OS9 interacts with DC-STAMP and modulates its intracellular localization in response to TLR ligation

doi:10.1016/j.molimm.2008.06.032

Molecular Immunology

Volume 46, Issue 4, February 2009, Pages 505-515

https://doi.org/10.1016/j.molimm.2008.06.032 Get rights and content

Abstract

Dendritic cell-specific transmembrane protein (DC-STAMP) has been first identified as an EST in a cDNA library of human monocyte-derived dendritic cells (DC). DC-STAMP is a multimembrane spanning protein that has been implicated in skewing haematopoietic differentiation of bone marrow cells towards the myeloid lineage, and in cell fusion during osteoclastogenesis and giant cell formation. To gain molecular insight in how DC-STAMP exerts its function, DC-STAMP interacting proteins were identified in a yeast-2-hybrid analysis. Herein, we report that amplified in osteosarcoma 9 (OS9) physically interacts with DC-STAMP, and that both proteins colocalize in the endoplasmic reticulum in various cell lines, including immature DC. OS9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. Interestingly, we now demonstrate that toll-like receptor (TLR)-induced maturation of DC leads to the translocation of DC-STAMP from the ER to the Golgi while OS9 localization is unaffected. Applying TLR-expressing CHO cells we could confirm ER-to-Golgi translocation of DC-STAMP following TLR stimulation and demonstrated that the DC-STAMP/OS9 interaction is involved in this process. Collectively, the data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering, suggesting a novel role for OS9 in myeloid differentiation and cell fusion.

Introduction

Dendritic cells (DC) play a pivotal role in the initiation of innate and adaptive immune responses. Immature DC capture foreign antigens in peripheral tissues and migrate to the T-cell areas of secondary lymphoid organs where they present these antigens to T- and B-cells. The capture of antigens in the steady state allows DC to control immunotolerance towards itself (Banchereau and Steinman, 1998, Mellman and Steinman, 2001). Antigen uptake in the context of inflammation or infection results in DC maturation. In the presence of inflammatory cytokines and through binding of so-called pathogen-associated molecular pattern (PAMP)² to one or more paralogues of the toll-like receptor (TLR) family, DC mature and acquire the capacity to induce potent immunity. They initiate both innate and adaptive immune responses (Akira and Takeda, 2004, Janeway and Medzhitov, 2002). As a consequence, DC have gained considerable interest as vaccine adjuvants and are currently exploited in the treatment of cancer after loading DC with tumor-cell derived antigens (Figdor et al., 2004, Schreurs et al., 2000).

Although much is now known about the cellular nature of DC, molecular insight in its function, although mounting, is fragmentary at best. In order to gain more insight into this aspect of DC, we and others have analyzed DC at the molecular level. One of the genes identified was dendritic cell-specific transmembrane protein (DC-STAMP), which appears to be preferentially expressed by myeloid DC (Eleveld-Trancikova et al., 2005, Eleveld-Trancikova et al., 2008, Hartgers et al., 2000, Hartgers et al., 2001). Others have reported the induction of DC-STAMP expression in macrophages stimulated with interleukin 4 (IL-4) (Staege et al., 2001), as well as its expression in osteoclasts (Nomiyama et al., 2005). DC-STAMP has 4– transmembrane regions, and its gene localizes to chromosome 8q23 (Hartgers et al., 2001). In monocyte-derived DC and HEK293 cells, DC-STAMP localizes to the endoplasmic reticulum (Eleveld-Trancikova et al., 2005). Biological roles for DC-STAMP are only recently emerging. We previously reported that DC-STAMP inhibits granulopoiesis, but promotes myeloid differentiation in murine bone marrow cells transduced with a retroviral construct expressing DC-STAMP fused to GFP (Eleveld-Trancikova et al., 2008). Also, there is evidence for a role of DC-STAMP in osteoclastogenesis, as DC-STAMP expression appears to facilitate osteoclast differentiation in a murine macrophage RAW cell line that is otherwise incapable of differentiating into osteoclasts (Kukita et al., 2004). These findings were further corroborated in homozygous DC-STAMP knock-out mice, as these mice have mild osteopetrosis and display debilitating defects in the formation of multinuclear osteoclasts and giant multinucleated cells in response to foreign bodies, showing that DC-STAMP is required for cell fusion. However, the molecular mechanism by which DC-STAMP exerts its effect in osteoclasts and DC remains unknown (Yagi et al., 2005).

In order to identify interacting partners of DC-STAMP, we have constructed a prey cDNA library of mature and immature DC, and used the cytoplasmic tail of DC-STAMP as a bait in a yeast-2-hybrid screening. Here, we report the identification of amplified in osteosarcoma 9 (OS9) as an interacting partner of DC-STAMP. The interaction of the two proteins is confirmed biochemically and by their colocalization in various cell types, including DC. Previous studies have implicated a role for OS9 in ER-to-Golgi transport (Friedmann et al., 2002, Litovchick et al., 2002). Interestingly, we now demonstrate that DC-STAMP enters the secretory pathway upon TLR stimulation in DC transduced with an adenovirus expressing DC-STAMP and CHO cells stably expressing TLR. The localization of OS9 in CHO cells and DC is not affected by maturation stimuli. Mutational analysis of DC-STAMP and OS9 suggest a critical role of the OS9/DC-STAMP interaction in the ER-to-Golgi transport of DC-STAMP.

Section snippets

Plasmids, adenoviral vectors and cloning

Plasmids pGADGH and pGBT9 were used for yeast-2-hybrid analysis, and pER-DsRed, which contains the ER-targeting sequence of calreticulin fused to DsRed, was used for co-localization studies (all from Clontech, Mountain View, CA). The plasmids and adenoviral plasmids encoding DC-STAMP fused to the GFP protein have been described elsewhere (Eleveld-Trancikova et al., 2005). The plasmid encoding DC-STAMP fused to a heamagglutinin (HA) tag was created by replacing the EGFP moiety of the pEGFP-N3

OS9 interacts with the cytoplasmic tail of DC-STAMP

To identify DC-STAMP binding partners, a yeast-2-hybrid analysis was performed. The predicted cytoplasmic carboxy-terminal tail of DC-STAMP, corresponding to amino acids 403–470, was used to screen a prey cDNA library derived from a mixture of immature and mature DC (Triantis et al., 2006). As shown in Table 1, of the 58 positive colonies identified, eight colonies (14%) contained a sequence derived from human OS9. Further sequence analysis revealed that both OS9 isoform 1 (6 clones) and an

Discussion

This study was carried out to gain insight in the molecular environment of DC-STAMP, a protein involved in myeloid differentiation and giant cell formation. By means of yeast-2-hybrid analysis and co-immunoprecipitation assays, we identified OS9 as a bona fide DC-STAMP-interacting protein (Fig. 1, Fig. 2). Furthermore, endogenous OS9 colocalizes with DC-STAMP in the ER of immature DC and in OS9 transfected HEK293 cells (Fig. 3). Intriguingly, upon TLR stimulation DC-STAMP, but not OS9

Acknowledgements

We gratefully acknowledge Dr. Jack Fransen, Department of Cell Biology, NCMLS, Radboud University Nijmegen Medical Centre, for providing the antibody against ERGIC53, and Angelique Lemckert and Menzo Havenga, Crucell NV, Leiden, The Netherlands, for providing the adenoviral DC-STAMP-GFP construct. Furthermore, we would like to thank Dr. Richard Janssen, BioFocus DPI BV, Leiden, The Netherlands, for helpful discussion. This work was financially supported by grant 912-02-34 and VICI grant

References (32)

J.H. Baek et al.
OS-9 interacts with hypoxia-inducible factor 1alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1alpha
Mol. Cell
(2005)
J.M. Beekman et al.
Modulation of FcgammaRI (CD64) ligand binding by blocking peptides of periplakin
J. Biol. Chem.
(2004)
E. Friedmann et al.
YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins
J. Biol. Chem.
(2002)
R.J. Klebe et al.
A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast
Gene
(1983)
L. Litovchick et al.
The carboxyl-terminal tail of kinase splitting membranal proteinase/meprin beta is involved in its intracellular trafficking
J. Biol. Chem.
(1998)
L. Litovchick et al.
A selective interaction between OS-9 and the carboxyl-terminal tail of meprin beta
J. Biol. Chem.
(2002)
I. Mellman et al.
Dendritic cells: specialized and regulated antigen-processing machines
Cell
(2001)
S. Akira et al.
Toll-like receptor signalling
Nat. Rev. Immunol.
(2004)
Ausubel, F.M., 1987. Current Protocols in Molecular Biology. Greene Publishing Associates; John Wiley, Order...
J. Banchereau et al.
Dendritic cells and the control of immunity
Nature
(1998)

J.M. Beekman et al.

Direct interaction between FcgammaRI (CD64) and periplakin controls receptor endocytosis and ligand binding capacity

Proc. Natl. Acad. Sci. U.S.A.

(2004)

R. Bernasconi et al.

A dual task for the Xbp1-responsive OS-9 variants in the mammalian ER: inhibiting secretion of misfolded protein conformers and enhancing their disposal

J. Biol. Chem.

(2008)

J.C. Christianson et al.

OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Nat. Cell Biol.

(2008)

I.J. de Vries et al.

Phenotypical and functional characterization of clinical grade dendritic cells

J. Immunother.

(2002)

D. Eleveld-Trancikova et al.

The dendritic cell-derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum

J. Leukocyte Biol.

(2005)

D. Eleveld-Trancikova et al.

The DC-derived protein DC-STAMP influences differentiation of myeloid cells

Leukemia

(2008)

Cited by (23)

Transcriptomic analyses reveal new genes and networks response to H5N1 influenza viruses in duck (Anas platyrhynchos)
2019, Journal of Integrative Agriculture
H5N1 influenza represents one of the great challenges to public health. Some H5N1 viruses (i.e., A/goose/Hubei/65/05, GS/65) are weakly pathogenic, while the others (i.e., A/duck/Hubei/49/05, DK/49) are highly pathogenic to their natural hosts. Here, we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus. We demonstrated that, compared to the GS/65 virus, the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways (i.e., RIG-I and MDA5) in ducks. We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes, while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus. We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis (WGCNA). Moreover, we first demonstrated that the duck BCL2L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently. These analyses, in combination with our comprehensive transcriptomic data, provided global view of the molecular architecture for the interaction between host and H5N1 viruses.
Targeted sequencing of DCSTAMP in familial Paget's disease of bone
2019, Bone Reports
Citation Excerpt :
TRACP+ cells with 4 or more nuclei were considered osteoclasts. The pEGFP N3 DC-STAMP wild type [WT] plasmid was generously provided by Professor Gosse Jansen (Jansen et al., 2009), with the DC-STAMP gene cloned into the vector prior to the N terminus of the EGFP sequence. The c.1189C > T p.L397F mutation was introduced into the wild-type sequence by site-directed mutagenesis (QuikChange kit; Stratagene) according to the manufacturer's protocol using primers designed in house as follows: Forward; 5’-TGGGACTGTTGTCCTCTATCTTTATGCAACTTAAAATCCTG, and reverse; 5’-CAGGATTTTAAGTTGCATAAAGATAGAGGACAAGAGTCCCA.
Paget's disease of bone (PDB) has a strong genetic component. Variants in SQSTM1 are found in up to 40% of patients with a family history of the disease, where a pattern of autosomal dominance with incomplete penetrance is apparent. By contrast, SQSTM1 variants are only found in up to 10% of patients with sporadic disease. It has been hypothesised that the remaining genetic susceptibility to PDB, particularly in familial cases, could be explained by rare genetic variants in loci previously identified by Genome Wide Association Studies. It is likely that polygenic factors are involved in many individuals. In this study we utilised whole exome sequencing to investigate predisposing genetic factors in an unsolved PDB kindred and identified a c.1189C > T p.L397F variant in DC-STAMP, also known as TM7SF4, that co-segregated with disease. DCSTAMP was identified as a gene of interest in PDB following Genome Wide Association Studies and has been previously shown to play critical roles in osteoclast fusion. The variant we identified has also been reported in association with PDB in a French-Canadian cohort however the significance of this variant was inconclusive. Targeted screening of DCSTAMP in our familial cohort of PDB patients revealed an additional 8 variants; however we did not find a significant association between any of these, including p.L397F, with PDB. Osteoclastogenesis assays from the affected proband and his unaffected brother demonstrated an increase in osteoclast number and nucleation, consistent with the pagetic phenotype. In converse to other established Paget's associated genetic variations such as SQSTM1, TNFRSF11A and OPTN, expression of the mutant DC-STAMP protein attenuated the activation of transcription factors NFκB and AP-1 when exogenously expressed. We found that the p.L397F variant did not influence the subcellular localization of the protein. Based on these findings we conclude that genetic variation in DCSTAMP is not a significant predisposing factor in our specific cohort of PDB patients and the p.L397F variant is unlikely to be a contributing factor in PDB pathogenesis.
OS9 protein interacts with Na-K-2Cl Co-transporter (NKCC2) and targets its immature form for the endoplasmic reticulum-associated degradation pathway
2016, Journal of Biological Chemistry
Citation Excerpt :
The function of OS9 remained mysterious until human OS9 and its yeast homolog Yos9p were implicated in ER-to-Golgi transport (35). Likewise, mammalian OS9 interacts with the C-terminal tails of meprin β (32) and DC-STAMP (33) to facilitate their transport from the ER. Besides its role in ER-to-Golgi transport, OS9 has been reported to interact with hypoxia-inducible factor 1-α in the cytosol to regulate its proteasome-mediated degradation in an O2-dependent manner (46).
Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.
MiR-7b directly targets DC-STAMP causing suppression of NFATc1 and c-Fos signaling during osteoclast fusion and differentiation
2014, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
Citation Excerpt :
It has been discovered that DC-STAMP is also expressed on OCPs and macrophages and plays a predominant role in cell-cell fusion for the formation of both mature osteoclasts and foreign body giant cells [29,30]. Since then, the mechanism and potential ligands of DC-STAMP have been studied and discussed intensively, but many questions remain unclear [31–37]. MicroRNAs (miRNAs) are non-coding small RNAs composed of approximately 22 nucleotides that bind to their target sites, which are mainly found in the 3′-untranslated regions (UTRs) of genes, and thereby repress translation by degrading mRNA or suppressing expression.
DC-STAMP is a key regulating molecule of osteoclastogenesis and osteoclast precursor (OCP) fusion. Emerging lines of evidence showed that microRNAs play crucial roles in bone metabolism and osteoclast differentiation, but no microRNA has yet been reported to be directly related to OCPs fusion. Through a microarray, we found that the expression of miR-7b in RAW264.7 cells was significantly decreased after induction with M-CSF and RANKL. The overexpression of miR-7b in RAW264.7 cells attenuated the number of TRAP-positive cells number and the formation of multinucleated cells, whereas the inhibition of miR-7b enhanced osteoclastogenesis. Through a dual luciferase reporter assay, we confirmed that miR-7b directly targets DC-STAMP. Other fusogenic molecules, such as CD47, ATP6v0d2, and OC-STAMP, were detected to be down-regulated in accordance with the inhibition of DC-STAMP. Because DC-STAMP also participates in osteoclast differentiation through the ITAM-ITIM network, multiple osteoclast-specific genes in the ITAM-ITIM network were detected to identify how DC-STAMP is involved in this process. The results showed that molecules associated with the ITAM-ITIM network, such as NFATc1 and OSCAR, which are crucial in osteoclastogenesis, were consistently altered due to DC-STAMP inhibition. These findings suggest that miR-7b inhibits osteoclastogenesis and cell-cell fusion by directly targeting DC-STAMP. In addition, the inhibition of DC-STAMP and its downstream signals changed the expression of other fusogenic genes and key regulating genes, such as Nfatc1, c-fos, Akt, Irf8, Mapk1, and Traf6. In conclusion, our findings indicate that miR-7b may be a potential therapeutic target for the treatment of osteoclast-related bone disorders.
Identification of cellular proteins interacting with equine infectious anemia virus S2 protein
2010, Virus Research
The macrophage-tropic lentivirus, equine infectious anemia virus (EIAV), encodes the small auxiliary protein S2 from a short open reading frame that overlaps the amino terminus of env EIAV S2 is dispensable for virus replication in cultured cells but is required for disease production. S2 is approximately 7 kDa and has no overall amino acid sequence homology to other cellular or viral proteins. Therefore it is likely that S2 plays a role as an adaptor protein. To further investigate S2 function we performed a yeast-2-hybrid screen to identify cellular proteins that interact with EIAV S2. The screen identified two human cellular proteins, amplified in osteosarcoma (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) that interact with S2. The equine homologues of these proteins were cloned and their interactions with S2 confirmed using co-immunoprecipitation assays. We identified two OS-9 isoforms that interact with S2 and a third splice variant that does not, indicating a region of OS-9 apparently required for the S2 interaction. The roles of these cellular proteins during EIAV infection have not been determined.
DC-STAMP interacts with ER-resident transcription factor LUMAN which becomes activated during DC maturation
2010, Molecular Immunology
Dendritic cells (DCs) are the professional antigen-presenting cells (APC) which efficiently prime the immune response or induce tolerance. We recently identified Dendritic Cell Specific TrAnsMembrane Protein (DC-STAMP), a novel 470 amino acid protein preferentially expressed by dendritic cells. Previously we demonstrated that DC-STAMP re-localizes towards the Golgi upon DC maturation. To identify proteins that interact with DC-STAMP, a yeast-2-hybrid analysis was performed. Here, we report a physically interacting partner of DC-STAMP in the endoplasmic reticulum (ER), called LUMAN (also known as CREB3 or LZIP). LUMAN was previously described as an ER-resident transcription factor with unknown function. It is activated in a process called regulated intramembrane proteolysis (RIP), which involves translocation to the Golgi and subsequent proteolytic cleavage. The proteolytically activated form of the protein then translocates to the nucleus. Our data indicate that DC-STAMP plays an important role in the modulation of LUMAN activation. Moreover, we demonstrate that LUMAN is endogenously expressed by DC and becomes activated by RIP upon DC maturation induced by various different stimuli. These data define LUMAN/DC-STAMP as a novel regulatory circuit in DC.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

OS9 interacts with DC-STAMP and modulates its intracellular localization in response to TLR ligation

Abstract

Introduction

Section snippets

Plasmids, adenoviral vectors and cloning

OS9 interacts with the cytoplasmic tail of DC-STAMP

Discussion

Acknowledgements

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

Gene

J. Biol. Chem.

J. Biol. Chem.

Cell

Toll-like receptor signalling

Nat. Rev. Immunol.

Dendritic cells and the control of immunity

Nature

Direct interaction between FcgammaRI (CD64) and periplakin controls receptor endocytosis and ligand binding capacity

Proc. Natl. Acad. Sci. U.S.A.

A dual task for the Xbp1-responsive OS-9 variants in the mammalian ER: inhibiting secretion of misfolded protein conformers and enhancing their disposal

J. Biol. Chem.

OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Nat. Cell Biol.

Phenotypical and functional characterization of clinical grade dendritic cells

J. Immunother.

The dendritic cell-derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum

J. Leukocyte Biol.

The DC-derived protein DC-STAMP influences differentiation of myeloid cells

Leukemia