Molecular characterization and in vitro biological activity of placentin, a new member of the insulin gene family.

Insulin and insulin-like growth factors belong to a family of polypeptides involved in essential physiological processes. Placentin, a new member of the insulin family, was recently identified as a 139-amino acid open reading frame from a cDNA clone isolated from a subtracted library of first trimester human placenta. Tris/Tricine/SDS-polyacrylamide gel electrophoresis and immunoblot analyses of histidine-tagged recombinant placentin indicate that it is composed of two peptide chains of apparent molecular masses of 4 and 13 kDa. Conditioned media produced by recombinant expression of placentin cDNA in the placental 3AsubE cell line were assayed for biological activity and found to stimulate tyrosine phosphorylation and DNA synthesis. While these effects closely mimicked those of insulin, they were not mediated by the insulin receptor as shown by the lack of tyrosine phosphorylation of this receptor upon placentin treatment. Moreover, in cytotrophoblast primary culture, production of chorionic gonadotropin, a marker of trophoblast differentiation, was increased upon treatment with placentin-conditioned media, while unaffected by insulin. These results suggest that placentin might participate in the cellular proliferation and/or differentiation processes during placental development.


Insulin and insulin-like growth factors belong to a family of polypeptides involved in essential physiological processes. Placentin, a new member of the insulin family, was recently identified as a 139-amino acid open reading frame from a cDNA clone isolated from a subtracted library of first trimester human placenta. Tris/ Tricine/SDS-polyacrylamide gel electrophoresis and immunoblot analyses of histidine-tagged recombinant placentin indicate that it is composed of two peptide chains of apparent molecular masses of 4 and 13 kDa.
Conditioned media produced by recombinant expression of placentin cDNA in the placental 3AsubE cell line were assayed for biological activity and found to stimulate tyrosine phosphorylation and DNA synthesis. While these effects closely mimicked those of insulin, they were not mediated by the insulin receptor as shown by the lack of tyrosine phosphorylation of this receptor upon placentin treatment. Moreover, in cytotrophoblast primary culture, production of chorionic gonadotropin, a marker of trophoblast differentiation, was increased upon treatment with placentin-conditioned media, while unaffected by insulin. These results suggest that placentin might participate in the cellular proliferation and/or differentiation processes during placental development.
Insulin and insulin-like growth factors belong to a family of polypeptides essential for proper regulation of physiological processes such as energy metabolism, cell proliferation, development, and differentiation (1,2). These polypeptides exert their effects through membrane receptors belonging to the superfamily of polypeptide growth factor receptor tyrosine kinases and their oncogenic analogs (3)(4)(5). The intracellular signal transduction pathway for these receptors is characterized by a tyrosine kinase activity that initiates a chain of phospho-rylation-related events. The insulin family consists of insulin, IGF 1 I and II, relaxin (6), and LEY I-L (7). The consensus elements defining their relationship are the positions of six half-cystine residues essential for proper tertiary structure formation (8). While receptors mediating the effects of relaxin and LEY I-L have not yet been described, insulin and IGF I act through distinct tyrosine kinase subclass II receptors (3), and the actions of IGF II appear to be mediated through several receptors, including those for insulin and IGF I (1).
The placenta is a rich source of growth factors and their receptors involved in the regulation of cell proliferation and differentiation, particularly during the early stages of gestation. Autocrine loops for IGF-II, EGF, and PDGF are implicated in the growth of highly proliferative trophoblastic cells (9 -11). Seeking novel secreted molecules involved in cell growth control, a subtracted cDNA library enriched in sequences from cytotrophoblast cells of first trimester placenta (12) was constructed and screened. One of the isolated clones, designated placentin, showed significant similarities to the insulin family (Genome Sequence Data Base accession number L34838, Koman et al., 1994, PCT 337 269, and Ref. 13). The present study describes the molecular characterization and biological activity of recombinant placentin. Growth factor-like properties of recombinant placentin were investigated by determining phosphorylation of cellular proteins, stimulation of thymidine incorporation and hCG production in trophoblast cells.

Recombinant Expression and Conditioned Media Preparation-Pla-
centin cDNA was reamplified from placental RNA and resequenced in order to ascertain that no polymerase chain reaction induced errors existed in the initial sequence. The following primers were used: 5Ј-AAGAAAGGCTGAGAACACC-3Ј and 5Ј-TATGAGAATGGATGAGATG-3Ј. The cDNA was inserted in the pBK-CMV (Stratagene) eukaryotic expression vector in the sense and antisense orientations. For expression of His-tagged placentin for purification, constructs were made in the cytomegalovirus promoter-based expression vector pRK5 where the cDNA sequence was modified adding a carboxyl-terminal six-residue histidine tag to the recombinant peptide putative A domain, in this case the downstream primer was: 5Ј-CTCTAGATATCAGTGATGGTGATG-GTGATGTGTACATAATTTAACTGAAGTT-3Ј. His-tagged product was purified by chromatography on a nickel-NTA-agarose column (Diagen, Germany), eluted with 5% acetic acid. The peptide was analyzed by Tris/Tricine/SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (14). 293 human embryonal kidney, COS-7 African green monkey kidney, or 3AsubE SV40-transformed trophoblast cells (ATCC (15)) were transfected using the CaPO 4 method. For preparation of conditioned media after transient expression, media was changed to MEM, 0.15% BSA 48 h posttransfection, changed again after 2 h, and conditioned for 24 h. Stable transfected cells selected for Geneticin resistance were conditioned for 48 h at 80% confluence. For prokaryotic expression, the placentin cDNA was inserted in-frame into a glutathione S-transferase fusion vector (pGEX, Pharmacia Biotech Inc.) with which Escherichia coli were transformed.
Antibody Production-Chinchilla bastard rabbits were immunized intradermally with incomplete Freund's adjuvant containing sonicated, boiled E. coli (5 ϫ 10 10 /ml) expressing the glutathione S-transferaseplacentin fusion protein. Most of the protein was found in inclusion * This work was supported by grants from the Centre National de la Recherche Scientifique, France and the Max-Planck-Institut fü r Biochemie, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

bodies.
Tyrosine Phosphorylation, Western Blotting, and Immunoprecipitation-Conditioned media or 170 nM insulin in MEM, 0.15% BSA were used to treat untransfected 3AsubE target cells grown to 80% confluence. Targets were preincubated with 0.5 mM sodium orthovanadate for 4 h and treated for 10 min at 37°C in the presence of 0.5 mM orthovanadate. For Western blot analysis, cells were harvested by washing rapidly with ice-cold phosphate-buffered saline, 0.5 mM orthovanadate, lysis in 2 ϫ SDS loading buffer, 0.5 mM orthovanadate and heating for 10 min at 98°C. Equal amounts of protein from each sample were subjected to SDS-PAGE (8% acrylamide) and Western blotted with monoclonal anti-phosphotyrosine antibodies (Upstate Biotechnology, Inc.) (16). After revelation with ECL (Amersham Corp.), blots were stripped by 10 min. treatment in 0.1 M glycine, pH 2.5, and reincubated with anti-␣-actinin antibodies in order to verify the equivalence of transferred protein amounts. Western blotting with polyclonal placentin antiserum at 1:1000 dilution was performed similarly after transfer from Tris/Tricine/SDS gels. Placentin antiserum was precleared on milk-blocked nitrocellulose before use in order to reduce background. Immunoprecipitation with anti-insulin receptor antibodies (Upstate Biotechnology, Inc.) was performed as described (17) and Western blotted with anti-phosphotyrosine antibodies.
[ 3 H]Thymidine Incorporation-3AsubE target cells were grown to confluence in 96-well plates, starved for 48 h in 100 l of serum-free MEM, 0.15% BSA, treated by adding 100 l of conditioned media from transfected cells, or 1 g/ml (170 nM) insulin in MEM, 0.15% BSA. After incubation for 8 h, 1 Ci of [ 3 H]thymidine in 30 l of MEM, 0.15% BSA was added. Following another 16-h incubation, cells were trypsinized, collected on a cell harvester, and the incorporated radioactivity was counted (18).
Trophoblast Primary Culture and hCG Analysis-Cytotrophoblasts were isolated from terminal placenta as described by Alsat et al. (19). Cells (1.2 ϫ 10 6 /3.5-cm dish) were allowed to adhere for 24 h in 1.5 ml of DMEM ϩ20% fetal calf serum, after which the medium was exchanged with conditioned media (prepared as described above but with DMEM, 0.1%BSA, 170 nM insulin in DMEM, 0.1%BSA or control media (DMEM, 0.1% BSA). This was repeated every 24 h up to 3 days. After the 3rd day, cells and media were harvested, cells were assayed for total protein (BCA, Pierce), and media were assayed for hCG (radioimmunoassay, Behring Hoescht) determination. No significant variations in protein content were found within each experiment, allowing direct comparison of hCG amounts. Under the growth and assay conditions utilized, no detectable amounts of hCG were secreted by control or placentin-transfected 3AsubE cells. The unstimulated production of hCG in primary culture varied from 0.7 to 3 IU/24 h/10 6 plated cells in different experiments.

Expression and Molecular Characterization of Recombinant
Placentin-Placentin displays a number of structural features resembling those of insulin (Fig. 1A), including the characteristic six half-cystine residues of the insulin family and 25% amino acid sequence identity to insulin A-and B-chains in corresponding domains. Dibasic recognition sites for putative enzymatic cleavage between C and A domains are also conserved, whereas no homology is found in respective cleavage sites between B and C domains. The consensus sequence Arg-X-Lys-Arg between the C and A domains is a furin recognition site that would allow processing to occur in most cells (20) (see Fig. 1A).
The molecular characterization of placentin was undertaken using recombinant placentin. Transfection experiments indicated that human kidney 293, monkey kidney COS-7, and human placental 3AsubE cells were able to produce bioactive placentin as assayed by induction of tyrosine phosphorylation of cellular proteins described below. In order to produce and purify significant amounts of placentin, the cDNA sequence was modified, adding a carboxyl-terminal six-residue histidinetag to the recombinant peptide, and was expressed in human kidney 293 cells known to show high transfection and production efficiency. The modified peptide was found to be secreted and was purified from conditioned media by nickel-agarose chromatography. Analysis of the recombinant product by Tris/ Tricine/SDS-PAGE (Fig. 1B) indicated the presence of two major peptides of 4 and 13 kDa that could correspond to A-chain and uncleaved B and C domains, respectively, produced upon furin cleavage. However, apparent molecular masses on these types of gels may differ from those in traditional Tris-HCl/ PAGE, as reported for insulin (14). Western blot analysis using antibodies generated by immunizing rabbits with recombinant glutathione S-transferase-placentin fusion protein revealed immunoreactive peptides of similar size (Fig. 1C).
Stimulation of Tyrosine Phosphorylation-The biological activity of recombinant placentin was investigated using 3AsubE cells derived from trophoblasts of terminal placenta by temperature-sensitive SV40 immortalization. They have been described as maintaining the trophoblast phenotype at nonpermissive temperature (37-40°C) (15). Treatment of 3AsubE cells with conditioned media from stable placentin sense cDNA-transfected 3AsubE cells or with insulin-induced rapid (10 min) tyrosine phosphorylation of several proteins of apparent molecular masses of about 60, 85, 95, 120 -140, and 170 -190 kDa (Fig. 2A). Densitometric scan analysis revealed that placentin-and insulin-induced similar increases in tyrosine phosphorylation of the 85-kDa phosphoprotein (2.4-and 2.9fold, respectively) (Fig. 2C) while some proteins phosphorylated after insulin treatment were not significantly affected by placentin (e.g. around 150 kDa). Comparable results were obtained with conditioned media from cells transiently expressing placentin; treatment with conditioned media from nontransfected or vector-transfected cells gave basal phosphorylation levels equivalent to that shown for media from antisense-transfected cells ( Fig. 2A). In addition, conditioned media from His-tagged placentin expressing 293 cells was found to have similar activity in stimulation of tyrosine phosphorylation as that shown for 3AsubE-produced placentin-conditioned media in Fig. 2A. In preliminary experiments, stimulation of tyrosine phosphorylation with purified His-tagged placentin was observed as well, indicating that the observed activity is a direct effect of placentin.
Lack of Insulin Receptor Activation-Since cross-reactivity on insulin receptors has been described for other members of the family (21), immunoprecipitation with anti-IR antibodies was performed in order to investigate the possibility that placentin effects were mediated by insulin receptors (Fig. 2D). While the IR ␤-subunit was found to be tyrosine-phosphorylated in response to insulin as expected, no detectable phosphorylation was observed upon placentin treatment, suggesting lack of IR activation in the response to placentin.

Increase of [ 3 H]Thymidine Incorporation in Placentin-treated 3AsubE
Cells-Increase of tyrosine phosphorylation is often involved in the stimulation of DNA synthesis in many different cell types. The biological effects of placentin were also investigated by measuring stimulation of [ 3 H]thymidine incorporation in quiescent 3AsubE cells after treatment with conditioned media from placentin cDNA-transfected cells (Fig. 3). Stimulation of similar magnitude to that induced by insulin was observed in response to placentin (increased incorporation by 1.8-fold for placentin and 1.7-fold for insulin).
Stimulation of hCG Production in Primary Cultures of Trophoblasts by Placentin-Further characterization of placentin effects was performed on trophoblasts in primary culture. Trophoblasts differentiate to form syncytiotrophoblasts throughout placental development until parturition and the production of hCG is considered as a marker of this differentiation, also in vitro (22). Treatment with conditioned media from sense cDNA-transfected 3AsubE cells increased hCG production 2.6fold, suggesting a stimulated differentiation process, while conditioned media from antisense transfected cells had no effect (Fig. 4). In agreement with previous investigations (23), insulin did not affect hCG production by trophoblasts in primary culture.

DISCUSSION
The present study describes the molecular characterization and the in vitro biological activity of placentin, a new member of the insulin gene family. Other members of the insulin family require various posttranslational modifications for bioactivity. For insulin and relaxin (6) the leading signal peptide as well as the connecting C peptide are excised by tissue-specific posttranslational processing from prepro-sequences, leaving two peptide chains A and B linked by disulfide bridges, whereas the insulin-like growth factors are processed without removal of the C peptide (21). For recombinant placentin expression, 293, COS-7, and 3AsubE cells were compared and found to secrete bioactive product after transfection. When placentin was expressed in 293 cells, the two immunoreactive peptide chains of 4 and 13 kDa observed may derive from cleavage between the C and A domains, as expected from the sequence data upon furin cleavage. 3AsubE cells derived from trophoblasts of terminal placenta were selected as host cells for transfection with unmodified cDNA when producing placentin-conditioned media for characterization of biological activity. These conditions presumably meet those for the native peptide most closely. Purification of bioactive His-tagged placentin was found to be difficult. Thus, purification on a larger scale of the peptide (whether recombinant or native) requires further optimization in terms of solubility and bioactivity.
Tyrosine phosphorylation of cellular proteins is recognized as a key factor in the processes leading to cell proliferation and/or differentiation. A large number of membrane receptors as well as cytosolic protooncogene products have been identified as tyrosine kinases mediating the biological activity of polypeptide growth factors, such as insulin. The tyrosine phosphorylation and [ 3 H]thymidine incorporation stimulating activity of recombinant placentin was investigated on 3AsubE cells, assuming that they might express receptors binding placentin. Autocrine regulation is one of the characteristic features of trophoblasts and placenta has been described to express several types of insulin-related receptors (24 -26). The effects of placentin-conditioned media closely mimicked those of insulin both qualitatively and in magnitude, strongly suggesting that placentin could activate signal transduction pathways generally associated to polypeptide growth factor receptors. However, in contrast to insulin, placentin did not induce the autophosphorylation of the insulin receptor ␤-subunit. Consistently, insulin did not affect hCG production under the experimental conditions used, nor was this described for any other member of the insulin family. These observations suggest that placentin might be acting through a receptor positively coupled to cellular tyrosine phosphorylation, distinct from the insulin receptor. The possibility exists, however, that some of the observed effects with conditioned media might be indirect effects of placentin, through specific induction of other compounds.
In summary, the biological activity on placental cells of a new member of the insulin family was demonstrated in vitro. Present results suggest that placentin is expressed by, and affects, trophoblasts and thus might be involved in an autocrine loop, joining IGF-II, EGF, and PDGF in concerted regu-lation of placental development and growth. The placenta is well defined as an endocrine organ producing several polypeptides with maternal and fetal targets. While the activity defined in this study was restricted to placental cells, preliminary data suggest possible effects on cells of other tissue origin. Placentin might thus have endocrine/paracrine effects in maternal or fetal compartments depending on its bioavailability, receptor distribution and selectivity.