether they act as transactivators that compete for binding to the same elements with nonhomeodomain-type repressor proteins. Although many homeodomain transcription factors function as transactivator proteins to stimulate transcription of target genes, it has been shown that a number of homeodomain proteins act as transcriptional repressors on the promoter of neural (40, 41, 42, 43, 44, 45) as well as nonneural genes (46, 47, 48). Evidence indicates that these proteins are composed of modular domains, some of which mediate activation whereas others mediate repression (46, 49, 50, 51). Thus, it appears that the overall transcriptional activity of a given homeodomain protein is the result of unique combinations of positive and negative acting regions operating according to determined molecular environments in different cell types.
It is possible that both positive- and negative-acting transcription factors bind to TAAT-containing elements of the somatostatin promoter so that the transcriptional activity imparted by these DNA-regulatory sequences is the result of the combined effects of both positive and negative trans-acting proteins. Thus, in pancreatic RIN-1027-B2 cells, binding of activator homeoproteins would predominate over repressor proteins, whereas the opposite would occur in neural RC2.E10 cells. Consistent with this model, our data generated by EMSA suggest that complex 2 on SMS-TAAT1 and SMS-TAAT2, present in nuclear extracts of RC2.E10 cells but almost absent in those of RIN-1027-B2 cells, may correspond to a repressor protein. Conversely, complex 1, more prominent in RIN-1027-B2 cells than in RC2.E10 cells, may correspond to a transcriptional transactivator. Notably, SMS-TAAT3 binds an additional complex (complex 4) not detected on SMS-TAAT1 or SMS-TAAT2. Since SMS-TAAT3 did not show a significant negative activity, it is possible that complex 4 corresponds to a transcriptional transactivator complex whose influence would counteract the overall repressor activity of the other complexes apparently shared with SMS-TAAT1 and SMS-TAAT2. Nevertheless, it is also possible that complexes with similar electrophoretic mobilities in pancreatic and in neural cells correspond to related, but not identical, proteins with small differences in their amino acid sequences that could account for their activities as activators or repressors (40, 52, 53), or even to identical proteins that may have bimodal properties, functioning as activators in some cell types and as repressors in others (41, 43, 51, 54). Efforts to elucidate the identities of the proteins that bind to somatostatin TAAT elements are underway in our laboratory.
That at least one of the TAAT-containing elements of the somatostatin gene promoter characterized in the present study is targeted by both positive and negative DNA-binding proteins is also supported by our data on cAMP-induced somatostatin gene transcription, which suggest that SMS-TAAT2 subserves a dual role as a negative modulator in basal conditions and as a positive facilitator of transcription under conditions of cAMP-dependent stimulation. Earlier studies have identified CREB, phosphorylated by protein kinase A in response to cAMP, as a major transactivator of somatostatin gene transcription in response to cAMP stimulation. However, the somatostatin CRE can also be recognized by other transcription factors such as CCAAT/enhancer-binding protein-ß (C/EBPß) and C/ATF, which function as potent transactivators of somatostatin gene transcription in basal conditions via CREB-independent mechanisms (6). Thus, it is possible to hypothesize that positive-acting proteins bound to SMS-TAAT2 could facilitate phospho-CREB-dependent transcription via functional or physical interactions with phosphorylated CREB or with certain coactivators such as CREB-binding protein or TATA box-associated factors. On the other hand, negative acting SMS-TAAT2-binding proteins would interact with a different set of CRE-binding or TATA box-associated proteins, as it has been shown that some amino acid sequences that act as repressor domains of homeodomain proteins can interact with specific components of the RNA polymerase II complex (55, 56, 57).
During development, somatostatin-expressing cells in the central nervous system appear first in the primordium of the E14 hypothalamus, and other telencephalic regions are recruited later (12, 13, 15, 16). As the central nervous system develops, somatostatin is expressed in differentiating neurons as well as in glial cells, but the number of somatostatin-positive cells fluctuates. Thus, in some areas the number of somatostatin-positive cells increases progressively and remains unchanged in the adult organism, whereas in other regions somatostatin gene expression is transient, increasing first and then decreasing until it disappears. This pattern of gene expression suggests a developmentally regulated dynamic interplay between positive- and negative-acting transcription factors, some of which may use TAAT-containing sites as cis-acting targets. Identification of these neurally expressed transcription factors and elucidation of their complex interactions on the somatostatin promoter will shed light on our understanding of the molecular mechanisms that regulate the expression of a single gene in tissues of different embryological origins.
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MATERIALS AND METHODS |
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Establishment of Immortalized Forebrain-Derived Cells
Primary cultures of cells from developing cerebral cortex and
hippocampus, two brain regions that contain somatostatin-producing
neurons (12), were prepared from E16 fetuses removed from
timed-pregnant Sprague Dawley rats. After careful removal of the
meningeal membranes, the cerebral cortex was separated from the rest of
the brain, and the developing hippocampi were dissected as described
(58). Cells from these structures were dispersed, seeded onto
polyornithine-coated plates, and infected 24 h later by a
replication-defective recombinant retrovirus derived from the Moloney
murine leukemia virus that contains a neomycin-resistant gene and an
SV40T oncogene encoding tsA58/U19, a temperature-sensitive mutant
allele (17, 18). Retroviral particles (titer 105 cfu/ml)
were produced by a -2 packaging fibroblast cell line (F4 subclone,
kindly provided by Dr. G. Almazan, McGill University, Montreal, Quebec,
Canada). Retrovirus-containing conditioned medium from
-2 cells was
applied directly onto primary cultured cells in the presence of 8
µg/ml polybrene (Sigma). Cells were incubated at 33 C in DMEM
supplemented with 10% FBS in the presence of the neomycin analog G418
(GIBCO Laboratories, Grand Island, NY). Resistant colonies appeared
35 weeks later and were individually picked using cloning rings and
expanded. After five to six passages, a fraction of cells from each
colony was frozen in liquid nitrogen.
Rat islet somatostatin-producing RIN-1027-B2 (59) cells were grown in DMEM supplemented with 10% FBS. All cell lines were cultured in the presence of penicillin (100 U/ml) and streptomycin (10 µg/ml).
RT-PCR, Immunocytochemistry, and RIA
Total RNA (10 µg) purified by CsCl gradient centrifugation
from individual cell lines was primed with poly-(dT)15 and
incubated with avian myeloblastosis virus (AMV) reverse
transcriptase (Boehringer Mannheim Biochemicals) to synthesize cDNA.
For PCR amplification, a forward primer that anneals to the
5'-untranslated region (5'-GACCCACCGCGCTCAAGCTCGGCTG-3') and a reverse
primer that anneals to the 3'-end of the coding region of exon 2
(5'-AACAGGATGTGAATGTCTTCCAGAA-3') of the rat somatostatin gene (60)
were used. PCR conditions were: 95 C for 5 min, followed by 30 cycles
of 94 C for 30 sec, 50 C for 30 sec, and 72 C for 1 min, after which a
5-min incubation at 72 C followed. After PCR, an aliquot of the
reaction was resolved in a 1% agarose gel, blotted onto a nylon
membrane, probed with a 32P-labeled internal primer that
anneals to the 5'-region of exon 2 of the somatostatin gene
(5'-TTCGAGTTGGCAGACCTCTGCAGCTCCAGCCT-3'), and autoradiographed at -70
C.
For immunocytochemistry, cells were plated into "Lab-Tek" culture chambers (Nunc Inc., Naperville, IL), fixed in 4% paraformaldehyde in PBS for 5 min, washed in PBS, and permeabilized with methanol for 2 min at -20 C. After blocking with normal goat serum, cells were incubated overnight with either normal rabbit serum or with a somatostatin-specific polyclonal antiserum (INCSTAR Co., Stillwater, MN) at 1:500 dilution. Immunodetection was carried out with a secondary biotinylated goat antirabbit antiserum (Bio-Rad Laboratories, Hercules, CA) using immunoperoxidase staining with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
For RIA, cells growing in 35-mm dishes were scraped in 2 M acetic acid and boiled for 5 min. After centrifugation, the supernatant was collected, adjusted to pH 7.5, and lyophilized. Somatostatin content in these samples was determined by RIA using an antiserum (dilution 1:75,000) generated in sheep against synthetic somatostatin as described (61). For iodination, 100 pmol Tyr1-somatostatin (Sigma) was reacted with 1 mCi [125I]sodium iodide (DuPont-New England Nuclear) using the method described by Greenwood et al. (62). The minimum detectable concentration was 7.8 pg/ml.
Western Immunoblots
RH1.C4 or RC2.E10 cells were plated in duplicate 35-mm dishes at
a density of 25 x 104 or 15 x 104
cells per dish, respectively. After an overnight incubation at 33 C,
half of the plates were transferred to a 39 C tissue culture incubator,
and incubations at both 33 C and 39 C proceeded for an additional
24 h. Cells were then lysed in buffer containing 125
mM Tris-HCl (pH 6.8), 4% SDS, 15% glycerol, 10%
ß-mercaptoethanol, and 10 mM dithiothreitol. Proteins
were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane.
SV40T immunoreactivity was detected with a monoclonal primary antibody
(1:1000 dilution) (Calbiochem, La Jolla, CA) and a horse antimouse
peroxidase-conjugated secondary antibody (1:5000 dilution) (Bio-Rad).
CREB immunoreactivity was detected with a polyclonal primary antiserum
(1:500 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) and a goat
antirabbit peroxidase-conjugated secondary antibody (1:20,000 dilution)
(Bio-Rad). Immunoreactive bands were visualized using an enhanced
chemiluminescence detection system (Amersham, Buckinghamshire,
U.K.).
Plasmid Constructions
The plasmid SMS550 was constructed using DNA fragments obtained
by PCR amplification of somatostatin gene sequences in the plasmid
SMS900 (4). The upstream amplimers incorporated a BamHI
restriction site in its 5'-end. The downstream amplimer annealed to the
sequence corresponding to the XbaI site at position +54. The
resulting fragment was digested with the appropriate restriction
enzymes, purified on an agarose gel, and ligated into the promoterless
plasmid pOCAT (63) that had been digested with BamHI and
XbaI. All other CAT reporter plasmids bearing 5'-deletions
of the somatostatin promoter have been described previously (4, 5).
The plasmid SMS900CRE was constructed by ligating a
BamHI-BglII fragment obtained from SMS900 into
the BamHI site of plasmid SMS65
CRE (5). The resulting
plasmid, SMS900
CRE, preserves all the somatostatin gene sequences
from positions -900 to +54, with the exception of a 4-base deletion
within the core CRE motif.
Plasmids SMS900T1M, SMS900T2M, and SMS900UEBM have been described previously (8). Plasmid SMS900T3M was constructed by oligonucleotide-directed mutagenesis using a DNA fragment obtained by PCR amplification of somatostatin sequences in plasmid SMS900. The upstream amplimer anneals to a sequence located immediately upstream of the BamHI site of SMS900. The downstream amplimer was designed to anneal to a region of the somatostatin promoter spanning nucleotides -377 to -341, which contains the SMS-TAAT3 sequence located upstream of a KpnI site. This primer contained a six-base mismatch to replace the sequence 5'-GTAATC-3' by 5'-ACGGCT-3', a similar change to the ones introduced in SMS-TAAT1, SMS-TAAT2, and SMS-UE-B (8). Reduced binding of nuclear proteins to the mutated SMS-TAAT3 sequence was confirmed by EMSA using synthetic oligonucleotides (data not shown). After PCR amplification and restriction enzyme digestion, this fragment was used to replace the wild-type fragment located between BamHI and KpnI in SMS900.
For the construction of T3SMS65, a synthetic double-stranded oligonucleotide corresponding to SMS-TAAT3 (nucleotides -377 to -355) with BamHI and BglII sites at the 5'- and 3'-ends, respectively, was ligated into the BamHI site of the plasmid SMS65 (4). The sequence of this oligonucleotide is (coding strand): 5'-GATCCAAGTCCAGTAATCTGAGT-ACAT-3'.
The correct sequence of all the newly made plasmids was verified by the enzymatic procedure (Sequenase, United States Biochemical Corp., Cleveland, OH).
Transfections and CAT Assays
Initially, DNA transfections of neural cells were tested using
three different methodologies: a modified
diethylaminoethyl-dextran procedure (5), a calcium phosphate-DNA
coprecipitation method, and lipofectin (GIBCO Laboratories, Grand
Island, NY). Two different CAT reporter plasmids were used in parallel
experiments, one under the control of the ß-actin promoter (a gift of
Dr. William Walker, University of Pittsburgh), and another under the
control of the Rous sarcoma virus (RSV) enhancer. The
diethylaminoethyl-dextran procedure was found to yield very low levels
of CAT activity and was discarded. The calcium phosphate-DNA
coprecipitation method yielded adequate levels of reporter plasmid
expression, and ß-actin-CAT was found to exhibit levels of CAT
activity higher than those of RSVCAT. However, the ß-actin-CAT/RSVCAT
activity ratio was highly variable from experiment to experiment, a
likely reflection that transfection efficiencies were not uniform. In
contrast, when lipofectin was used, ß-actin-CAT was consistently
found to be 8 to 10 times more potent than RSVCAT (observed in at least
five independent experiments carried out in duplicate). This
information was used as an indication that transfection efficiencies
were relatively uniform among different dishes in each experiment. This
was confirmed in a different set of experiments in which cells were
stained for ß-galactosidase after transfection of a plasmid bearing
an RSV-ß-galactosidase fusion gene.
RH1.C4 and RC2.E10 cells were transfected with lipofectin following instructions provided by the manufacturer. Cells growing as monolayers up to 80% confluency were trypsinized and plated at a density of 5 x 105 cells per 60-mm plate. After an overnight incubation, 20 µg of reporter plasmid DNA mixed with lipofectin were added to the cells in 1 ml serum-free DMEM and incubated for 4 h. After this, 3 ml DMEM supplemented with 10% FBS was added. CAT activity was measured by a solution assay (64) 48 h after transfection. For cAMP-induction studies, 8-Br-cAMP was added to cells 16 h before harvesting. All the values are expressed as mean ± SEM of at least three independent experiments carried out in duplicate.
DNA Protein-Binding Assays
EMSAs were carried out with nuclear extracts (65), in the
presence of the protease inhibitors pepstatin A (1 mg/ml), leupeptin
(10 mg/ml), aprotinin (10 mg/ml), and p-aminobenzamidine
(0.1 mM). Protein concentrations were determined by the
Bio-Rad protein assay with BSA as a standard. Synthetic complementary
oligonucleotides with 5'-GATC overhangs were annealed and labeled by a
fill-in reaction using -32P-dATP and Klenow enzyme.
Binding reactions were carried out in the presence of 2 µg of
poly(deoxyinosinic-deoxycytidylic)acid, and specific
competitors, as indicated, using nuclear extracts (10 µg protein)
incubated with 20,000 cpm of radiolabeled probe (
610 fmol) in a
total volume of 20 µl containing 20 mM potassium
phosphate (pH 7.9), 70 mM KCl, 1 mM
dithiothreitol, 0.3 mM EDTA, and 10% glycerol. The
sequences of the oligonucleotides corresponding to SMS-TAAT1,
SMS-TAAT2, SMS-UE-B, and nonspecific competitor have been published
previously (6, 7, 8). The sequence of the SMS-TAAT3 oligonucleotide is
identical to the one used to construct the plasmid T3SMS65 (see
above).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by NIH Grant DK-49670 and by a grant from the Whitehall Foundation Inc. P.S. was supported by fellowships from the University of Hamburg (Germany) and from Deutsche Forschungsgemeinschaft.
Received for publication February 25, 1998. Revision received May 22, 1998. Accepted for publication June 5, 1998.
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REFERENCES |
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