A Novel Neuroendocrine Intracellular Signaling Pathway

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

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. (Molecular Endocrinology 11: 1846–1857, 1997)

INTRODUCTION 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 un0888-8809/97/$3.00/0 Molecular Endocrinology Copyright © 1997 by The Endocrine Society

folded 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. 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-kB, 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 sterolregulatory 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 1846


Fig. 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.

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

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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 (8–10). 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 (11–13). 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] (14–17). 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.

RESULTS AND DISCUSSION 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.

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Generation and Characterization of Inducible RESP18 and Inducible Peptidylglycine aAmidating 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 ascorbatedependent oxidation of peptide C-terminal glycyl residues into amides, releasing glyoxylate (22). AtT-20 cells were first transfected with the pUHD17–2 vector that directs constitutive expression of a tetracyclinecontrolled 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). 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-20inducible(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 0–6 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

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addition of Dox, and staining was maximal from 2–4 days; we chose 2 days of induction for all experiments. 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 6 fold (n 5 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 6 3 fold (n 5 3). In previously characterized AtT-20RESP cells constitutively overexpressing RESP18, a 24-fold increase in RESP18 biosynthesis was observed when compared with wild type AtT-20 cells (12). 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 R11 to R16) 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 R16 cDNA probe, an enrichment of up-regulated cDNAs was observed after each round of subtraction (R11 to R16; Fig. 4A, upper). For transcripts down-regulated by induction of RESP18 (R-1 to R-3), no enriched cDNAs were observed with the R16 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


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Fig. 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 mg 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 mM.

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Fig. 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 mg 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.

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 R16 probe did not hybridize to plasmids encoding S26 or POMC (Fig. 4A; pBS.S26, pBS.POMC). As expected, the R16 probe recognized RESP18 plasmid digested with the same restriction endonucleases used to prepare the enriched cDNA samples, indicating the presence of RESP18 in the R16 cDNA. Fragments of the RESP18 cDNA should be highly enriched in R16 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 R16 probe (Fig. 4A, lower). Northern blot analysis was used to determine whether the enriched R16 cDNAs reflected transcripts regulated with RESP18 levels in iRESP cells; induction of PAM in iPAM cells was analyzed as a control (Fig. 4B). The R16 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.


Fig. 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 R16 (A, upper) or RESP18 (A, lower) probes. Amplified cDNA fragments from noninduced and subtracted iRESP samples (1 mg) 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 mg). The digested pBS.RESP sample showed the expected 464-bp fragment deduced from restriction sites in the RESP18 cDNA. (B) Total RNA (10 mg) from iRESP or iPAM cells treated with 0 (2) or 500 ng/ml Dox (1) for 2 days was fractionated on a denaturing agarose/formaldehyde gel and probed with [32P]dCTP-labeled probes created from the R-3 or R16 enriched cDNAs, PAM-1 cDNA, or RESP18 cDNA. The migration of DNA standards and rRNA (18S and 28S) are indicated.

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

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iRESP cells. The R16 cDNAs were cloned into a pBS vector yielding the R16 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 mg 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). 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

Table 1. Summary of R 1 6 Library Screening Category a

Clones (total) Clones with insertsa RESP18 clonesb Clones sequencedc Clones regulated with RESP18 (RRTs) Novel Homology to known genes Clones not regulated with RESP18 Novel Homology to known genes Clones sequenced but not detected by Northern blot analysis of total RNA a

No. of clones

;3500 ;3220 ;1015 33 19 10 9 10 2 8 4

Estimated number of colonies deduced from a blue/white screen. b Estimated from colony lifts. c These 33 transcripts were multiply represented in the library and accounted for ;2200 colonies identified by colony lift.

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Fig. 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 mg/ml Dox (1) in the absence or presence of two different RESP18 antisera (JH1162 or JH1163; 20 ml antisera/ml medium) as indicated; noninduced iRESP cells are shown for a comparison (2). Total RNAs (10 mg) from these samples were analyzed by Northern blot using probes to RRT4, RRT38, RESP18, and S26 as indicated.

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-kB-dependent ER to nuclearsignaling 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-kB-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 1 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 mg 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 RESP18dependent 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. 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


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Fig. 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 mg 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).

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-kB dependent pathway is present in other cell types, we cannot completely rule out the possibility that RESP18 activates the NF-kB 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 100–500 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 visu-

alized 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). 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 biosyn-

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Fig. 7. Identification and Tissue Distribution of Select RRTs A, Northern blot analysis of total RNA (10 mg) 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, 46–51). 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.

thetic 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 (150–200 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 59-overhang (39), and digestion with EcoRI. The RESP18 cDNA was ligated into the pUHD10–3 vector (gift from Dr. Manfred Bujard) prepared by digesting with XbaI, filling in the 59-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 pUHD10–3 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 pUHD17–2.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 pUHD17–2.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 23 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 A1 RNAs were prepared directly from cells using the Microfasttrack mRNA Isolation Kit (Invitrogen; San Diego, CA). Poly A1 mRNAs (0.5 mg) 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 ligated to linkers. Linker A was prepared from synthetic oligonucleotides (59-TAGTCCGAATTCAAGCAAGAGCACA-39 and 59-CTCTTGCTTGAATTCGGACTA-39) 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, 59-ATGTCCGGATCCGCGAAGCTTCACA-39 and 59-AAGCTTCGCGGATCCGGACAT-39 and ligated to the fragmented cDNAs derived from the noninduced iRESP cells. This twolinker 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 manufacturer’s protocol (Qiagen). The fragmented cDNA samples were amplified by PCR using oligo-

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nucleotides 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 mg of the amplified cDNAs from induced iRESP cells were hybridized with 9 mg 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 ml of subtracted cDNAs, diluting to 150 ml with Tris-EDTA buffer, and amplifying 3 ml of the sample by PCR as above, yielding the R11 cDNAs [iRESP cells for up-regulated transcripts (1), 1 round of subtraction]. The same experimental paradigm was used to generate enriched cDNAs for transcripts downregulated 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 (R12 to R16) or down-regulated by RESP18 (R-2 to R-3). A plasmid library of the R16 cDNAs was prepared. The R16 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 b-D-galactopyranoside (X-gal) and isopropyl b-thiogalactopyranoside (IPTG). Colony lifts were used to eliminate colonies containing RESP18 inserts (;1015 of 3500). Each round of screening was accomplished by DNA sequencing 8–16 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.

Received April 23, 1997. Revision received August 21, 1997. Accepted August 25, 1997. 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.

REFERENCES 1. Nunnarri J, Walter P 1996 Regulation of organelle biogenesis. Cell 84:389–394 2. Kozutsumi Y, Segal M, Normington K, Gething M-J, Sambrook J 1988 The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332:462–464 3. Mori K, Ma W, Gething M-J, Sambrook J 1993 A transmembrane protein with a cdc21/CDC28-related kinase

MOL ENDO · 1997 1856

4.

5. 6.

7.

8. 9. 10.

11.

12.

13. 14.

15.

16.

17.

18.

19. 20. 21. 22.

activity is required for signaling from the ER to the nucleus. Cell 74:743–756 Cox JS, Shamu CE, Walter P 1993 Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73:1197–1206 Pahl HL, Baeuerle PA 1997 Endoplasmic-reticulum-induced signal transduction and gene expression. Trends Cell Biol 7:50–55 Srivastava CH, Davies MV, Kaufman RJ 1995 Calcium depletion from the endoplasmic reticulum activates the double-stranded RNA-dependent protein kinase (PKR) to inhibit protein synthesis. J Biol Chem 270:16619–16624 Oyarce AM, Hand TA, Mains RE, Eipper BA 1996 Dopaminergic regulation of secretory granule-associated proteins in the rat intermediate pituitary. J Neurochem 67:229–241 Guest PC, Rhodes CJ, Hutton JC 1989 Regulation of the biosynthesis of insulin-secretory-granule proteins. Biochem J 257:431–437 Guest PC, Bailyes EM, Rutherford NG, Hutton JC 1991 Insulin secretory vesicle biogenesis. Biochem J 274: 73–78 Alarcon C, Lincoln B, Rhodes CJ 1993 The biosynthesis of the subtilisin-related proprotein convertase PC3, but not that of the PC2 convertase, is regulated by glucose in parallel to the proinsulin biosynthesis in rat pancreatic Islets. J Biol Chem 268:4276–4280 Bloomquist BT, Darlington DN, Mains RE, Eipper BA 1994 RESP18, a novel endocrine secretory protein transcript, and four other transcripts are regulated in parallel with pro-opiomelanocortin in melanotropes. J Biol Chem 269:9113–9122 Schiller MR, Mains RE, Eipper BA 1995 A neuroendocrine-specific protein localized to the endoplasmic reticulum by distal degradation. J Biol Chem 270:26129– 26138 Schiller MR, Darlington DN 1996 The stage-specific expression of RESP18 in the testes. J Histochem Cytochem 44:1489–1496 Lan MS, Lu J, Goto Y, Notkins AL 1994 Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 13:505–514 Lu J, Notkins AL, Lan MS 1994 Isolation, sequence, and expression of a novel mouse brain cDNA, mIA-2, and its relatedness to members of the protein tyrosine phosphatase family. Biochem Biophys Res Commun 204: 930–936 Passini N, Larican JD, Genovese S, Appella E, Sinigaglia F, Rogge L 1995 The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2. Proc Natl Acad Sci USA 92:9412–9416 Wasmeier C, Hutton JC 1996 Molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes. J Biol Chem 271:18161–18170 Solimena M, Dirkx RJ, Hermel J, Pleasic-Willliams S, Shapiro JA, Caron L, Rabin DU 1996 ICA512, an autoantigen of type 1 diabetes, is an intrinsic membrane protein of neurosecretory granules. EMBO J 15:2102–2114 Streuli M 1996 Protein tyrosine phosphatases in signaling. Curr Opin Cell Biol 8:182–188 Gossen M, Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551 Gossen M, Bolin AL, Bujard H 1993 Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci 18:471–475 Milgram SL, Johnson RC, Mains RE 1992 Expression of individual forms of peptidylglycine a-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and

Vol 11 No. 12

routing to secretory granules. J Cell Biol 117:717–28 23. Paquet L, Zhou A, Chang EY, Mains RE 1996 Peptide biosynthetic processing: distinguishing prohormone convertases PC1 and PC2. Mol Cell Endocrinol 120:161– 168 24. Bloomquist BT, Darlington DN, Mueller GP, Mains RE, Eipper BA 1994 Regulated endocrine specific protein-18: a short-lived novel glucocorticoid-regulated endocrine protein. Endocrinology 135:2714–2722 25. Milgram SL, Chang EY, Mains RE 1996 Processing and routing of a membrane-anchored form of proneuropeptide Y. Mol Endocrinol 10:837–846 26. Wang Z, Brown DD 1991 A gene expression screen. Proc Natl Acad Sci USA 88:11505–11509 27. Borjigin J, Wang MM, Snyder SH 1995 Diurnal variation in mRNA encoding serotonin N-acetyltranferase in pineal gland. Nature 378:783–785 28. Vincent S, Marty L, Fort P 1993 S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eukaryotic cells and tissues. Nucleic Acids Res 21: 1498 29. Pahl HL, Baeuerle PA 1995 A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factor NF-kB. EMBO J 14:2580–2588 30. Shamu CE, Cox JS, Walter P 1994 The unfolded-proteinresponse pathway in yeast. Trends Cell Biol 4:56–60 31. Bloomquist BT, Eipper BA, Mains RE 1991 Prohormoneconverting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol 5:2014–2024 32. Beaulieu M, Goldman ME, Miyazaki K, Frey EA, Eskay RL, Kebabian JW, Cote E 1984 Bromocriptine-induced changes in the biochemistry, physiology, and histology of the intermediate lobe of the rat pituitary gland. Endocrinology 114:1871–1884 33. Scammell JG, Burrage TG, Dannies PS 1986 Hormonal induction of secretory granule in a pituitary tumor cell line. Endocrinology 119:1194–1543 34. Nunoue K, Ohashi K, Okano I, Mizuno K 1995 LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family. Oncogene 11:701–710 35. Sanchez-Garcia I, Rabbitts TH 1994 The LIM domain: a new structural motif found in zinc-finger-like proteins. Trends Genet 10:315–320 36. Hiraoka J, Okano I, Higuchi O, Yang N, Mizuno K 1996 Self-association of LIM-kinase 1 mediated by the interaction between LIM domain and a C-terminal kinase domain. FEBS Lett 399:117–121 37. Kuroda S, Tokunaga C, Kiyohara Y, Higuchi O, Konishi H, Mizuno K, Gill GN, Kikkawa U 1996 Protein-protein interaction of the zinc finger LIM domains with protein kinase C. J Biol Chem 271:31029–31032 38. Eikmanns BJ, Rittmann D, Sahm H 1995 Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum acid gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177:774–782 39. Sambrook I, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 40. Bhat RV, Cole AJ, Baraban JM 1992 Chronic cocaine treatment suppresses basal expression of zif268 in rat forebrain: in situ hybridization studies. J Pharmacol Exp Ther 263:343–249 41. Ouafik L’, Stoffers DA, Campbell TA, Johnson RC, Bloomquist BT, Mains RE, Eipper BA 1992 The multifunctional peptidylglycine a-amidating monooxygenase gene: exon/intron organization of catalytic, processing and routing domains. Mol Endocrinol 6:1571–1584 42. Patel M, Sive H 1994 Current Protocols in Molecular Biology. John Wiley, New York 43. Sive HL, St John T 1988 A simple subtractive hybridization technique employing photoactivatable biotin and


phenol extraction. Nucleic Acids Res 16:10937 44. Cox JS, Walter P 1996 A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87:391–404 45. Sidrauski C, Cox JS, Walter P 1996 tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell 87:405–413 46. Okano I, Hiraoka J, Otera H, Nunoue K, Ohashi K, Iwashita S, Momoki H, Mizuno K 1995 Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs. J Biol Chem 270:31321–32330 47. Bernard O, Ganiatsas S, Kannourakis G, Dringen R 1994 Kiz-1, a protein with LIM zinc finger and kinase domains, is expressed mainly in neurons. Cell Growth Differ 5:1159–1171 48. Osada H, Hasada K, Inazawa J, Uchlida K, Ueda R, Takahashi T 1996 Subcellular localization and protein interaction of the human LIMK2 gene expressing alternative transcripts with ion. Biochem Biophys Res Commun 229:582–589

1857

49. Mizuno K, Okano I, Ohashi K, Nunoue K, Kuma K, Miyata T, Nakamura T 1994 Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif. Oncogene 9:1605–1612 50. Cheng AK, Robertson EJ 1995 The murine LIM-kinase gene (LIMK) encodes a novel serine threonine kinase expressed predominantly in trophoblast giant cells and the developing nervous system. Mech Dev 52:187–197 51. Proschel C, Blouin MJ, Gutowski NJ, Ludwig R, Noble M 1995 LIMK1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro. Oncogene 11:1271–1281 52. Fukunaga N, Imagagawa S, Sahara T, Ishii A, Suzuki M 1992 Purification and characterization of monomeric isocitrate dehydrogenase with NADP1-specificity from Vibrio parahaemolyticus Y-4. J Biochem 849–855 53. Ishii A, Suzuki M, Sahara T, Takada Y, Sasaki S, Fukunaga N 1993 Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 175:6873–6880