A Novel Neuroendocrine Intracellular Signaling Pathway
Martin R. Schiller,
Richard E. Mains and
Betty A. Eipper
The Departments of Neuroscience and Physiology The Johns
Hopkins University School of Medicine Baltimore, Maryland
21205-2105
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ABSTRACT
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Expression of many components of the secretory
pathway in peptidergic neuroendocrine cells is precisely controlled in
response to secretagogues. Regulated endocrine-specific protein
(RESP18) was identified as a dopamine-regulated intermediate pituitary
transcript. Although the amino acid sequence of RESP18 initially
suggested that it might be a novel preprohormone, its widespread
expression in peptide-producing neurons and endocrine cells and its
localization to the lumen of the endoplasmic reticulum suggested that
it subserves a unique function. Subtractive hybridization of a
pituitary corticotrope AtT-20 cell line engineered for inducible RESP18
expression demonstrated a RESP18-dependent induction of several
transcripts. Regulation of RESP18 expression in vitro and
in vivo was accompanied by changes in the same transcripts.
Several cDNAs encoding transcripts up-regulated by RESP18 were analyzed
by DNA sequencing, searching the GenBank databases for homologous
proteins, and Northern blotting. One novel clone showed a tissue
distribution nearly identical to that of RESP18. One clone was
identical to rat LIMK2, a protein kinase containing modular
protein-protein interaction LIM (lin-11, isl-1, mec-3) domains. Another
clone was similar to monomeric bacterial isocitrate dehydrogenases.
Like the unfolded protein response, these data demonstrate a novel
signaling pathway from the secretory pathway lumen to the nucleus.
RESP18 acts as a lumicrine peptide (an intracellular luminal autocrine
hormone) inducing this pathway.
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INTRODUCTION
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The ability of cells to adjust to hormonal stimulation and varying
extracellular conditions involves tight intracellular control over
metabolism and intracellular communication between the cytosol,
nucleus, and various subcellular organelles (1). One of the best
understood intracellular signaling pathways is the unfolded protein
response (UPR), where accumulation of unfolded proteins in the lumen of
the endoplasmic reticulum (ER) induces the expression of
protein-folding chaperones (2). In the UPR model, conditions that
promote the accumulation of unfolded proteins in the ER cause the
dimerization of the transmembrane protein kinase IRE1/ERN1 (Refs. 3 and
4; Fig. 1A
). Upon activation of this
kinase, the transcription factor HAC1u is alternatively
spliced to yield Hac1i; the Hac1i protein has a
longer half-life and activates transcription of several ER resident
protein-folding chaperones. The alternative splicing is believed to be
catalyzed by RGL1.

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Figure 1. UPR and RESP18-Dependent Signaling Pathways
A, Schematic representation of the ubiquitous UPR ER to nuclear
signaling pathway and the neuroendocrine-specific secretory pathway
lumen to nuclear signaling pathway identified here. Conditions that
lead the accumulation of unfolded proteins in the ER activate a
transmembrane protein kinase, IRE1/ERN1, by an unknown mechanism. This
transmembrane kinase then activates RGL1, a RNA-splicing enzyme that
mediates the alternative splicing of the mRNA encoding
HAC1u, producing HAC1i. HAC1i binds
to DNA elements upstream of several protein-folding chaperones and
induces transcription of these genes (5, 44, 45). CGN,
cis-Golgi network; TGN, trans-Golgi
network; UPRE, UPR element. B, Experimental design for testing the
RESP18-signaling hypothesis.
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In addition to the UPR pathway, at least three other ubiquitous
signaling pathways allow communication between the ER lumen and the
cytosol or nucleus affecting cytokine production, sterol metabolism,
and translation (5, 6). In the ER overload response, an increase of
proteins in the ER triggers the activation of Nf-
B, a transcription
factor that is known to stimulate transcription of mRNAs encoding
cytokines. In the sterol-regulatory element binding protein (SREBP)
pathway, sterol depletion causes cleavage of the transmembrane ER
protein SREBP-1, releasing a fragment that translocates to the nucleus,
binds sterol-regulatory elements, and affects transcription of genes
involved in fatty acid synthesis, cholesterol synthesis, and
cholesterol uptake. Protein translation can be altered by the
release of ER calcium stores, which activates double-stranded
RNA-dependent protein kinase, an enzyme that phosphorylates
eukaryotic initiation factor-2 and inhibits translation. Many
studies suggest the presence of other intracellular signaling pathways.
For example, several genes mediating the proliferation of peroxisomes
are activated by peroxisomal proliferators and controlled through a
common nuclear transcription factor, peroxisome proliferator-activated
receptor (5). Mitochondrial proteins involved in electron transport are
regulated by binding of heme to the transcription factor HAP1p (1). The
presence of such pathways signifies the importance of controlling and
coordinating organelle function.
Neurons and endocrine cells that produce and store bioactive
peptides for regulated secretion can devote up to 9% of their protein
synthesis to the production of these peptides, which are stored in
specialized organelles called secretory granules or large dense-core
vesicles (7, 8). Synthesis, storage, and secretion of these bioactive
peptides involve many different proteins, and little is known about the
mechanism through which the cell controls the many elements that
contribute to the peptidergic phenotype. Treatment of rats with
dopaminergic drugs or exposure of Xenopus laevis to light or
dark backgrounds was known to affect POMC production in intermediate
pituitary melanotropes, a homogeneous population of peptide-producing
cells. In addition, levels of transcripts encoding many peptide
biosynthetic enzymes and approximately 1% of all proteins were altered
by these treatments; numerous studies support this observation in other
peptide-producing systems (8, 9, 10).
While exploring the function of regulated endocrine-specific
protein (RESP18), a novel 18-kDa protein expressed exclusively in
peptidergic neurons and endocrine cells, several attributes of RESP18
suggested that it might play a role as an intracellular signaling
molecule (11, 12, 13). First, RESP18 mRNA levels are hormonally regulated
in several in vivo and in vitro systems. Second,
RESP18 protein turns over rapidly with a half-life of less than 20 min
in AtT-20 corticotrope tumor cells. Third, RESP18 protein is normally
confined to the lumen of the ER by its rapid degradation in a distal
compartment; elevated expression allows RESP18 to traverse the ER into
distal secretory pathway compartments. Finally, the sequence of RESP18
is homologous to a short region in the luminal domain of several newly
identified neuroendocrine-specific receptor type protein tyrosine
phosphatases [28% identity over 70 amino acids to mouse, human, and
rat IA-2] (14, 15, 16, 17). The neuroendocrine-specific protein tyrosine
phosphatases (IA-2s and phogrins) are localized to the Golgi and
secretory granules (17, 18). Other receptor type protein tyrosine
phosphatases are known to participate in cellular transformation,
migration, and proliferation (19). These observations suggested that
RESP18 might participate in a neuroendocrine intracellular signaling
pathway (Fig. 1A
). In this study we have identified a novel signaling
pathway from the lumen of the secretory pathway to the nucleus that is
induced by RESP18 expression.
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RESULTS AND DISCUSSION
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To determine whether RESP18 might play a role in intracellular
signaling, we combined two known methodologies into a novel approach
that yielded a cDNA library enriched for transcripts potentially
regulated in response to RESP18 expression (Fig. 1B
). Our earlier
in vitro transcription/translation and subcellular
fractionation studies demonstrated that the N-terminal signal peptide
of RESP18 is cleaved cotranslationally and that RESP18 is contained
within the lumen of the secretory pathway (11). Therefore, any
regulation of transcripts in response to elevated RESP18 expression
would indicate the occurrence of communication from the luminal space
to the nucleus, as seen in the UPR.
Generation and Characterization of Inducible RESP18 and Inducible
Peptidylglycine
-Amidating Monooxygenase (PAM) Cell Lines
First, an AtT-20 cell line was engineered for inducible RESP18
expression using the reverse tetracycline repressor system (rTet) of
Gossen and co-workers (Fig. 2
) (20, 21).
A similar inducible cell line for a prohormone-processing enzyme, PAM,
was also created and used as a control for possible nonspecific effects
of the transfected rTet expression system or nonspecific effects from
overexpressing an exogenous mRNA or protein. PAM catalyzes the oxygen
and ascorbate-dependent oxidation of peptide C-terminal glycyl residues
into amides, releasing glyoxylate (22). AtT-20 cells were first
transfected with the pUHD172 vector that directs constitutive
expression of a tetracycline-controlled transactivator, a fusion
protein consisting of a mutant Escherichia coli tetracycline
repressor and the C-terminal transactivator domain of virion protein
VP16 from herpes simplex virus. Stable AtT/pUHD cell lines expressing
the chimeric protein were selected by growth in G418 and characterized
by analysis of total RNA. The positive lines were subcloned, and
Northern blot analysis confirmed expression of the correct size
tetracycline-controlled transactivator mRNA (
1 kb) in the lane
containing AtT/pUHD RNA (Fig. 2B
). This transcript was not detected in
wild-type AtT-20 cells. The clonality of the AtT/pUHD cell lines was
assessed by in situ hybridization analysis using
digoxigenin-labeled riboprobes generated from the
tetracycline-controlled transactivator cDNA. All cells of clone 3B
exhibited intense staining when the antisense riboprobe was used; cells
stained at background levels using a sense probe (Fig. 2C
).

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Figure 2. The Reverse tet Expression System for Inducible
Expression of RESP18 and PAM-1 in AtT-20 Cells
A, The rTet-inducible expression system (20, 21) was established
in AtT-20 cells. Transfected AtT-20 cells constitutively expressing the
tetracycline-responsive transactivator under control of the
cytomegalovirus (CMV) promoter were transfected with pUHD.RESP or
pUHD.PAM constructs. These constructs encode the target protein under
control of seven bacterial tet operator sequences
proximal to the minimal CMV promoter. Under basal conditions (OFF), the
tetracycline-controlled transactivator does not bind to the
tet operator sequences and exogenous RESP18 or PAM-1
expression remains turned off. Tetracycline or Dox promotes the
dimerization of the tetracycline-controlled transactivator on the
tet operator sequences, bringing the viral
transactivator domain in proximity to the CMV promoter and initiating
transcription of RESP18 or PAM-1 (ON). B, Northern blot analysis of RNA
prepared from wild type AtT-20 cells and AtT/pUHD cells (10 µg total
RNA); the blot was hybridized with a [32P]dCTP-labeled
rtTA cDNA probe and visualized by autoradiography. The migration of 18S
and 28S rRNA are indicated. C, In situ hybridization
analysis of AtT/pUHD cells using sense and antisense random primed
digoxigenin-UTP riboprobes for the tetracycline-controlled
transactivator. Hybridized riboprobe was visualized using an
anti-digoxigenin-alkaline phosphatase conjugate and alkaline
phosphatase staining as described. Bar, 50 µM.
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The genes to be expressed, rat RESP18 and rat PAM-1, were placed
downstream of repeated tet operator sequences (pUHD.RESP and
pUHD.PAM) to allow inducible expression of these genes (Fig. 2A
). The
clonal AtT/pUHD cell line was then cotransfected with either the
pUHD.RESP or pUHD.PAM construct, and pSCEP, a vector conferring
resistance to hygromycin B (23), to generate AtT-20-inducible(i)
iRESP and iPAM cells, respectively. Under basal conditions the
tetracycline-controlled transactivator does not bind to the
tet operator sequences and the target cDNA is not expressed.
Addition of tetracycline or doxycycline (Dox) to the culture medium
activates transcription of RESP18 or PAM-1. iRESP and iPAM cell lines
were screened by indirect immunofluorescence using antiserum directed
against RESP18 or PAM-1, respectively (24, 25). Clones exhibiting
intense immunostaining after induction and low basal expression were
chosen for further study. The kinetics of RESP18 induction in the iRESP
cell line were examined by evaluating indirect immunofluorescence
staining 06 days after addition of 500-4000 ng/ml Dox (data not
shown). No detectable induction was observed after 4 h, but
increased immunostaining was apparent 20 h after addition of Dox,
and staining was maximal from 24 days; we chose 2 days of induction
for all experi-ments.
The induction of RESP18 expression in iRESP cells was assessed by
Northern blotting and biosynthetic labeling (Fig. 3
). Northern blot analysis showed a
dose-dependent induction of RESP18 mRNA in iRESP cells after growth for
2 days in medium containing Dox (Fig. 3A
). Maximal induction of RESP18
mRNA [20 ± 6 fold (n = 3)] was observed with Dox
concentrations of 1000 ng/ml or higher. Biosynthetic labeling of wild
type AtT-20 cells showed that the synthetic rate of RESP18 protein in
wild type AtT-20 cells was not affected by growth in medium containing
4000 ng/ml Dox, a concentration higher than that used for subsequent
experiments (Fig. 3B
). Addition of 4000 ng/ml Dox did not affect the
synthesis of total cellular protein as judged by scintillation counting
of total radiolabeled cellular protein precipitated with
trichloroacetic acid (not shown). The synthetic rate of RESP18 protein
in iRESP cells grown under basal conditions was similar to that of wild
type AtT-20 cells (Fig. 3B
). The iRESP cells were treated with
increasing concentrations of Dox for 2 days; enhancement of RESP18
protein synthesis was observed starting at 10 ng/ml Dox. Dox
concentrations of 500-2000 ng/ml induced a maximal increase in RESP18
biosynthesis of 45 ± 3 fold (n = 3). In previously
characterized AtT-20-RESP cells constitutively overexpressing RESP18, a
24-fold increase in RESP18 biosynthesis was observed when compared with
wild type AtT-20 cells (12).

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Figure 3. Inducible Expression of RESP18 in iRESP Cells
AtT-20 and iRESP cells were treated with Dox for 2 days at the
concentrations (ng/ml) indicated (panels A and B). Cells were analyzed
by Northern blotting (A) or biosynthetic labeling (B). For Northern
blot analysis, 10 µg total RNA were loaded in each lane; the blot was
hybridized with a [32P]dCTP-labeled RESP18 cDNA probe.
Endogenous and exogenous RESP18 mRNA (0.8 kb) comigrate on this
Northern blot. For biosynthetic labeling, cells were harvested after a
15-min incubation in CSFM-Air medium containing
[35S]-Met/Cys (pulse), and equal amounts of labeled cell
extracts were immunoprecipitated. Wild type AtT-20 cells (treated with
0 and 4000 ng/ml Dox) were analyzed as a control.
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Use of Subtractive Hybridization to Analyze the Effect of RESP18
Induction
We then compared noninduced and induced iRESP cells using
PCR-based subtractive hybridization (26, 27). For iRESP cells, cDNA
fragments derived from noninduced cells were subtracted from those of
induced cells for six successive rounds (samples R+1 to R+6) to yield
cDNA samples enriched for transcripts potentially up-regulated by
RESP18 expression. A similar experimental paradigm was used to generate
a cDNA sample enriched for transcripts potentially down-regulated by
RESP18 expression (samples R-1 to R-3). To monitor the efficacy of the
subtraction, equal amounts of cDNAs from each round of subtraction were
analyzed by Southern blot (Fig. 4A
). When
the blot was hybridized with R+6 cDNA probe, an enrichment of
up-regulated cDNAs was observed after each round of subtraction (R+1 to
R+6; Fig. 4A
, upper). For transcripts down-regulated by
induction of RESP18 (R-1 to R-3), no enriched cDNAs were observed with
the R+6 probe, indicating that the enriched cDNA populations for
up-regulated and down-regulated transcripts do not cross-react. The
levels of S26, a ribosomal protein (28), are unaffected by secretagogue
treatment, and levels of POMC biosynthesis are unaffected by induction
of RESP18 expression (not shown). Thus both transcripts should be
absent from the enriched cDNA samples; consistent with this, the R+6
probe did not hybridize to plasmids encoding S26 or POMC (Fig. 4A
;
pBS.S26, pBS.POMC). As expected, the R+6 probe recognized RESP18
plasmid digested with the same restriction endonucleases used to
prepare the enriched cDNA samples, indicating the presence of RESP18 in
the R+6 cDNA. Fragments of the RESP18 cDNA should be highly enriched in
R+6 since RESP18 expression was induced upon treatment of the cells
with Dox. Probing the same Southern blot with labeled RESP18 cDNA
demonstrated enrichment of RESP18 in the same samples recognized by the
R+6 probe (Fig. 4A
, lower).

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Figure 4. Southern and Northern Blot Analyses Show Regulation
of Transcripts in Parallel to Induction of RESP18 Expression in iRESP
Cells
Southern blot analysis of cDNA subtraction using R+6 (A,
upper) or RESP18 (A, lower) probes.
Amplified cDNA fragments from noninduced and subtracted iRESP samples
(1 µg) were fractionated on a 1.4% agarose gel and analyzed by
Southern blot with the probes indicated. The number of subtractions for
transcripts up-regulated by RESP18 expression (noninduced cell cDNAs
subtracted from induced cell cDNAs) or transcripts down-regulated by
RESP18 expression (induced cell cDNAs subtracted from noninduced cell
cDNAs) are indicated. Plasmids (pBS.S26, pBS.POMC, pBS.RESP) and
plasmids digested with AluI and RsaI
(*) were analyzed as controls (1 µg). The digested
pBS.RESP sample showed the expected 464-bp fragment deduced from
restriction sites in the RESP18 cDNA. (B) Total RNA (10 µg) from
iRESP or iPAM cells treated with 0 (-) or 500 ng/ml Dox (+) for 2 days
was fractionated on a denaturing agarose/formaldehyde gel and probed
with [32P]dCTP-labeled probes created from the R-3 or R+6
enriched cDNAs, PAM-1 cDNA, or RESP18 cDNA. The migration of DNA
standards and rRNA (18S and 28S) are indicated.
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Northern blot analysis was used to determine whether the enriched R+6
cDNAs reflected transcripts regulated with RESP18 levels in iRESP
cells; induction of PAM in iPAM cells was analyzed as a control (Fig. 4B
). The R+6 probe showed increased hybridization with several mRNAs in
iRESP cells treated with Dox to induce expression of RESP18 protein but
not in iPAM cells treated with Dox to induce expression of PAM protein.
This suggests that the levels of several transcripts were specifically
responsive to expression of RESP18 in AtT-20 cells. Analysis of the
same Northern blots with the R-3 cDNA probe, which should be enriched
for transcripts potentially down-regulated by RESP18 expression, showed
no significantly altered hybridization in the iPAM or iRESP cell lines
upon induction of the exogenous protein; this observation indicates
that there were no sufficiently abundant transcripts whose levels
decreased in response to expression of RESP18. Hybridization of the
same Northern blots with RESP18 or PAM probes verified a 7.5- and
6.0-fold increase in the levels of RESP18 mRNA and PAM mRNA,
respectively, upon treatment of the inducible cell lines with Dox.
Immunoprecipitable RESP18 and PAM-1 protein each account for about
0.5% of the total protein synthesized in a 15-min pulse sample from
the two induced cell lines. Thus these results indicate that the
transcripts up-regulated upon induction of RESP18 expression were
specific to induction of RESP18 in iRESP cells and were not associated
with the inducible expression system or with the overexpression of
another secretory pathway protein.
The Effect of RESP18 Expression on Levels of Several Transcripts
Demonstrates Signaling from the Secretory Pathway Lumen to the
Nucleus
We then investigated individual transcripts that were responsive
to induction of RESP18 expression in iRESP cells. The R+6 cDNAs were
cloned into a pBS vector yielding the R+6 cDNA library. Of the 3500
colonies analyzed, approximately 8% lacked an insert or contained a
small insert in frame with lacZ; as expected, RESP18 was the
most abundant cDNA in the library (29% of the colonies, Table 1
). Screening of 3500 colonies revealed
33 distinct cDNA fragments: 19 cDNA fragments hybridized with mRNAs
specifically regulated by RESP18 levels in iRESP cells; 10 cDNA
fragments hybridized detectably with mRNAs not regulated by RESP18
levels; four cDNA fragments did not detectably hybridize to mRNAs when
10 µg total RNA were analyzed, presumably due to their low abundance.
Figure 5
shows representative Northern
blots probed with four cDNA fragments that recognize transcripts
regulated by RESP18 induction in iRESP cells and not by PAM induction
in iPAM cells (clones 1, 4, 12, 22) and with a cDNA fragment that
recognizes a transcript that was not responsive to RESP18 induction
(clone 31).

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Figure 5. Northern Blot Analysis Shows Several Transcripts
are Responsive to Induction of RESP18 Expression in iRESP Cells
A, Northern blots identical to those shown in Fig. 4B were probed with
[32P]dCTP-labeled cDNA probes generated from selected
clones. The migration of 18S and 28S rRNA are indicated; an
asterisk indicates transcripts regulated upon induction
of RESP18 expression in iRESP cells, but not by PAM-1 levels in iPAM
cells. B, iRESP cells were induced with 2 µg/ml Dox (+) in the
absence or presence of two different RESP18 antisera (JH1162 or JH1163;
20 µl antisera/ml medium) as indicated; noninduced iRESP cells are
shown for a comparison (-). Total RNAs (10 µg) from these samples
were analyzed by Northern blot using probes to RRT4, RRT38, RESP18, and
S26 as indicated.
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To determine whether RESP18 induction of selected transcripts required
secretion of RESP18 into the medium, we employed antisera directed to
the N- and C-terminal regions of RESP18 (JH1162 and JH1163,
respectively; Ref.24) (Fig. 5B
). When RESP18 antisera were added to
the media of iRESP cells during induction of RESP18 with Dox, RRT4 and
RRT38 mRNAs were still induced. Probing parallel Northern blots with
S26 verifies equal loading of RNA in each lane, and probing with RESP18
verifies that the presence of RESP18 antisera does not diminish RESP18
induction in iRESP cells. These observations indicate that the actions
of RESP18 occur from within the cell and that RESP18 is not acting
after secretion as an autocrine hormone.
Since the levels of RESP18, a secretory pathway luminal protein,
affected the levels of numerous transcripts, our hypothesis is that a
signal is sent from the lumen to the nucleus. Criteria similar to those
presented here were involved in the initial discovery of the UPR (2).
Several conditions known to stimulate the unfolded protein or
NF-
B-dependent ER to nuclear-signaling pathways had no affect on the
synthesis or half-life of RESP18 protein, suggesting that the signal
involving RESP18 is distinct (12, 29, 30). Furthermore, the unfolded
protein and NF-
B-dependent signaling pathways are thought to be
ubiquitous while RESP18 expression is limited to neuroendocrine tissues
(12, 29, 30). Since the actions of RESP18 are similar to those of a
hormone or neurotransmitter but acting from within the lumen of the
secretory pathway, RESP18 would be a lumicrine (lumen + autocrine)
protein. The transcripts responsive to RESP18 induction will be
referred to hereafter as RESP18-responsive transcripts (RRTs).
Some RRTs are Regulated by Dopaminergic Drugs in Melanotropes
The iRESP cells were engineered for inducible RESP18 expression;
we sought to determine whether the RRTs were coordinately regulated
with RESP18 in vivo. Since RESP18 mRNA levels are increased
in rat pituitary melanotropes by haloperidol (a dopamine antagonist)
treatment and decreased by bromocriptine (a dopamine agonist)
treatment, we thought that regulation of RRTs might also be under
dopaminergic control in melanotropes. Rats were treated daily with
vehicle, bromocriptine, or haloperidol for 3 weeks. Total RNA from the
neurointermediate pituitaries was analyzed by Northern blot using
several labeled RRT cDNA probes (Fig. 6A
). As previously reported, levels of
POMC and RESP18 mRNA in haloperidol-treated animals were slightly
higher than control animals, while POMC and RESP18 mRNA levels were
greatly reduced in rats treated with bromocriptine (11, 31, 32). We
examined 10 RRTs and found that the two RRTs that were detectable by
Northern blot analysis using 2 µg total neurointermediate pituitary
RNA were also regulated by dopaminergic drugs. Probing a parallel blot
with S26 shows equal loading of RNA in all lanes. Levels of some RRTs
also paralleled the response of RESP18 to dexamethasone treatment of
AtT-20 cells and to insulin, estradiol, and epidermal growth factor
treatment of GH3 cells (11, 33); these physiological
stimuli brought about parallel changes in RESP18 and RRT expression in
these two endocrine cell lines (not shown). Taken together, the
observation of a RESP18-dependent signal in iRESP cells, and the
parallel responses of RESP18 and several RRTs in melanotropes and
RESP18 with some RRTs after hormonal treatment of AtT-20 and
GH3 cells, suggest that RESP18 itself may play a role in
signaling in vivo.

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Figure 6. Selected Transcripts Are Responsive to RESP18
Induction in an in Vivo System
A, Rats were treated daily with bromocriptine, haloperidol, or vehicle
for 3 weeks. Rats were killed, total RNA was prepared from pituitary
neurointermediate lobes, fractionated on a denaturing agarose gel,
transferred, and analyzed by Northern blot with
[32P]dCTP-labeled cDNA probes as indicated. Each lane
contains 2 µg total RNA. B, Northern blots from Fig. 5A were probed
with [32P]dCTP-labeled cDNA probes specific for several
transcripts known to be regulated by dopaminergic drugs in the
intermediate pituitary (11).
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Since some RRTs and RESP18 were under dopaminergic control in
intermediate pituitary melanotropes in vivo, we examined the
iRESP cells for changes in the levels of several other transcripts
whose levels are known to be regulated by dopaminergic drugs in
melanotropes [Ref. 31 and Fig. 6B
). Northern blot analysis
demonstrated that none of the eight transcripts examined was noticeably
altered upon induction of iRESP or iPAM cells; the S26 probe
demonstrates equal loading of RNA samples. This observation suggests
that, in the response of intermediate pituitary melanotropes to
dopamine, RESP18 is coordinately expressed with some RRTs or is a
downstream regulator of a subset of dopamine-regulated transcripts. In
addition, the fact that the levels of BiP are not affected by the
levels of RESP18 or PAM suggests that RESP18 does not influence the UPR
(Fig. 6B
) (2). Although RESP18 is a neuroendocrine-specific protein and
the NF-
B dependent pathway is present in other cell types, we cannot
completely rule out the possibility that RESP18 activates the NF-
B
dependent pathway.
Sequence Homology and Tissue Localization of Three RRTs
We investigated the nature of individual RRTs by DNA
sequencing, examining for homology to known proteins, and by
determining sites of expression by Northern blot analysis of several
rat tissues; samples of these analyses are shown in Fig. 7
. The subtractive hybridization protocol
involves generation of small cDNA fragments; RRTs ranged from 100500
bp in length. Several RRTs had amino acid homology to sequences in the
EST database but did not have significant homology to genes of known
function. Northern blot analysis of rat tissues visualized with a cDNA
probe specific for RRT35 exhibited a distribution of a 1.3-kb mRNA very
similar to that of RESP18, showing strong expression in neuroendocrine
tissues (Fig. 7A
). Clone 40 (RRT40) encoded a 22-amino acid peptide
with exact identity to a LIM domain in rat LIMK2 (Fig. 7B
and Ref.34).
LIM domains are double zinc finger domains that mediate protein-protein
interactions and are found in homeobox proteins, cytoskeletal proteins,
protein kinases, and LIM-only proteins (35). LIMK2 is a kinase
containing LIM domains that exhibits heterotypic interactions with
another family member LIMK1; LIMK1 exhibits homotypic interactions and
also binds some protein kinase C isoforms (36, 37).

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Figure 7. Identification and Tissue Distribution of Select
RRTs
A, Northern blot analysis of total RNA (10 µg) from selected
rat tissues using labeled RRT35 probe. RRT35 is not homologous to any
protein in the nonredundant database (8/97). B, The primary sequence of
RRT40 is identical to that of the kinase rat LIMK2
(shaded) in the second LIM domain; gray
letters indicate amino acids conserved among the second LIM
domain in all LIMKs (34, 4651). C, RRT17 shows 59% amino acid
identity to the N terminus of three bacterial isocitrate
dehydrogenases; bold letters are those residues
conserved among RRT17 and the three monomeric isocitrate dehydrogenases
(38, 52, 53). The single letter amino acid code is used.
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Another cDNA, RRT17, encoded 110 amino acids showing a 59%
identity to three bacterial monomeric isocitrate dehydrogenases (Fig. 7C
). Levels of RRT17 transcripts are regulated by dopaminergic drugs in
the intermediate pituitary (Fig. 6A
), and RRT17 may represent a novel
inducible mammalian isocitrate dehydrogenase. Isocitrate dehydrogenase
is part of the biosynthetic pathway for the production of glutamate in
Corynebacterium glutamicium, an organism used industrially
because it secretes glutamate (38). One could postulate such a
biosynthetic role in the production of several neurotransmitter
precursors and amino acid building blocks for the production of
prohormones. Furthermore, it is interesting that this protein is under
dopaminergic control in melanotropes (probe 17), as is cytochrome C
oxidase subunit III, proteins that may alter the utilization of carbon
sources (31).
In summary, subtractive hybridization of an inducible cell line
has demonstrated a novel role for RESP18 in signaling from the
secretory pathway lumen to the nucleus in neuroendocrine cells. The
approach described here should be broadly applicable to the study of
proteins involved in signal transduction, identification of the
regulated transcripts of signaling pathways, and the study of novel
cDNA sequences, especially those involved in signal transduction. The
coordinate regulated expression of RESP18 with some RESP18-regulated
transcripts in several in vitro and in vivo
systems suggests physiological relevance of the RESP18-dependent
signal. Since RESP18 acts as an autocrine hormone from within the lumen
of the secretory pathway, RESP18 is a lumicrine hormone. The homology
of RESP18 to the N terminus of a select group of protein tyrosine
phosphatases will direct future studies toward unraveling the mechanism
of the RESP18 signaling response.
 |
Materials and Methods
|
---|
Animals
Adult male Sprague-Dawley rats (150200 g) obtained from
Charles River (Raleigh, NC) were treated with bromocriptine,
haloperidol, or vehicle for 3 weeks as previously described (11). Total
RNA was prepared from neurointermediate pituitaries. All animal
procedures were approved by the Johns Hopkins University Institutional
Animal Care and Use Committee.
Generation of pUHD Constructs
The RESP18 cDNA insert was prepared from pBS.RESP by digestion
with Bgl2, treatment with the Klenow polymerase to fill in
the 5'-overhang (39), and digestion with EcoRI. The RESP18
cDNA was ligated into the pUHD103 vector (gift from Dr. Manfred
Bujard) prepared by digesting with XbaI, filling in the
5'-overhang with Klenow polymerase, and digesting with EcoRI
(pUHD.RESP). To generate the rat PAM-1 (rPAM-1) cDNA insert, pBS.rPAM-1
was digested with ApaI, treated with the Klenow polymerase,
and digested with XbaI and PvuI. The prepared
rPAM-1 cDNA was ligated into the pUHD103 vector prepared by digestion
with SacII, treatment with Klenow polymerase, and digestion
with EcoRI (pUHD.PAM). The rat RESP18 and rPAM-1 cDNA
insertion sites were confirmed by DNA sequencing using the dideoxy
chain termination method (Sequenase System 2; United States
Biochemical, Cleveland, OH). Plasmids for transfection were prepared
according to the Qiagen maxiprep protocol (Chatsworth, CA).
Cell Culture/Transfection and in Situ
Hybridization
Wild type AtT-20/D-16v cells were grown and transfected as
previously described (22). AtT-20 cells were transfected with the rTet
expression system devised by Gossen et al. (21) for
inducible expression of RESP18 or rPAM-1. AtT-20 cells were first
transfected with pUHD172.neo, and lines containing plasmid were
selected with G418 (0.5 mg/ml). Clones constitutively expressing the
tetracycline-controlled transactivator (rtTA) were screened by slot
blot and Northern blot for RNA using a rtTA cDNA probe prepared from
pUHD172.neo plasmid by random primed labeling with
[32P]dCTP.
Clonal pUHD cell lines were obtained by subcloning and screened by
in situ hybridization. In situ hybridization was
as previously described with minor modifications (40). A 5-min wash
with 2x saline-sodium citrate/50% formamide at 52 C was added after
the RNase digestion step. Also, digoxigenen-UTP-labeled sense and
antisense rtTA riboprobes were synthesized, quantified, and used
(instead of [35S]-labeled probes) as described by
Boehringer Mannhiem (Indianapolis, IN).
The pUHD.RESP and pUHD.PAM vectors were separately cotransfected into
AtT/pUHD cells with pSCEP (23). Cells containing plasmid were selected
by growth in medium containing hygromycin B (200 U/ml; Sigma) and G418
and subcloned as required. The resulting iRESP and iPAM lines were
evaluated by immunostaining with RESP18 and PAM antisera, respectively.
When grown in medium containing FCS and NuSerum (used for transfection,
screening, and subcloning), all clones exhibited significant expression
of RESP18 or PAM-1. Growth of cell lines in DMEM:F12 medium containing
sera from donor herds (10% donor horse serum and 10% donor FCS with
iron; GIBCO/BRL; Gaithersburg, MD) resulted in low basal and highly
inducible expression of RESP18 or PAM-1 in several lines; donor herds
are not treated with tetracycline.
Northern Blotting, Southern Blotting, Biosynthetic Labeling, and
Immunoprecipitation
Preparation of RNA and Northern blot analysis were described
previously (13). For Southern and Northern blot analyses (12, 41),
radiolabeled [32P]dCTP probes were prepared by random
priming using the Stratagene PRIME-IT II kit (La Jolla, CA).
Biosynthetic labeling and immunoprecipitation were carried out as
previously described with minor modifications (12, 24). AtT-20, iRESP,
and iPAM cells were biosynthetically labeled by incubation with
CSFM-Air (complete serum-free media without bicarbonate buffer)
medium containing [35S]Met/Cys for 15 min. The indicated
concentration of Dox was added to all media used for metabolic labeling
studies of induced cells. Cell extracts were prepared in acetic acid,
and spent media were centrifuged to remove cell debris. For RESP18
immunoprecipitation, rabbit polyclonal antiserum directed toward the N
terminus (JH1162) was used (24).
Generation of Subtracted cDNAs and Libraries
Subtracted libraries were prepared by modifying previously
described procedures (26, 27). iRESP cells maintained in
tetracycline-free media (noninduced) were induced with 500 ng/ml Dox
for 48 h. For noninduced and induced iRESP cells, poly
A+ RNAs were prepared directly from cells using the
Microfasttrack mRNA Isolation Kit (Invitrogen; San Diego, CA). Poly
A+ mRNAs (0.5 µg) from each sample were used to prepare
single-strand cDNAs using the cDNA cycle kit and priming with oligo-dT
(Invitrogen); the second strands were synthesized by conventional
means. The double-strand cDNAs were fragmented using
AluI and RsaI and then li-gated to linkers.
Linker A was prepared from synthetic oligonucleotides
(5'-TAGTCCGAATTCAAGCAAGAGCACA-3' and 5'-CTCTTGCTTGAATTCGGACTA-3') and
ligated to fragmented cDNAs derived from induced iRESP cells as
previously described (26). Linker B was prepared in a similar manner
from the synthetic complementary oligonucleotides,
5'-ATGTCCGGATCCGCGAAGCTTCACA-3' and 5'-AAGCTTCGCGGATCCGGACAT-3' and
ligated to the fragmented cDNAs derived from the noninduced iRESP
cells. This two-linker approach is a modification suggested by Patel
and Sive (42). Free nucleotides and excess linkers were removed from
ligation reactions using Qiaquick PCR purification cartridges according
to the manufacturers protocol (Qiagen). The fragmented cDNA samples
were amplified by PCR using oligonucleotides from the appropriate
linker, and amplified cDNAs were isolated using Qiaquick cartridges as
above. To generate biotinylated DNAs for subtraction, cDNA samples were
photobiotinylated twice, as previously described (43).
To enrich cDNAs for transcripts up-regulated by RESP18 expression, 3
µg of the amplified cDNAs from induced iRESP cells were hybridized
with 9 µg of amplified biotinylated cDNAs from noninduced iRESP
cells; biotinylated cDNAs were removed using streptavidin as described
(43). The first round of subtraction was completed by removing 15 µl
of subtracted cDNAs, diluting to 150 µl with Tris-EDTA buffer, and
amplifying 3 µl of the sample by PCR as above, yielding the R+1 cDNAs
[iRESP cells for up-regulated transcripts (+), 1 round of
subtraction]. The same experimental paradigm was used to generate
enriched cDNAs for transcripts down-regulated by RESP18 expression in
iRESP cells (R-1). As previously described (26) and as modified above,
the same experimental paradigm was used to generate enriched cDNAs
for two to six rounds of subtraction for any transcripts up-regulated
by RESP18 (R+2 to R+6) or down-regulated by RESP18 (R-2 to R-3).
A plasmid library of the R+6 cDNAs was prepared. The R+6 cDNAs were cut
at the EcoRI site in the linker used for subtractive
hybridization. The cDNA fragments were ligated to pBS(SK) vector
prepared by digestion with EcoRI and treated with shrimp
alkaline phosphatase (New England Biolabs, Beverly, MA). The library
was titered and plated at 800 colonies per 150-mm plate. Clones
containing no insert or small inserts (
270 of 3500) were eliminated
by a blue/white screen using 5-bromo-4-chloro-3-indolyl
ß-D-galactopyranoside (X-gal) and isopropyl
ß-thiogalactopyranoside (IPTG). Colony lifts were used to eliminate
colonies containing RESP18 inserts (
1015 of 3500). Each round of
screening was accomplished by DNA sequencing 816 of the remaining
clones and eliminating further analysis of similar clones by colony
lift. Of the 4000 clones plated, 3500 are represented by the clones
reported here.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Jimo Borjigin for the subtractive hybridization
protocol, Jennifer Brakeman for assistance with in situ
hybridization, Drs. Manfred Bujard and Hermann Gossen for the rTet
inducible expression system, and Carla Berard, Rich Johnson, Andrew
Quon, Marie Bell, Dr. Dan Darlington, Adnan Malik, and Dr. Giuseppe
Ciccotosto for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Betty A. Eipper, The Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205-2105.
This work was supported by Grants DA-00266, DA-05540, and DA-10478 from
the National Institute of Drug Abuse.
Received for publication April 23, 1997.
Revision received August 21, 1997.
Accepted for publication August 25, 1997.
 |
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