A Novel Hematopoietic Granulin Induces Proliferation of Goldfish (Carassius auratus L.) Macrophages*

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

The mammalian progranulin genes are ubiquitously expressed in various tissues (9 -10, 26 -29) and have been detected in epithelial and hematopoietic cell lines (26 -29) and neoplastic cells (30 -36). Progranulin was shown to be highly expressed in epithelial cells that exhibit rapid turnover, such as the columnar epithelium of the gastrointestinal tract (29) and the cells of the immune and nervous systems (21)(22)37).
In general, granulin gene sequences that encode for functional peptides are progranulin genes. There are a number of published granulinlike sequences identified in lower vertebrates as well as invertebrates (e.g. zebrafish, GenBank TM accession numbers AF273479 and AF273480). Although a number of granulin genes have been identified in lower vertebrates and invertebrates, many of the peptides encoded by these genes have yet to be functionally characterized.
We report on a unique granulin-like gene of the goldfish. Northern blot, real-time PCR, and RT-PCR 3 analyses revealed that this granulin gene was expressed exclusively in the hematopoietic tissues of the goldfish. Recombinant goldfish granulin induced dose-dependent proliferation of primary goldfish macrophages in vitro, which was abrogated by an affinity-purified anti-granulin IgG. Our findings indicate that granulin was present in macrophage culture supernatants and that it promoted growth of cells at discrete stages of myeloid differentiation pathway.

EXPERIMENTAL PROCEDURES
Fish-Goldfish (Carassius auratus) were purchased from Mt. Parnell Fisheries Inc. (Mercersburg, PA) and maintained at the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The fish were kept at 20°C in a flow-through water system and fed to satiation daily with trout pellets. The fish were acclimated to this environment for at least 3 weeks prior to use in experiments.
Isolation of Primary Macrophages from Goldfish and RNA Isolation-Isolation of goldfish kidney leukocytes, and the generation of primary kidney macrophages (PKM) and peripheral blood mononuclear cells were performed as previously described (38 -42). The kinetics of PKM growth in culture were similar to those reported for mammalian macrophages derived from bone marrow cultures in the presence of conditioned medium from the L-929 fibroblast cell line (43). Three distinct macrophage subpopulations are a feature of PKM cultures: the early progenitors, the monocytes, and mature macrophages (44,45). PKM cultures were incubated at 20°C until the cells were at a stage of active proliferation (proliferative phase) or nonproliferation (senescence phase), typically 6 and 10 days post-cultivation, respectively. PKM from the proliferative and senescence phases were isolated, flash-frozen using liquid nitrogen, and stored at Ϫ80°C until used. The mRNA for the two macrophage subpopulations was isolated using TRIzol TM reagent (Invitrogen) and the Oligotex mRNA isolation kit (Qiagen) according to the manufacturers' specifications.
Generation of Macrophage-activating Factor (MAF) Supernatants-MAF supernatants were prepared using protocols described previously (41). These supernatants contain a complex mixture of factors that have been functionally characterized and shown to induce antimicrobial responses of goldfish macrophages (41)(42).
Construction of cDNA Libraries of Primary Kidney Macrophages of Goldfish-Complementary DNA libraries were constructed from 2 g of proliferative or senescence phase PKM poly (A)ϩ RNA by directional ligation of PKM cDNA into ZAP bacteriophage using a ZAP cDNA synthesis kit, and the ZAP-cDNA Gigapack III Gold cloning kit (Stratagene) as described previously (46). Non-amplified PKM proliferative and senescence phase cDNA libraries were screened using standard procedures described previously (46). Following the tertiary PCR-based screen, individual clones were PCR-amplified, sequenced, confirmed to encode for a single-sized insert, and stored individually at 4°C in 500 l of SM buffer (50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO 4 , pH 7.5) and chloroform.
DNA Sequencing and Analysis-The PCR-amplified clone inserts corresponding to each of the confirmed granulin positive clones were purified using the QIAquick PCR purification kit (Qiagen) and sequenced using a DYEnamic TM ET terminator cycle sequencing kit (Amersham Biosciences) and a PE-Applied Biosystems 377 automated sequencer. Sequences were analyzed using Genetool TM (Biotools) and subsequent gene annotations were conducted using BLAST programs (www.ncbi.nlm.nih.gov/BLAST/). Conserved motifs were identified, and predictions were based on analytical tools provided in the ExPASy proteomics server (www.expasy.org) (47). Sequence alignments were performed using ClustalX, version 1.83.
Real-time PCR Analysis of Granulin Expression-Real-time PCR analysis was carried out using the Applied Biosystems 7500 Fast realtime PCR system. The relative expression of goldfish granulin in relation to ␤-actin was assessed using primers generated with Primer Express software (Applied Biosystems). The primers used for expression analysis of goldfish granulin were: 5Ј-TTGATGTTACTCATGGCA-GCTCTT-3Ј and 5Ј-GGGCCTGAGAGATCCATCATT-3Ј. The primers used for expression analysis of goldfish ␤-actin were: 5Ј-GCACGC-GACTGACACTGAAG-3Ј and 5Ј-GAAGGCCGCTCCGAGGTA-3Ј. Analysis of the relative tissue expression data from five fish was carried out using the 7500 Fast software (Applied Biosystems).
RT-PCR Analysis of Goldfish Granulin Expression in Macrophages-Cultured PKM were sorted into early progenitor, monocyte, and mature macrophage subpopulations using a FACSCalibur flow cytometer (BD Biosciences) as described previously (39,44,45), and the RNA was isolated immediately after sorting. First-strand synthesis was done using an oligo(dT) primer (Stratagene, La Jolla, CA) with 2.5 g of total RNA according to manufacturer's protocols. The primers used to amplify goldfish granulin by RT-PCR were: sense 5Ј-AAGATGGTTCCAGTG-TTGATGTTAC-3Ј, antisense 5Ј-ACCCCACTGGCCGGCTGCT-GT-3Ј.
Northern Blot Analysis-Twenty five g of total RNA was subjected to electrophoresis on a 1.5% agarose, 20% formaldehyde gel and transferred overnight to Genescreen Plus nylon membranes (PerkinElmer Life Sciences). Blots were screened using 200 ng of a goldfish granulin probe created using RT-PCR. The probe was singly labeled using [␣-32 P]dCTP and purified using QIAquick gel extraction columns (Qiagen). Hybridization with the probe was allowed to proceed overnight at 42°C, and then unbound probe was removed by three consecutive washes of 2ϫ SSC, 0.1% SDS for 5 min each and three times with 0.1ϫ SSC, 0.1% SDS for 20 min. Blots were then exposed to Kodak X-Omat film and stored at Ϫ80°C for 24 h before being developed.
Prokaryotic Expression of Goldfish Granulin-Goldfish granulin was expressed using a prokaryotic protein expression system. PCR amplification of the protein expression construct insert was performed as fol- . PCR amplification was conducted in an Eppendorf Mastercycler Gradient TM thermal cycler. Amplification was confirmed by agarose gel electrophoresis.
The granulin amplicon was cloned into the pET SUMO TA expression vector (Invitrogen) and transformed into chemically competent TOP10 Escherichia coli (Invitrogen) according to the manufacturer's specifications. Cells were plated onto LB-ampicillin (100 g/ml) plates and incubated overnight at 37°C. Randomly selected colonies were amplified by PCR, and positive clones were grown overnight in 5 ml of LB medium containing 100 g/ml ampicillin. Plasmids were isolated using a QIAprep Spin Miniprep kit (Qiagen). Once positive clones were isolated, restriction digests followed by gel electrophoresis verified the presence of insert and vector DNA. Plasmids were sequenced, as described above, to confirm that inserts were ligated into the expression vector in the proper orientation and in-frame. Sequence data were analyzed using Genetool (Biotools).
Production of Recombinant Granulin-Plasmid DNA containing the granulin expression construct was transformed into BL21 Star TM (DE3) One Shot E. coli (Invitrogen) for recombinant protein expression. 10 ng of plasmid DNA was transformed into the bacteria, which was then grown overnight at 37°C in LB medium containing 50 g/ml kanamycin. Induction of recombinant protein expression was performed in a pilot expression experiment by the addition of isopropyl-␤-D-thiogalactopyranoside according to the manufacturer's protocols. The expression of recombinant granulin was evaluated at 1, 2, 4, and 6 h post-induction with isopropyl-␤-D-thiogalactopyranoside. Individual samples were then analyzed by SDS-PAGE and Western blotting for the presence of recombinant protein expression.
For large scale expression and purification of the target proteins, 50 ml of LB medium containing 100 g/ml carbenicillin was grown overnight at 30°C with shaking to an A 600 of ϳ1.0 to 2.0. Ten milliliters of this culture was then inoculated into 250 ml of LB (100 g/ml carbenicillin), and a total of four flasks were prepared (1 liter total). Cultures were incubated until mid-log phase of growth was achieved followed by the induction of target protein expression with 0.1 mM isopropyl-␤-Dthiogalactopyranoside. Cultures were then grown for 2 h prior to the purification of the recombinant molecules.
Recombinant granulin was engineered to contain a N-terminal His 6 tag to facilitate subsequent detection and purification. Bacteria were removed by centrifugation at 2000 ϫ g, and supernatants were collected. Granulin was purified from culture supernatants using MagneHIS beads (Promega) according to the manufacturer's specifications. Purified proteins were eluted in a solution containing 100 mM HEPES and 500 mM imidazole and then were dialyzed overnight against 1ϫ phosphate-buffered saline. Protein samples were then filter-sterilized in preparation for immunodetection and analysis of biological activity. Total protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce) according to the manufacturer's protocols.
Immunodetection of Recombinant Goldfish Granulin (rgfGrn)-rgf-Grn was used as a source of antigen for rabbit immunizations. The primary immunization was performed by combining an equal volume of purified recombinant granulin (100 g), with 750 l of Freund's complete adjuvant. Booster injections were done exactly as the primary immunizations but substituted with Freund's incomplete adjuvant. The IgG fraction was purified by precipitation using saturated ammonium sulfate, solubilization of precipitate in phosphate-buffered saline, and purification using a HiTrap protein A HP column (Amersham Biosciences) according to the manufacturer's protocol. Fractions containing IgG were pooled and filter-sterilized (0.22 m filter, Millipore). The specificity of the antibody was determined by immunoblot using as a target rgfGrn under native and denaturing conditions. rgfGrn and native goldfish granulin (4ϫ concentrated macrophage culture supernatants) were detected by immunoblot analysis using an anti-His 6 monoclonal antibody (Invitrogen) or with affinity-purified rabbit anti-goldfish rgfGrn IgG. Briefly, proteins were separated by SDS-PAGE under reducing conditions using 12.5% polyacrylamide gels, transferred to 0.2-m nitrocellulose membranes (Bio-Rad), and incubated overnight at 4°C in the presence of the primary antibody. Membranes were subsequently washed, incubated with a horseradish peroxidase-conjugated monoclonal antibody, and developed using the ECL Advance TM Western blotting detection kit (Amersham Biosciences) according to the manufacturer's specifications.
Induction of Proliferation of Macrophages by Recombinant Granulin-PKM cultures were established, and distinct differentiation stages were sorted by fluorescence-activated cell sorting and seeded at a density of 1 ϫ 10 4 cells well Ϫ1 in 96-well culture plates (Falcon). Cells were seeded in 50 l of complete culture medium and treated with 5, 50, 100, 250, and 500 ng of recombinant goldfish granulin suspended in 50 l of incomplete cell culture medium and incubated for 52 h at 20°C. Fifteen l of bromodeoxyuridine labeling reagent (BrdUrd, Roche Applied Science) per well was added, and cells were incubated for an additional 24 h at 20°C. The reaction was developed according to the manufacturer's specifications, and optical densities were determined at 450 nm using a microplate spectro-photometer (Biotek). The colorimetric reaction was directly proportional to the number of proliferating PKM in culture (data not shown). The induction of macrophage proliferation by rgfGrn was determined after the addition of different amounts (1, 10, 50, 100, 300, 500 ng) of anti-rgfGrn to cultures.

RESULTS
The most common transcript identified in differential cross-screening of proliferative and senescence phase goldfish macrophage cDNA libraries was granulin (Fig. 1). Thirty-one partial granulin-like transcripts were identified, and all exhibited higher expression in proliferating macrophages. All of the transcripts were sequenced and found to be identical. The fully sequenced cDNA transcript of goldfish granulin is 947 nucleotides in length with an open reading frame of 477 nucleotides. The predicted protein is 159 amino acids long and had 18 conserved cysteine residues, 12 of which represent a full granulin cysteine motif common for all known granulin proteins. The remaining 6 cysteine residues make up one-half of this motif (Fig. 2). The granulin sequence has been submitted to GenBank TM (accession number DQ369750).
fish granulins of carp, zebrafish, and goldfish intestine show highly conserved cysteine-rich motifs (Fig. 3B). Goldfish granulin was most similar to carp granulin 3, with an amino acid identity of 56%. Of all the granulins analyzed, goldfish granulin shared the highest identity with other fish granulins (Fig. 3A), a finding that was supported by phylogenetic analysis that grouped the goldfish granulin in close proximity to carp granulins 2 and 3 (Fig. 4). Phylogenetic analysis also suggested that all fish granulins share distinct features that separate them from the granulins of mammals. Although the granulin proteins identified in carp and from goldfish intestine have no corresponding mRNA transcript sequences, zebrafish granulins 1 and 2 and zebrafish hybrid granulin had transcript organization similar to that of goldfish granulin.
The expression of goldfish granulin transcript was analyzed by Northern blot, RT-PCR, and real-time PCR. Analysis of transcript expression in the heart, brain, spleen, kidney, gill, liver, and intestine revealed that goldfish granulin was expressed primarily in the kidney and the spleen (Fig. 5). Real-time PCR and RT-PCR analyses of granulin expression were also done using non-activated and activated macrophages and sorted goldfish macrophage subpopulations. Goldfish granulin expression was up-regulated in activated macrophages and activated peripheral blood mononuclear cells compared with non-activated controls (Fig. 6, A and B). Interestingly, goldfish granulin was expressed primarily in the monocyte subpopulation, with lower expression evident in mature macrophages and the early progenitor cells (Fig.  6, C and D). FIGURE 4. Phylogenetic tree of selected granulin peptides. Goldfish granulin groups closely to carp granulins 3 and 2, which are closely associated with the zebrafish granulins 1 and 2 and hybrid as well as with carp granulin-1 and the goldfish granulin identified from intestinal exudates. All of the progranulin peptides from Xenopus, human, mouse, and rat are closely grouped, and all are out-grouped by granulin-like peptides identified in C. elegans. The tree was bootstrapped 10,000 times to ensure accuracy. Abbreviations are the same as for Fig. 3.

FIGURE 5.
A, Northern blot of goldfish granulin transcript expression in various tissues (L ϭ liver, K ϭ kidney, H ϭ heart, G ϭ gill, S ϭ spleen, I ϭ intestine, Br ϭ brain); kidney and spleen show the highest level of transcript expression. 18 S ribosomal RNA was used as a loading control. B, real-time PCR analysis of granulin expression in different tissues of the goldfish. The data are from five independent experiments (n ϭ 5). -Fold difference relative to ␤-actin expression is shown. RQ, relative quantification.

FIGURE 6. Real-time (B and D) and RT-PCR (A and C) analysis of granulin expression in goldfish macrophages.
A, lanes 1 and 2 represent granulin expression in non-activated and LPS/MAF-activated goldfish macrophages, respectively; lanes 3 and 4 represent granulin expression in non-activated and LPS/MAF-activated peripheral blood mononuclear cells, respectively. C, lanes 1, 2, and 3 represent granulin expression in progenitor cells, monocytes, and macrophages, respectively. ␤-Actin was used as a loading control in RT-PCR analyses. The data for real-time quantitative PCR are from five independent experiments (n ϭ 5). RT-PCR data are from a representative experiment of five that were performed. RQ, relative quantification.
To examine the effect(s) of granulin on fish macrophage development in vitro, we generated recombinant goldfish granulin using a prokaryotic expression system. The ability of rgfGrn to induce a proliferative response of goldfish macrophages was tested by adding different amounts (5-500 ng) of the rgfGrn to newly established cultures of sorted early progenitor cells, monocytes, and mature macrophages. The proliferation assays were done using eight separate PKM cultures established from individual fish (n ϭ 8). All optical density values were normalized to those of untreated control cells. The proliferation responses depended on the differentiation stage of macrophages treated with recombinant granulin. For example, rgfGrn induced significant proliferative response in progenitor cells (3-fold increase over crude cell conditioned medium by day 8) (Fig. 7A) and lower but significant proliferation in monocytes cultures (1.6-fold increase over cell conditioned medium by day 8) (Fig. 7B). In contrast, the mature macrophage subpopulation did not proliferate in the presence of rgfGrn (Fig. 7C). No  increase in proliferation of cultured macrophages was observed in the presence of the vector control (Figs. 7 and 8A).
The rgfGrn was used to generate an affinity-purified anti-rgfGrn rabbit IgG. The anti-His 6 (Fig. 8B, lane 1) and anti-rgfGrn IgG recognized rgfGrn (21 kDa; granulin plus additional vector sequence) as well as an unknown bacterial protein of an approximate molecular mass of 31 kDa (Fig. 8B, lane 2). Interestingly, the immunoblot analysis using anti-rgf-Grn IgG as a probe showed that the native goldfish granulin was present in goldfish macrophage culture supernatants, indicating that the molecule was secreted by actively growing macrophages (Fig. 8B, lane 3). The addition of known amounts of anti-rgfGrn IgG abrogated the proliferative response of macrophages in a dose-dependent manner (Fig. 8C).

DISCUSSION
In this study, we report on a novel granulin gene of the goldfish. Granulin was the most common transcript identified during differential cross-screening of the goldfish macrophage proliferative and senescence phase libraries (47). Granulins were first purified from the extracts of human inflammatory cell exudates, and from rat bone marrow (1). To date, seven granulin peptides (A to G) have been characterized (9,10,26,27), and it has been shown that they are generated following proteolytic cleavage of progranulin (7). The two main differences between goldfish granulin and mammalian progranulin were: (a) the goldfish granulin gene encoded for a much smaller protein (159 amino acids); and (b) unlike mammalian progranulin, which has been shown to be expressed ubiquitously in different tissues, the goldfish granulin expression was limited to hematopoietic tissues (kidney and spleen) and blood mononuclear cells. Furthermore, goldfish granulin was found to be differentially expressed in different macrophage subpopulations and to promote growth of macrophages; this was inversely related to their stage of maturation/differentiation.
The presence of granulin proteins in hematopoietic tissues of the carp has been reported: granulin-1, which was found mainly in extracts of the spleen; and granulins 1, 2, and 3, which were present in extracts of the head kidney (21). Furthermore, antibodies generated against carp granulin-1 appeared to recognize mononuclear cells in the head kidney of carp (21)(22). Sequence data from zebrafish granulins (GenBank TM accession numbers AF273479 and AF273480) suggest that this fish species possesses two genes that encode granulin proteins. Similar to the goldfish granulin, zebrafish granulins 1 and 2 possess 1.5 cysteine repeats, which may be the possible orthologs of goldfish granulin (49).
Given that the expression of goldfish granulin was up-regulated in proliferating macrophages and that granulin was present in macrophage culture supernatants, we hypothesized that granulin may play a role in cell proliferation. Indeed, the recombinant granulin induced a significant and dose-dependent proliferative response in early progenitor and monocyte subpopulations in vitro, indicating that this molecule may contribute to the regulation of goldfish macrophage hematopoiesis.
Progranulin was shown to be involved in different stages of embryonic development (48 -51) and in sexual differentiation of the rat brain via actions on the ventromedial hypothalamus (52)(53)(54), and it may be a trigger for rat copulatory behavior (55). Progranulin induced the proliferation of embryonic fibroblasts (R-cells) obtained from mice that lack functional insulin-like growth factor-1 receptor (IGF-1). Progranulin was shown to be the only growth factor capable of inducing the proliferation of R-cells in the absence of IGF-1 and platelet-derived growth factor (56), through the activation of the p44/42 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways and induction of cyclin D1 and cyclin B. Interestingly, these pathways are involved in the signaling cascade for IGF-1 and thus may be the reason that progranulin can act in place of IGF-1 (8,57,58). Progranulin was shown to participate in inflammatory responses by inducing cellular migration during wound healing (4,59,60). Although the multifunctional nature of the progranulin was well characterized, a receptor for progranulin has yet to be identified.
Progranulin cannot only exert its biological effects as an intact protein but also can generate multiple functions as a result of proteolytic cleavage and production of functional smaller granulin peptides. For example, epithelin 1/granulin A (Epi1/GrnA) was shown to induce the proliferation of murine keratinocytes as well as rat kidney cells NRK-SA6 in the presence of transforming growth factor ␤. However, Epi1/ GrnA was also shown to inhibit DNA synthesis and thus proliferation of the epidermoid cell line A431 and the human colon carcinoma cell line HCT116 (2,9). Interestingly, Epi2/GrnB was shown to antagonize the proliferative effects of Epi1/GrnA, as well as having growth inhibitory effects on A431 cells, albeit not to the same extent as Epi1/GrnA (2,9).
The structure, distribution, and function of the goldfish granulin transcript identified in this study set it apart from known mammalian granulins. Its obvious association with the hematopoietic organs of the goldfish and up-regulation in cells that are undergoing proliferation suggest that it may be an important growth factor during hematopoiesis in goldfish. Furthermore, the up-regulation of granulin expression after activation of macrophages and high expression in monocytes suggest that goldfish granulin, like mammalian granulin, may be involved in inflammation and wound repair events. Whether goldfish granulin can modulate the inflammation and wound healing events is currently under investigation in our laboratory.