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J Biol Chem, Vol. 274, Issue 35, 24766-24772, August 27, 1999


A Novel Oncostatin M-inducible Gene OIG37 Forms a Gene Family with MyD118 and GADD45 and Negatively Regulates Cell Growth*

Koh NakayamaDagger , Takahiko HaraDagger , Masahiko Hibi§, Toshio Hirano§, and Atsushi MiyajimaDagger

From the Dagger  Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan and the § Osaka University School of Medicine, Suita-shi 565-0871, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oncostatin M (OSM) is a member of the IL-6 family cytokines that use gp130 as a common signal transducer and exhibits both growth stimulatory as well as growth inhibitory activity depending on the cells. To analyze the mechanism of OSM function, we isolated immediate early responsive genes upon OSM stimulation. Here we describe the novel OSM-inducible gene OIG37 that is related to MyD118 and GADD45. The MyD118 gene has been described as an immediate early gene induced by IL-6 in M1 monocytic cells, and GADD45 was identified as a gene induced by UV or gamma -ray irradiation. Both are considered to function in growth arrest and/or DNA repair. Although the expression of OIG37, MyD118, and GADD45 was rather ubiquitous, it was differentially regulated. As the gp130 mutant defective for activating the STAT3 pathway showed the reduced induction of OIG37 by cytokine stimulation and expression of dominant negative STAT3 inhibited the induction of OIG37 by OSM, STAT3 is involved in OIG37 induction by IL-6 family cytokines. To examine the function of OIG37, we expressed it in NIH3T3 and IL-3-dependent BaF3 cells and found that OIG37 suppressed cell growth without any evidence of apoptosis. Whereas both MyD118 and OIG37 suppressed cell growth in both cell lines, suppression by OIG37 was more efficient than by MyD118. Immunoprecipitation experiments indicated that OIG37 associates with p21, a cyclin-dependent kinase inhibitor, and proliferating cell nuclear antigen.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oncostatin M (OSM)1 was originally identified as a growth inhibitor of the A375 human melanoma cells and molecularly cloned from the U937 human hematopoietic cell line (1, 2). It was subsequently found to exhibit various biological functions such as growth stimulation of Kaposi's sarcoma (3), endothelial cells (4), and muscle cells (5). It is a member of the IL-6 family cytokines that include IL-6, IL-11, OSM, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and cardiotrophin-1 (6-10). These cytokines exhibit very similar biological activities when they act on the same cells. This functional similarity is now explained by gp130, the common signaling subunit of the receptors for these cytokines (11). The OSM receptor (OSMR) consists of gp130 and the OSM-specific subunit that is structurally similar to gp130. In the human, the LIF receptor (LIFR), which consists of gp130 and the LIF-binding protein (known as LIFRbeta ), also serves as an OSMR (12, 13). Thus, OSM and LIF exhibit the same effects through the LIFR, and OSM induces its unique activity through the OSM-specific receptor. Although the human OSM gene was isolated a decade ago, mouse OSM had not been isolated until we cloned the mouse OSM cDNA as a cytokine-inducible gene in hematopoietic cells (14). OSM expression is regulated by the STAT5 transcription factor that is activated by various cytokines including IL-3, GM-CSF, EPO, and IL-2 (14-16). Biological as well as biochemical analysis of mouse OSM and its receptor revealed that unlike human OSM, mouse OSM utilizes only the OSM-specific receptor but not the LIFR, indicating that the functions of LIF and OSM are segregated in the mouse (17). Curiously, however, human OSM functions through the mouse LIFR but not the mouse OSMR.

Although OSM mRNA is most abundantly expressed in the bone marrow cells, the role of OSM in bone marrow hematopoiesis still remains unknown. However, we recently found that OSM stimulates development of hematopoietic cells in the aorta/gonad/mesonephros region of developing mouse embryos (18). It is now known that definitive hematopoietic stem cells arise in the aorta/gonad/mesonephros region before they massively generate various lineages of hematopoietic cells in the fetal liver (19). In an in vitro culture of the aorta/gonad/mesonephros cells, OSM was demonstrated to markedly stimulate development of hematopoietic cells and also endothelial cells. Recent results suggest that hematopoietic cells and endothelial cells are derived from a common precursor termed hemangioblast and OSM appears to stimulate hemangioblasts rather than the hematopoietic stem cells. To understand the role of OSM in hematopoiesis, we attempted to clone OSM-inducible genes from a hemangioblastic cell line, LO, that we recently established.2 Here we describe one of the OSM-inducible genes, OIG37, which exhibits a significant homology with two known sequences, GADD45 and MyD118.

GADD45 and MyD118 are known as growth suppressive genes. The GADD45 gene was originally cloned as a UV-inducible gene in Chinese hamster ovary cells (20). Overexpression of GADD45 in HeLa cells effectively suppressed their growth (21). MyD118 was first identified as an immediate early responsive gene induced by IL-6 in the murine myeloid cell line M1 (22). IL-6 and some of its family members are known to suppress the growth and to induce differentiation of M1 cells to macrophages (23, 24). Overexpression of MyD118 also resulted in growth suppression of the H1299 human carcinoma cells (21). Although the precise mechanism of growth arrest by these genes remains unknown, it was demonstrated that both GADD45 and MyD118 associate with p21, a cyclin-dependent kinase inhibitor, and proliferating cell nuclear antigen (PCNA) (25), which are implicated in the cell cycle regulation (26-28). In the present study we describe that OSM-inducible OIG37 also binds with p21 and PCNA and that it suppresses cell growth.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Murine IL-3-dependent BaF3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 ng/ml IL-3, 50 µM beta -mercaptoethanol, and antibiotics. NIH3T3OR cells were maintained in Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics. M1 cells, a murine myeloid leukemia cell line, were cultured in RPMI 1640 containing 10% FBS, 50 µM beta -mercaptoethanol, and antibiotics. The murine hemangioblast-like cell line LO was maintained in Dulbecco's modified Eagle's medium with 15% FBS, 10 ng/ml of OSM, 1 mM sodium pyruvate, and antibiotics. COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and antibiotics.

Construction and Subtraction of cDNA Libraries-- LO cells cultured without OSM for 24 h were stimulated with or without 100 ng/ml OSM for 1 h. Messenger RNA was extracted from LO cells by using the Fast track kit (Invitrogen) according to the manufacturer's instructions. 1 µg of poly(A+) RNA was used for constructing a cDNA library primed by oligo(dT) using the Time Saver kit (Amersham Pharmacia Biotech). The OSM-unstimulated LO cDNA library was subtracted from the OSM-stimulated library as described previously (29). Candidate OSM-inducible cDNA clones were randomly isolated from the subtracted library and sequenced by an automated sequencer (PE Biosystems). Among the several unknown clones, OIG37 was chosen for subsequent analyses.

Isolation of Full-length OIG37 cDNAs-- The partial cDNA fragment of OIG37 obtained from the subtraction was labeled with Digoxigenin (DIG) using the high-prime kit (Roche Molecular Biochemicals) and was used to isolate the full-length cDNA clones from a randomly primed LO cDNA library. The isolated clone was sequenced and the 5' end of the cDNA was determined using a 5'-rapid amplification of the cDNA ends using the kit (Life Technologies, Inc.).

Northern Hybridization-- LO cells, NIH3T3OR cells, and BaF3 cells transfected with the OSMR were deprived of OSM and serum (LO), serum (NIH3T3OR), or IL-3 (BaF3) for several hours, respectively, and stimulated with OSM. M1 cells were directly stimulated with IL-6 without factor depletion. Cells were harvested at 0, 0.5, 1, 2, and 4 h after the stimulation. Stress-inducible expression was tested by the addition of methyl methane sulfonate (MMS) at 100 µg/ml to these cells under the same condition as cytokine stimulation. Total RNA was extracted using the Trizol reagent (Life Technologies, Inc.) according to the manufacturer's protocol. 10 µg of total RNA for each sample were analyzed on a 1.2% agarose electrophoresis gel containing 2.4% formaldehyde. RNA was then transferred to a positively charged nylon membrane and hybridized with DIG-labeled probe in high SDS buffer (50% formamide, 5× SSC, 0.05 M phosphate buffer, pH 7.0, 2% DIG blocking reagent, 0.1% lauroylsarcosine) at 50 °C for overnight. After washing the filter to remove the nonspecifically bound probe, the blot was incubated with the alkaline phosphatase-conjugated anti-DIG antibody. Bound alkaline phosphatase was detected by a chemiluminescent substrate according to the manufacturer's instructions (Roche Molecular Biochemicals). The DNA probes used for blotting were prepared from the DNA fragments encoding the regions that are most diverged among the GADD45 family members (amino acids 1-30), and therefore the probes did not cross-hybridize each other.

Expression of G-CSFR/gp130 Chimeric Receptors and the Dominant Negative Form of STAT3-- The DNA fragment encoding the chimeric receptor consisting of the extracellular domain of the G-CSF receptor and the intracellular domain of gp130 was inserted into a retroviral vector (30, 31). The vector was transfected into the retrovirus packaging cells Bosc23 by using LipofectAMINE (Life Technologies, Inc.). The supernatant of these cells was used to infect NIH3T3. Two days after the infection, the cells expressing the G-CSFR/gp130 chimeric receptor were selectively sorted by fluorescence-activated cell sorter using anti-G-CSFR antibody. Thus, the levels of chimeric receptor expression were not significantly different among various chimeric receptors. The NIH3T3 transfectants expressing the dominant negative form of STAT3 (32) were also established by the retrovirus infection carrying the dn STAT3 cDNA with internal ribosomal entry site (IRES) and green fluorescent protein (GFP) (33). The cell sorting was done by using GFP as a marker.

Immunoblotting-- COS7 cells transfected with the hemagglutinin (HA)-tagged OIG37, MyD118, and GADD45 expression vectors were lysed in immunoprecipitation lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, and a mixture of protease inhibitors (phenylmethylsulfonyl fluoride and leupeptin)). 2 µg of anti-HA antibody (Roche Molecular Biochemicals) was added to the lysate, followed by the addition of protein A (Pierce). Proteins in the immune complexes were subjected to SDS-polyacrylamide gel electrophoresis on a 12% gel and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The blot was incubated with anti-p21 antibody or anti-PCNA antibody (Santa Cruz Biotechnology), followed by the secondary antibody conjugated with horseradish peroxidase. The immunecomplex was detected by the ECL system (Amersham Pharmacia Biotech).

Colony Forming Assay-- LO cells, NIH3T3OR cells, and BaF3 cells were infected with retrovirus carrying OIG37 cDNA. The OIG37 cDNA was placed upstream of the IRES sequence followed by GFP so that GFP positive cells are expected to express OIG37. GFP-positive cells sorted by using FACSVantage (Becton Dickinson) were plated in 96-well plates at 1 cell/well. Cells in the 96-well plates were cultured for 20 days, and the wells with the growing cells (colonies) and the number of cells in each colony was counted.

Cell Growth Assay-- NIH3T3OR cells were transfected with pMAM-OIG37 vector and cultured in G418 selection medium (0.4 mg/ml) for 3 weeks to establish the stable transfectants. NIH3T3OR cells or NIH3T3OR-OIG37 transfectants were plated in a 6-well plate at a starting cell number of 5 × 104. These cells were cultured with 5 × 10-7 M dexamethasone (Dex) or without it, and the cell number was counted for every 12 h up to 72 h when cells became confluent. The cell number at each time point was counted in three different wells, and the average of the three wells are presented.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of OIG37 as a Novel OSM-inducible Gene-- To isolate OSM-inducible genes, we prepared two cDNA libraries from OSM responsive LO cells stimulated with or without OSM for 1 h. The cDNA inserts excised from the cDNA library of the OSM-unstimulated LO cells were subtracted with those from the OSM-stimulated LO library by the subtraction method that we developed previously (29). The subtracted cDNA inserts were recloned in a plasmid vector, and the cDNA clones randomly isolated from the subtracted cDNA library were subjected to sequence analysis. Among these genes, the OSM-inducible gene OIG37 encoded a novel protein and showed a clear response to OSM stimulation in LO cells as well as in NIH3T3OR cells (see below) (Fig. 1A). The OIG37 cDNA encodes a protein of 143 amino acids that shows 55% identity with GADD45 and MyD118 at the amino acid level and 60% at the nucleotide level. Although these three proteins do not possess any protein motifs to predict their function, they clearly form a new family of proteins (Fig. 1B). The family members have highly conserved regions in the middle part of the protein at around amino acids 40-100. There is a putative PCNA-binding motif in the conserved region (Fig. 1B) (34). The predicted secondary structure showed at least two alpha  helices and a few small beta  sheets in that region.


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  		Fig. 1.   The nucleotide sequence and the deduced amino acid sequence of OIG37. A, the numbers on the left side are the nucleotide numbers starting from the first A of the initiation codon. The numbers on the right side are the amino acid numbers from the first methionine. The asterisk shows the stop codon. The coding region sequence is shown in capital letters. B, sequence alignment of OIG37, MyD118, and GADD45. The dots show the identical sequences among these three genes. Conserved regions in these sequences are surrounded by boxes. The bar above the sequence indicates the putative PCNA-binding region. OIG37 is 55% identical to those genes at the amino acid level and encodes a ~20-kDa protein.

Expression of OIG37, MyD118, and GADD45-- Northern blot analysis of mouse tissues with the OIG37 probe demonstrated that OIG37 mRNA is expressed in all tissues we tested: brain, liver, heart, bone marrow, thymus, spleen, testis, kidney, small intestine, muscle, and lung (Fig. 2A). Expression of GADD45 and MyD118 was also detected in most tissues, although the levels of their expression were different in different tissues, suggesting that expression of the three family members is differentially regulated. (Fig. 2A).


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  		Fig. 2.   Expression of OIG37. A, tissue distribution. Total RNA was extracted from adult mouse tissues by the AGPC method. 20 µg of total RNA from each tissue was applied on the gel, and the blot was hybridized with the OIG37, MyD118, and GADD45 probes as described under "Experimental Procedures." OIG37 was expressed in all the tissues examined as indicated. B, cytokine and induction of OIG37, MyD118, and GADD45. LO, NIH3T3OR, and BaF3/OSMR cells that were deprived of factor and/or serum for 24, 8, and 6 h, respectively, were stimulated with 100 ng/ml of OSM. M1 cells were stimulated with 100 ng/ml of IL-6. RNA was extracted at various time points as indicated after the stimulation and analyzed by Northern blot with the OIG37, MyD118, and GADD45 probes as shown. C, to compare the induction pattern of OIG37, MyD118, and GADD45 by stress stimulation with cytokine stimulation, NIH3T3OR cells were deprived with serum for 8 h and then stimulated with MMS at 100 µg/ml. Total RNA was extracted from the cells to perform the Northern blotting as in B.

We also examined the induction of OIG37 in various OSM responsive cells. The LO cell line used for the cDNA subtraction is an OSM-dependent hemangioblast-like cell line derived from the aorta/gonad/mesonephros region of mouse embryos. Depletion of OSM from the LO cells for 24 h eliminated the OIG37 mRNA, and readdition of OSM resulted in a rapid increase of the OIG37 mRNA within 30 min. Although MyD118 mRNA was not completely eliminated by depletion of OSM for 24 h, it was induced by OSM within 30 min and returned to the basal level after 1 h of OSM stimulation (Fig. 2B). In contrast, GADD45 mRNA was constitutively expressed in LO, and its level was not affected by OSM.

NIH3T3OR is a subline of NIH3T3, and its growth is suppressed by OSM as reported previously (17). Addition of OSM induced the OIG37 mRNA expression within 30 min, and the OIG37 level was decreased to the basal level within 2 h. In contrast, although the basal levels of MyD118 and GADD45 mRNA expression were detected, neither MyD118 nor GADD45 was significantly induced by OSM.

M1 is a murine myeloid cell line that is induced to differentiate into macrophages in response to IL-6 and was originally used to isolate the MyD118 gene. M1 cells are unresponsive to mouse OSM because of the lack of OSMR (17). IL-6 induced OIG37 mRNA in 1 h, and the mRNA level was maintained at least 4 h after the stimulation. However, the level of OIG37 expression was lower than those of OSM-stimulated LO and NIH3T3OR cells. In contrast, induction of MyD118 by IL-6 was faster than OIG37 and was clearly detected in 30 min, whereas the GADD45 mRNA level was only slightly decreased in 1 h after the IL-6 stimulation and returned to the basal level after 4 h.

BaF3/OSMR, a transfectant of the murine IL-3-dependent hematopoietic cell line BaF3, expresses the mouse OSMR and proliferates in response to OSM (35). The cells deprived of cytokines for 6 h were stimulated with OSM. Interestingly, no OIG37 mRNA was expressed at any time points in the cells under this condition. In contrast, MyD118 expression was slightly induced in 30 min in response to OSM, and GADD45 mRNA was present at a constant level regardless of the presence or absence of OSM. These results indicate that expression of the three members of this novel gene family is differentially regulated and that while signaling by gp130 plays a major role in expression of OIG37 as well as MyD118 by OSM and IL-6, expression of these genes requires an additional cell type-specific factor(s).

GADD45 family genes including OIG37 were also induced by stresses, such as UV, gamma -ray, and genotoxic agents. In NIH3T3OR cells, OIG37 was weakly induced in 4 h after MMS treatment, whereas GADD45 was induced in 1 h after stimulation (Fig. 2C). This is also consistent with M1 cells in which OIG37 mRNA appeared 8 h after the MMS stimulation, whereas GADD45 mRNA was induced in 2 h after the MMS stimulation (data not shown). Thus, the induction of GADD45 family members in these cells is regulated by stress signals as well as cytokine stimulation, although the levels of induction and kinetics of these family members seemed to be different depending on the cell types. In addition, induction of the GADD45 family by MMS was rather slow compared with very rapid induction by OSM, suggesting that the mechanism of induction by cytokines and stress are different.

A Possible Involvement of STAT3 in OIG37 Expression-- To dissect signaling pathway emanating from gp130 that leads to OIG37 expression, we utilized the chimeric receptor consisting of the extracellular domain of the human G-CSFR and the intracellular domain of gp130. The chimeric receptor was previously shown to be activated by homodimerization induced by G-CSF (30). There are 6 tyrosine residues in the intracellular domain of gp130, and among them the tyrosine residues at 118 and 126 from the transmembrane domain are critical for signaling. The truncation mutant lacking the C-terminal part from 133 is still capable of inducing growth promoting signals, whereas the truncation mutant at the 25th amino acid is totally inactive. G25 and G133 are hGCSFR/gp130 chimeric receptors with truncation at the 25th and 133rd amino acid residue of gp130, respectively. G133F2 and G133F3 are substitution mutants of G133 in which tyrosine residues at 118 and 126 are substituted with phenylalanine, respectively. Both tyrosine residues at 118 and 126 are substituted with phenylalanine in G133F2/3. G-CSF activates STAT3 but does not activate the Ras pathway through G133F2. Conversely, G133F3 activates the Ras pathway but not STAT3 in response to G-CSF. G133F2/3 fails to activate both signaling pathways.

The chimeric receptors were expressed in NIH3T3OR cells by the retrovirus vector, and the receptor expressing cells were sorted by fluorescence-activated cell sorter; the expression levels of the G-CSFR/gp130 chimera were comparable in all the chimeric receptors examined. The induction of OIG37 in response to G-CSF was observed in a mutant G133 expressing NIH3T3OR cells. Expression of OIG37 by the G133F2 mutant was rather enhanced compared with G133, whereas OIG37 expression by G133F3 was remarkably reduced, indicating that induction of OIG37 is dependent on the STAT3 pathway (Fig. 3A). Consistently, expression of the dominant negative form of STAT3, a truncation mutant lacking the transactivation domain (32), in NIH3T3 cells reduced the expression level of OIG37 in response to OSM stimulation. These results suggest that expression of OIG37 is regulated by STAT3 (Fig. 3B).


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  		Fig. 3.   Signal transducing pathway of OIG37 induction. A, expression of OIG37 in G-CSFR/gp130 chimeric receptor transfectants. Northern blot was performed using NIH3T3OR cells transfected with the G-CSFR/gp130 chimeric receptor mutants. The chimeric receptor consists with the extracellular domain of the human G-CSF receptor and the intracellular domain of gp130 and is activated by G-CSF. The NIH3T3OR transfectants starved for 8 h without serum were stimulated with 100 ng/ml of G-CSF. 10 µg of total RNA was used for Northern blotting, and the OIG37 band was detected by the DIG-labeled DNA probe. The G133 and G277 mutants show the basal level of OIG37 expression. Although OIG37 expression by G133F3 mutant was significantly reduced, it was higher by G133F2 compared with that by G133. B, effect of dominant negative STAT3 in OIG37 induction. NIH3T3OR cells transfected with a dominant negative form of STAT3 showed the reduced expression of OIG37 when stimulated with 100 ng/ml of OSM.

Growth Suppression of NIH3T3OR Cells by OIG37-- To analyze the cellular function of OIG37, the cDNA was introduced in three cell lines, LO, NIH3T3OR, and BaF3. As MyD118 was induced by OSM and IL-6, the MyD118 function was also examined. We utilized the retrovirus expression vector that has IRES derived from polioma virus followed by the GFP gene (36). The OIG37 and MyD118 cDNAs were inserted upstream of the IRES so that the inserted genes are coexpressed with GFP. GFP positive cells were sorted from the retrovirus-infected cells by fluorescence-activated cell sorter, and the GFP positive sorted cells were seeded in three 96-well plates at one cell/well. After 20 days of culture, the numbers of the wells with growing cells (colonies) were counted. The numbers of the positive wells of the cells infected with the OIG37 expression virus vector were reduced to 60-70% of the cells infected with the empty vector (Fig. 4A). This effect was observed in all three cell lines tested. Likewise, expression of MyD118 also reduced the colony forming efficiency, but the inhibitory effect was rather less than that of OIG37. We also examined the size of each colony by counting the cell number in each positive well. Interestingly, the colony size (the number of cells in each colony) was also reduced in the cells infected with the OIG37 or MyD118 cDNA (Fig. 4B). The growth inhibitory effect was more profound in NIH3T3OR and BaF3 than in LO cells. These results indicate that enforced expression of OIG37 and MyD118 results in suppression of cell growth.


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  		Fig. 4.   Suppression of proliferation by OIG37 and MyD118. A, colony formation. LO, NIH3T3OR, and BaF3 cells were infected with the retrovirus vector in which the OIG37 or MyD118 cDNA was linked to the IRES followed by GFP. After infection, GFP-positive cells were sorted and were plated in 96-well plates, and the numbers of wells with growing cells were counted after 20 days. B, colony sizes of the virus-infected cells. The numbers of cells in each well that contains growing cells were counted. Colony size is classified into four ranges: 1 × 104 to 1 × 105; 1 × 105 to 2 × 105; 2 × 105 to 4 × 105; and more than 4 × 105. Averages of three independent plates are shown. S.E. is shown by the bars. White bars, mock; black bars, MyD118; hatched bars, OIG37.

To further demonstrate the growth suppressive function of OIG37, we inducibly expressed it in NIH3T3OR cells by using the Dex-inducible mouse mammary tumor virus promoter (37). To monitor the expression of OIG37, the FLAG peptide was attached to OIG37. We established two independent transfectants in which OIG37 expression was inducible by Dex (Fig. 5A). Both clones expressed almost the same level of OIG37. The level of OIG37 expression by Dex was comparable with that induced by OSM (data not shown). The cells were grown in the presence or absence of Dex, and the cell numbers were monitored. The growth rates of both NIH3T3OR transfectants expressing OIG37 were clearly reduced when Dex was added. The levels of growth suppression were almost the same between the two clones, because both expressed the same level of OIG37 (Fig. 5B). The growth suppressive effect was also seen in the BaF3 transfectants when OIG37 is induced by Dex (data not shown). Growth suppression induced by OIG37 expression did not accompany apoptotic cell death based on trypan blue exclusion and DNA ladder formation (data not shown).


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  		Fig. 5.   Suppression of proliferation by OIG37 in NIH3T3. A, inducible expression of FLAG-tagged OIG37 in NIH3T3OR cells resulted in reduction of the growth rate. NIH3T3OR cells were stably transfected with the expression vector that contains the OIG37 cDNA linked to the Dex-inducible mouse mammary tumor virus promoter. Two independent clones and the parental cells were cultured in the presence () or absence () of Dex, and the cell numbers were counted. B, expression of OIG37 by Dex. The two clones and the parental cells were cultured with 5 × 10-7 M Dex for 12 h, and the total lysates were analyzed by Western blotting using the anti-FLAG antibody as the FLAG tag was attached to OIG37.

Association of OIG37 with p21 and PCNA-- Because GADD45 and MyD118 were previously shown to associate with p21 and PCNA, we wished to know whether OIG37 also interacts with these cell cycle regulators. COS7 cells were transiently transfected with the expression vectors with HA-tagged OIG37, MyD118, or GADD45 cDNA, and anti-HA antibodies were used to immunoprecipitate these gene products from the extracts. The immunoprecipitates were analyzed by Western blotting with anti-p21 and anti-PCNA antibodies, and both proteins were clearly detected (Fig. 6), suggesting that the binding affinities of GADD45 family members to p21 or PCNA may be comparable.


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  		Fig. 6.   Association of OIG37 with p21 and PCNA. COS7 cells were co-transfected with the expression vector carrying the HA-tagged GADD45 family member cDNA and that of p21 cDNA, both of which are linked to the SRalpha promoter. Cell extracts were used to immunoprecipitate with anti-HA antibody. The immunoprecipitates were applied on a 12% polyacrylamide gel, and the blot was prepared. The blots were incubated with anti-HA antibody (lanes 1-4), anti-p21 antibody (lanes 5-8), or anti-PCNA antibody (lanes 9-12). Lanes 1, 5, and 9, transfected with the empty vector and p21 expression vector; lanes 2, 6, and 10, transfected with OIG37 and p21; lanes 3, 7, and 11, transfected with MyD118 and p21; lanes 4, 8, and 12, transfected with GADD45 and p21.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have isolated OIG37 as an OSM-inducible gene in a hemangioblastic cell line and found that OIG37 is related to two growth suppressive genes, MyD118 and GADD45. During preparation of this manuscript, Takekawa and Saito (38) published isolation of human GADD45-related genes, GADD45alpha (GADD45), GADD45beta (MyD118), and GADD45gamma as MTK1/MEKK4-binding proteins. OIG37 appears to be mouse GADD45gamma based on the sequence homology with human GADD45gamma (96%). In addition, the CR6 gene identified as an IL-2-inducible gene might be identical to OIG37/GADD45gamma (39). These three genes show extensive sequence homology and clearly form a new gene family. The middle part of these proteins, which potentially forms an alpha  helix and a beta  sheet, is particularly well conserved. Because Takekawa and Saito (38) demonstrated that all members of the GADD45 family bind to MTK1/MEKK4 and we and others show that they interact with p21 and PCNA, the conserved motifs of these proteins may be involved in the interaction with MTK1/MEKK4, PCNA, and p21. PCNA-interacting proteins such as p21 (40, 41), XPG (42), Fen1 (43), and cytosine methyl transferase (44) have the motif, QXXhXXaa, (where h indicates a hydrophobic amino acid and a indicates an aromatic amino acid), and OIG37 contains the sequence QGCLTAGVY (between 36th and 44th amino acid residues), which is similar to the PCNA-binding motif (45). This motif is also well conserved in GADD45 and MyD118 (Fig. 1B). It was also reported that the N-terminal half of GADD45, which contained this motif, was sufficient for the PCNA binding (27). These results suggest that binding of the GADD45 family members to PCNA is mediated by the conserved motif around amino acids 30-45.

OIG37 was highly inducible by OSM in LO and NIH3T3OR cells but was completely absent in BaF3/OSMR cells. MyD118 expression was rather constitutive and was slightly induced by OSM in LO and BaF3/OSMR but not in NIH3T3OR. GADD45 expression was constitutive in all these cells. In M1 cells, MyD118 was highly inducible by IL-6, but the OIG37 induction was much weaker, and expression of GADD45 was rather suppressed by IL-6. These results indicate that expression of OIG37 and MyD118 is regulated by signaling through gp130, the common signaling subunit of the OSMR and the IL-6 receptor, whereas GADD45 expression is independent of gp130 signaling. We have also tested stress-induced expression of OIG37 by the addition of MMS to NIH3T3OR and M1 cells. In contrast to the rapid induction of OIG37 by OSM, MMS-induced expression of OIG37 required a much longer time and the extent of expression was lower than that by cytokine stimulation. These results indicate that the mechanism of OIG37 induction by MMS is quite different from that by cytokines.

We have investigated cytokine signals that lead to OIG37 expression by using the chimeric receptors consisting of the extracellular domain of the G-CSF receptor and the intracellular domain of gp130. By expressing mutant chimeric receptors in which the cytoplasmic domain is mutated, we found that STAT3 is involved in the OIG37 induction. This possibility was further supported by suppression of OSM-induced expression of OIG37 by the dominant negative form of STAT3. On the other hand, the mutant receptor G133F3 defective for Ras activation showed a slightly higher induction, and Ras is known to inhibit activation of STATs such as STAT3 and STAT5 in certain cells (46-48); Ras may negatively regulate OSM-induced OIG37 expression. However, because expression of OIG37 by OSM is cell type-specific, a cell type-specific factor(s) may be involved in OIG37 induction in addition to the signaling molecules activated by gp130.

Inducible expression of OIG37 using the mouse mammary tumor virus promoter clearly established that OIG37 reduces the growth rate of NIH3T3OR, although it does not completely inhibit proliferation. Although OIG37 clearly suppresses proliferation of LO, NIH3T3OR, and BaF3 when it is expressed, growth suppressive activity of OIG37 is not necessarily correlated with the expression pattern in untransfected cells. Further, cell cycle analysis using the NIH3T3 transfectants showed that the transition from G1 to G2/M phase was delayed when OIG37 was induced (data not shown). OSM stimulates proliferation of LO and induces OIG37 expression, whereas it inhibits proliferation of NIH3T3OR, which expresses OIG37. In contrast, OSM does not induce OIG37 and stimulates proliferation of BaF3/OSMR. Likewise, MyD118 is expressed regardless of whether cell proliferation is stimulated (LO and BaF3/OSMR) or suppressed (NIH3T3OR and M1) by OSM or IL-6. Growth suppressive activity of OIG37 and MyD118, as clearly shown in transfected cells, may depend on the levels of their expression; perhaps OIG37 and MyD118 suppress cell proliferation only when their expression levels reach to a certain threshold.

Takekawa and Saito (38, 49) recently described that stress induces expression of the members of the GADD45 family, which in turn induce p38/JNK activation and apoptosis through MTK1/MEKK4 activation in HeLa cells. However, controlled expression of OIG37 in NIH3T3 (Fig. 5A) and BaF3 (data not shown) by using the Dex-inducible mouse mammary tumor virus promoter clearly indicated that expression of OIG37 only reduced the growth rate, but we never observed apoptosis induced by OIG37 (data not shown). These apparently contradictory results may be due to the difference in the expression levels, because Takekawa and Saito observed apoptosis when GADD45 members were transiently overexpressed, whereas our results were obtained with stably transfected cell lines. We therefore tested trypan blue staining and DNA ladder formation in COS7 cells transiently overexpressed OIG37 and found apoptotic cells. Thus, the physiological significance of apoptosis induced by OIG37 remains to be established.

Induction of apoptosis by the GADD45 family members through MEKK4 and p38/JNK is an attractive hypothesis to elucidate the stress-induced responses (38, 49). It was further demonstrated that the GADD45 family members interact with MEKK4 in transiently transfected COS7 cells (38). Likewise, we were able to demonstrate the association of OIG37 with p21 and PCNA in transiently transfected COS7 cells. However, it still remains uncertain whether MEKK4, p21, and PCNA interact with GADD45 family members under physiological conditions. It is possible that GADD45 family members interact with these proteins only when their expression reaches a certain level. This partly explains why the effect of OSM was variable depending on cells. Roles of MEKK4, p21, and PCNA in the function of the GADD45 family members await further investigation.

    ACKNOWLEDGEMENT

We thank Takashi Sekiguchi for excellent support for cell sorting.

    FOOTNOTES

* This work was partly supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, a grant from Core Research for Evolutionary Science and Technology, and research grants from The Uehara Foundation, The Foundation for Promotion of Cancer Research, and The Institute of Physical and Chemical Research (RIKEN).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB021884.

To whom correspondence should be addressed. Tel.: 81-3-5841-8474; Fax: 81-3-5841-8475; E-mail: miyajima@ims.u-tokyo.ac.jp.

2 T. Hara, Y. Mukouyama, and A. Miyajima, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: OSM, oncostatin M; IL, interleukin; LIF, leukemia inhibitory factor; OSMR, OSM receptor; LIFR, LIF receptor; G-CSF, granulocyte colony-stimulating factor; G-CSFR, G-CSF receptor; PCNA, proliferating cell nuclear antigen; FBS, fetal bovine serum; DIG, digoxigenin; MMS, methyl methane sulfonate; IRES, internal ribosomal entry site; GFP, green fluorescent protein; HA, hemagglutinin; Dex, dexamethasone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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