Oncostatin M A. Gregory Bruce and Timothy M. Rose * Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Box 357238, Seattle, WA 98195, USA *corresponding author tel: 206-616-2084, fax: 206-543-3873, e-mail: [email protected] DOI: 10.1006/rwcy.2000.06005. SUMMARY Structure Oncostatin M (OSM) is a pleiotropic cytokine within Human OSM is a secreted glycoprotein which is the IL-6 family of cytokines which regulates cell initially translated as a 252 amino acid polypeptide growth and differentiation in a wide variety of bio- with a 25 residue hydrophobic signal sequence at the logicalsystems,includinghematopoiesis,neurogenesis, N-terminus that is removed during the secretion pro- and osteogenesis. cess.Anadditionalposttranslationalcleavageremoves 31 C-terminal residues, resulting in a 192 amino acid disulfide-linked mature protein. OSM adopts a four BACKGROUND (cid:11)-helical bundle structure with up-up-down-down topology similar to that determined for other cyto- Discovery kines and growth factors (Rose and Bruce, 1991; Robinson et al., 1994). Oncostatin M (OSM) was originally discovered as a protein which inhibited the proliferation of a number of human tumor cell lines, but not normal human Main activities and fibroblasts. It was isolated from supernatants of U- pathophysiological roles 937 human histiocytic leukemia cells that were induced to differentiate into macrophage-like cells by treatment with phorbol 12-myristate 13-acetate OSM has been shown to be a pleiotropic cytokine (PMA)(Zarlingetal.,1986).Human,simian,bovine, which regulates cell growth and differentiation in a and murine forms of OSM have been cloned and wide variety of biological systems including hemato- characterized. OSM has been shown by sequence poiesis, neurogenesis, and osteogenesis (Bruce et al., homology and structural and functional similarities 1992b). The elaboration of the biological activities of to be closely related to leukemia inhibitory factor OSM has been confounded by the presence of (LIF) and other members of the IL-6 family of different OSM receptor signaling systems in humans cytokines, including IL-6, IL-11, ciliary neurotropic and mice. In humans, OSM signals through two factor (CNTF), and cardiotropin 1 (CT-1) (Bazan, different receptors complexes; the LIF/OSM shared 1991; Rose and Bruce, 1991). receptor (Gearing and Bruce, 1992), which shares high-affinity binding with LIF, an evolutionarily related protein with structural similarity to OSM Alternative names (Rose and Bruce, 1991), and the OSM-specific receptor which binds OSM uniquely (Bruce et al., A number of abbreviations for oncostatin M have 1992b).Inmice,OSMsignalsonlythroughthemurine been used in the literature, including OSM, OM, homologoftheOSM-specificreceptor(Ichiharaetal., Onco M, and ONC, although OSM is the preferred 1997; Lindberg et al., 1998). To confuse matters, abbreviation. There are no other factors that have human OSM, used historically for in vitro and in vivo subsequently been shown to be OSM. studies in mice, binds uniquely to the murine LIF 586 A. Gregory Bruce and Timothy M. Rose Table 1 OSM gene sequences Species Accession number Source Type Size (bp) References Human M27286 Brain Gene, exon 1/intron 1 junction 65 Malik et al., 1989 M27287 Brain Gene, exon 2/intron 1 and 2 junctions 155 Malik et al., 1989 M27288 Brain Gene, exon 3/intron 2 junction 1,643 Malik et al., 1989 AC004264 Gene locus 47,188 Unpublished Mouse D31942 Pre-B cell mRNA, complete 1,848 Yoshimura et al., 1996 Not deposited Promoter region 867 Yoshimura et al., 1996 Bovine S78434 Gene, exon 1 67 Malik et al., 1995 S78487 Gene, exon 2 152 Malik et al., 1995 S78435 Gene, exon 3 903 Malik et al., 1995 Table 2 OSM gene structure receptorandthusexhibitsonlythebiologicalactivities Properties Human Murine Bovine of LIF in mice and not that of OSM receptor OSM OSM OSM (Ichihara et al., 1997; Lindberg et al., 1998). Therefore, the biological activities for OSM are Gene size (cid:24)5kbp nd (cid:24)5kbp derived from signaling through two different recep- mRNA size (cid:24)2kbp (cid:24)2kbp nd torsandoverlapthoseofLIFinhumanbutnotmurine Intron number 2 2 2 systems. The ability of OSM to function through the OSM-specific and LIF/OSM shared receptors in Exon 1, noncoding (cid:24)25bp (cid:24)52bp nd humans and only the OSM-specific receptor in mice Exon 1, coding 34bp 31bp 34bp suggests that there may be significant differences in Intron 1 1,620bp nd 1,418bp the biological roles played by OSM in different Exon 2, coding 143bp 137bp 134bp species. Furthermore, although the biological activity ofOSMhasbeenstudiedinvariousinvitroassays,its Intron 2 534bp nd 572bp exact role in vivo has not been established. As such, Exon 3, coding 582bp 621bp 567bp the literature on OSM should be reviewed with Exon 3, noncoding 1,054bp 1,018bp nd careful consideration of these findings. nd,notdetermined. GENE AND GENE REGULATION portion of the mature molecule. The last exon con- Accession numbers tains the remaining coding sequences and all of the 30 noncoding sequences. The 30 ends ofthe OSM The OSM genes and mRNA transcripts have been cDNAs contain an A(cid:135)T-rich region with several cloned and characterized from human, simian, ‘ATTTA’ pentamer motifs, which are involved in the murine, and bovine sources (Table 1). The OSM regulation of the stability of many cytokine and genes span approximately 5kb and transcripts of lymphokine transcripts. The intron–exon structure is approximately 2kb have been detected in various identical between the human, murine, and bovine tissues (see below). Three exons have been identified genes (Table 2). in the OSM gene and the intron–exon junction se- quences conform to the GT...AT rule for nucleotides Sequence flanking eukaryotic exon boundaries. The first exon containsthe50 noncodingDNAandtheDNAencod- ing the initiating methionine and 10 amino acids of For the nucleotide sequence of human OSM, see the signal sequence. The second exon encodes the Figure1, ofmurineOSM,seeFigure2, andofbovine remainder of the signal sequence and the first OSM, see Figure 3. Oncostatin M 587 Figure 1 The nucleotide and encoded amino acid sequence of human OSM. Thesignalsequenceisboxedingreen.(Fullcolourfiguremaybeviewedonline.) Chromosome location related LIF gene (Rose et al., 1993). The OSM gene hasalsobeencolocalizedwiththeLIFgeneonmouse In humans, the OSM gene has been localized to chromosome 11, which is syntenic in this region with chromosome 22 at 22q12 just upstream of the closely human chromosome 22 (Yoshimura et al., 1996). 588 A. Gregory Bruce and Timothy M. Rose Figure 2 The nucleotide and encoded amino acid sequence of murine OSM. Thesignalsequenceisboxedingreen.(Fullcolourfiguremaybeviewedonline.) Relevant linkages telomeric to centromeric, with the OSM gene located upstream of the LIF gene. The OSM and LIF genes The OSM and LIF genes are tandemly arranged on were also found to be within 2cM of each other on human chromosome 22 in the same transcriptional murine chromosome 11. These findings provide orientation separated by approximately 10kb (Rose strong evidence that OSM and LIF genes resulted et al., 1993). The direction of gene transcription is from duplication of a common ancestral gene. Oncostatin M 589 Figure 3 The nucleotide and encoded amino acid sequence of bovine OSM. Thesignalsequenceisboxedingreen.(Fullcolourfiguremaybeviewedonline.) Regulatory sites and corresponding Cells and tissues that express transcription factors the gene ThemajorinitiationsiteofthemurineOSMgenewas There are only a few studies describing cell typesthat found at (cid:255)52 with a minor initiation site at (cid:255)81. No produceOSM.OSMwasfirstidentifiedinthehuman TATA box was found; however, there is a GC-rich myelocytic leukemia cell line U937, after treatment region near the transcription initiation site. A IFN(cid:13)- with phorbol esters. Subsequent studies identified activated site (GAS) like sequence, ‘TTCCCAGAA’, activated T lymphocytes and macrophages, cell cul- found at (cid:255)100 is identical to that of the human Fc(cid:13) tures derived from acquired immunodeficiency virus receptorgenepromoterandthe rat(cid:11) -macroglobulin (AIDS)-related Kaposi’s sarcoma (Nair et al., 1992) 2 promoter. This sequence appears to be involved in and retrovirus-infected CD4(cid:135) T cells (Radka et al., activation of the murine OSM promoter by binding 1993) as sources of OSM mRNA. Murine OSM STAT5, which is activated by IL-2, IL-3, and EPO. mRNAhasbeendetectedinbonemarrowandspleen Another GAS-like sequence, ‘TTCGAAGAA’, was but was not detectable in liver, lung, ovary, small found at (cid:255)180 but does not appear to be involved in intestine, kidney, or brain (Yoshimura et al., 1996). promoter activation by STAT5. Additionally, murine OSM has been detected in 590 A. Gregory Bruce and Timothy M. Rose the gonads of developing embryos (Hara et al., Sequence 1998). For the amino acid sequence of human OSM, see PROTEIN Figure1,ofmurineOSM,seeFigure2,andofbovine Accession numbers OSM, see Figure 3. A comparison of the human, simian, murine, and bovine sequences is shown in See Table 3. Figure 4. Table 3 OSM protein sequences Species Accession no. Source Type Size (aa) References Human 129168 U937 cells Complete 252 Malik et al., 1989 2935614 Complete 252 Unpublished 246727 Kaposi’s sarcoma cells Partial, N-terminal 20 Nair et al., 1992 Simian Not deposited CV1 cells Partial, mature 196 Bruce et al., 1992b Mouse 1709466 BF-EGFR/EPORH cells Complete 263 Yoshimura et al., 1996 Bovine 1709463 Complete 245 Malik et al., 1995 Figure 4 Comparison of human, simian, murine, and bovine OSM sequences. Undetermined residues in the simian sequence are indicated (.). The predicted (cid:11) helical and turn domains are shown (Kitchen et al., 1998). The N- and C-terminal cleavage sites are indicated with an arrow and the signal peptide is indicated. Oncostatin M 591 Description of proteins Human, simian, murine, and bovine OSM contain four conserved cysteine residues. The human and simian sequences contain an additional cysteine in The proposed structures of human, bovine, and helix B that is absent from the murine and bovine murineOSMarecomparedinTable4.TheOSMgene sequences. Mutational studies have shown that a encodes a pre-pro OSM precursor which is processed disulfide bond between the second and fifth cysteine posttranslationallyatboththeN-andC-termini.The residuesofhumanOSMisrequiredforactivity,while N-terminal hydrophobic signal sequence, (cid:24) 23–25 a bond between the first and fourth cysteine residues residues, is removed during the secretory process. An is not (Kallestad et al., 1991). The mature, natural additional cleavage occurs after a pair of basic resi- forms of hOSM and bOSM migrate in SDS-PAGE dues in the C-terminal domain, removing 31–57 resi- with an apparent molecular weight of 28kDa under dues, depending on the species, yielding the mature both reducing and nonreducing conditions (Zarling formofthesecretedprotein(Linsleyetal.,1990).The et al., 1986; Malik et al., 1992). This agrees with the 196 amino acid mature human OSM is the predomi- results from size-exclusion chromatography, which nant form isolated from PMA-treated U937 cells and indicate that the active form of the molecule is a from CHO cells transfected with the hOSM cDNA. monomer. The227aminoacidformofOSM(pro-hOSM)has been isolated from COS cells transfected with the hOSMcDNA. Thispro-OSMform isasactiveasthe Discussion of crystal structure 196-residue mature hOSM in competition binding assays, but is 5-fold to 60-fold less active in growth inhibition assays on A375 cells (Linsley et al., 1990). Although a crystal structure for OSM has not yet While transfection of the full-length human OSM been described, the structures for related proteins, cDNA in COS cells appears to produce primarily the including LIF, G-CSF, and growth hormone (GH) active,matureform,similarstudieswithmurineOSM have been determined (Betzel et al., 1993; Robinson cDNA have resulted in only inactive forms of the et al., 1994). Using the structural similarities between molecule(Yoshimuraetal.,1996).Thismaybedueto these proteins and OSM and NMR structural data improper cleavage of the C-terminus of murine OSM obtained from OSM, a homology model of OSM has in these cells. Although the 227 amino acid pro- been derived (Hoffman et al., 1996; Kitchen et al., hOSM has not yet been isolated from a natural 1998). This model demonstrates that OSM adopts source, tissue-specific processing could result in two a four (cid:11)-helical bundle structure similar to that forms of hOSM having distinct biological functions determinedforothercytokines,ashadbeenpredicted in vivo. High and low molecular weight forms of rat earlier (Rose and Bruce, 1991). The proposed struc- OSM have been detected in developing testis (de ture for OSM is supported by mutational analysis, Miguel et al., 1997). which demonstrated that discontinuous regions, Table 4 OSM protein structure Properties Human Simian Murine Bovine OSM (aa) OSM (aa) OSM (aa) OSM (aa) Precursor 252 nd 263 245 Signal peptide 25 nd 23 23 Pro-protein 227 nd 240 222 Excised C-terminal peptide 31 nd 57 39 Mature protein 196 196 183 183 Cysteine residues 5 5 4 4 Disulfide linkages 2 2 2 2 N-linked glycosylation sites 3 3 3 1 Identity to human OSM 100% 93% 48% 58% nd,notdetermined. 592 A. Gregory Bruce and Timothy M. Rose primarilyintheexceptionallyamphipathicC-terminal CELLULAR SOURCES AND helix, are involved in receptor binding (Kallestad TISSUE EXPRESSION et al., 1991). Cellular sources that produce Important homologies Human OSM has been purified from supernatants of U937 histiocytic lymphoma cells induced to differ- An alignment of the amino acid sequences of OSM entiate with phorbol 12-myristate 13-acetate (PMA) from derived from four different species is shown in (Zarling et al., 1986) and T-lymphocytes treated with Figure 4. Significant amino acid similarity is detected phytohemagglutinin or mAb 9.3 (anti-human CD28) throughout the coding regions and obvious con- (Brown et al., 1987). In addition, using monoclonal served sequence motifs are observed. The amino antibodies, human OSM has been detected in cell acid sequence of human OSM is 93% identical to culturesderivedfromAIDS-relatedKaposi’ssarcoma simian OSM, 58% identical to bovine OSM, and (Nair et al., 1992) and retrovirus-infected CD4(cid:135) T 48% identical to murine OSM (Table 4). Murine cells (Radka et al., 1993). OSM is also produced by OSM is 42% identical to bovine OSM. The most human synovial tissue macrophages (Okamoto et al., important conserved motif is found within the C- 1997).MurineOSMisexpressedatsignificantlevelsin terminal helix and contains the sequence pattern bonemarrowinmaturemice(Yoshimuraetal.,1996) ‘GYHRFM’. This region is believed to be a major and has been detected in the gonads of developing binding site with the OSM receptor. OSM shares embryos(Hara et al., 1998). OSMis highly expressed sequence homology and structural similarities with inthelatefetalandearlyneonatalrattestis,aswellas LIF and other members of the IL-6 family of inthematuringandadulttestis(deMigueletal.,1997). cytokines, including IL-6, G-CSF, CNTF, IL-11, andCT-1 (RoseandBruce, 1991;Bazan,1991;Bruce et al., 1996). Eliciting and inhibitory stimuli, including exogenous and Posttranslational modifications endogenous modulators As described above, the OSM gene encodes a pre- EarlystudiesinU937histocyticleukemiacellsshowed pro OSM precursor which is processed posttransla- that phorbol esters (PMA) induced the general tionally atboththeN- andC-termini. AnN-terminal expression of human OSM. Murine OSM has been hydrophobic signal sequence is removed during shown to be an immediate early gene induced by the secretion process and a C-terminal peptide is multiple cytokines, including IL-2, IL-3, and EPO cleavedatatrypsin-likerecognitionsequence(RSRR) (Yoshimura et al., 1996). (Table 4; Figure 4). OSM is a glycoprotein which is There are no known exogenous or endogenous modified by N-linked and, possibly, O-linked glyco- modulators of OSM. sylation (Linsley et al., 1990). The human, bovine, andsimiansequencescontainaconservedaminoacid RECEPTOR UTILIZATION site which may be N-linked glycosylated. A second potential N-linked glycosylation site is conserved in the human and simian sequences, but is absent from High- and low-affinity receptors for OSM have been the bovine sequence. There is evidence from hOSM detectedonawidevarietyofcelltypes(Linsleyetal., (natural-, CHO-, and COS-derived) that this second 1989;Hornetal.,1990).Thelow-affinityreceptorfor siteisnotused(Linsleyetal.,1990;Maliketal.,1989). OSM has been identified as gp130, a molecule pre- There are three potential glycosylation sites in the viously shown to be the signaling subunit of the IL-6 murine OSM but none of these sites is positionally receptor complex (Hibi et al., 1996). Structural and conserved in the other sequences. Recombinant functionalcharacterizationofgp130hasshownthatit human OSM expressed in bacteria is fully active belongs to a family of related cytokine receptors in vitro, indicating that glycosylation is not required (Bazan, 1991). Receptor binding in this family is for biological activity. Human, simian, bovine, and characterized by low-affinity binding to an ‘alpha’ murine OSM each contain two conserved intramole- receptor which is converted to high affinity in the cular disulfide bonds, one of which is required for presence of an additional related ‘beta’ receptor activity (Kallestad et al., 1991). subunit/subunits. Because gp130 is the low-affinity Oncostatin M 593 receptor for OSM, it is herein designated as OSMR(cid:11) and maintenance of murine ES cells in an undiffer- (gp130). Two different high-affinity heterodimeric entiated state. receptor complexes have been identified in humans; an OSM-specific receptor complex composed of OSMR(cid:11) (gp130) and a high-affinity converting IN VIVO BIOLOGICAL receptorsubunit,OSM‘beta’receptor(OSMR(cid:12)),and ACTIVITIES OF LIGANDS IN a receptor complex shared with LIF composed of ANIMAL MODELS OSMR(cid:11) (gp130) and the LIF ‘alpha’ receptor (LIFR(cid:11)). This differs from the binding seen in the Normal physiological roles mouse, where murine OSM only binds the murine OSM-specific receptor complex and not the LIF receptorcomplex.Itshouldbepointedoutthathuman The normal physiological roles of OSM are not yet OSM,usedinearlyinvitroandinvivoexperimentsin known. There is evidence in mice that mOSM may mice, binds only the murine LIF receptor complex play a role in germ-cell development as a stage- and and not the murine OSM receptor complex and thus sex-specific autocrine growth factor for Sertoli cells mimics the activity of LIF in mice. (Hara et al., 1998). Species differences IN VITRO ACTIVITIES In vitro findings The differences between the biological roles of OSM in different species are not yet understood. The findings that human OSM acts through both the While a number of biological activities have been human OSM-specific receptor and the shared LIF/ defined for OSM from in vitro studies, its primary OSM receptor while murine OSM acts through the biological role is still not clearly defined. Current murine OSM-specific receptor but not the murine evidence suggests that OSM plays a role in hemato- LIF receptor, suggests that there will be signifi- poiesis, neuropoiesis, bone growth, and regulation of cant differences between mouse and human OSM immune responses (Table 5). bioactivities. Regulatory molecules: Inhibitors Transgenic overexpression and enhancers Bovine OSM has been overexpressed in transgenic Soluble forms of OSMR(cid:11) (gp130) which bind OSM mice using various tissue-specific promoters. While a at low affinity act as antagonists (Montero-Julian wide variety of developmental abnormalities were et al., 1997). detected, the results are not interpretable since it is likelythatbovineOSM,likehumanOSM,mimicsthe effects of murine LIF in the transgenic mice rather Bioassays used than the effects of murine OSM. It will be necessary to use murine OSM transgenes to determine the bio- Human OSM bioactivity has been determined by a logicaleffectsofOSMinmice.However,thiswillstill numberofbioassays(Table5)includinggrowthinhib- not completely determine the function of OSM in ition of human melanoma A375 cells (Malik et al., humans and other species in which OSM can signal 1992), differentiation of human TH-1 (Liu et al., throughboththeOSM-specificreceptorandtheLIF/ 1994)ormurinemyeloidleukemiaM1cells(Roseand OSM shared receptor. Bruce, 1991) and maintenance of murine ES cells in anundifferentiatedstate(Roseetal.,1993).Itshould Interactions with cytokine network be noted that the assays on mouse cells are based on interaction of human OSM with the mouse LIF receptor, not the OSM receptor. A hOSM-specific OSM induces expression of IL-6 and the IL-6 recep- ELISA is commercially available. tor in certain cell types. Several studies have Murine OSM bioactivity has been determined by demonstrated a synergy with TGF(cid:12). In mice, OSM growth inhibition of murine myeloid leukemia M1 or is an immediate early gene induced by IL-2, IL-3, NIH 3T3 cells, support of murine DA-1 cell growth and EPO. 594 A. Gregory Bruce and Timothy M. Rose Table 5 In vitro properties of OSMa Property Species Cell type References (cid:11) -Antichymotrypsin induction Human HepG2 hepatoma Richards et al., 1992 1 Human Astrocytes Kordula et al., 1998 Human Lung epithelial Cichy et al., 1995 (cid:11) -Antitrypsin induction Human A549 epithelial Boutten et al., 1998 1 (cid:11) -Proteinase inhibitor induction Human A549 epithelial Sallenave et al., 1997 1 Human HTB58 lung epithelial Cichy et al., 1997a Cell survival enhancement Mouse Primordial germ Hara et al., 1998 Differentiation induction Human U937 leukemia Bruce et al., 1992a Human Breast cancer Douglas et al., 1998 EGR-1, c-jun, c-myc induction Human Fibroblasts Liu et al., 1992 Endothelin 1 induction Human HUVEC endothelial Saijonmaa et al., 1998 G-CSFb and GM-CSFc induction Human HEC endothelial Brown et al., 1993 Growth inhibition Human Various tumor Horn et al., 1990 Human Melanoma Brown et al., 1987; Gibbs et al., 1998 Human Endothelial Takashima and Klagsbrun, 1996 Haptoglobin induction Human HepG2 hepatoma Richards et al., 1992 IL-6 induction Human Lung fibroblast Richards and Agro, 1994 Human HEC endothelial Brown et al., 1991 LDLc modification Human U937 leukemia Maziere et al., 1994 LDL receptor induction Human HepG2 hepatoma Grove et al., 1991 Matrix metalloproteinase 1 induction Human Dermal fibroblasts Korzus et al., 1997 P21 kinase inhibitor induction Human MG63 osteosarcoma Bellido et al., 1998 p42MAPK/ERK-2 kinase induction Human Kaposi’s sarcoma Amaral et al., 1993; Faris et al., 1996 p62yes tyrosine kinase induction Human HEC endothelial Schieven et al., 1992 Plasminogen activator induction Human Brown et al., 1990 Human Synovial fibroblasts Hamilton et al., 1991 Human HepG2 hepatoma Okada et al., 1996 Polykaryon formation Human Bone marrow Heymann et al., 1998 Proliferation induction Human Kaposi’s sarcoma Miles et al., 1992; Nair et al., 1992; Cai et al., 1994 Human Various normal Horn et al., 1990 P-selectin induction Human Endothelial Yao et al., 1996 Soluble IL-6 receptor induction Human HepG2 Cichy et al., 1997b TIMP-1d induction Human Articular cartilage Nemoto et al., 1996 Human Synovial lining Gatsios et al., 1996 Mouse NIH 3T3 fibroblasts Richards et al., 1997 Human Fibroblasts Korzus et al., 1997