EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 3, 208-216, March 2009 B cell activation factor (BAFF) is a novel adipokine that links obesity and inflammation Yu-Hee Kim1, Bong-Hyuk Choi1, pression of TACI was distinct from that of BAFF-R and Hyae-Gyeong Cheon2 and Myoung-Sool Do1,3 BCMA under TNF-α and BAFF ligand treatment. BAFF-R and BCMA expression levels were upregu- 1School of Life and Food Sciences lated under pro-inflammatory conditions, but TACI Handong Global University was reduced. Conversely, BAFF-R and BCMA ex- Pohang 791-708, Korea pression levels were downregulated by rosiglitazone 2Center for Metabolic Syndrome Therapeutics treatment, but TACI was increased. Taken together, Drug Discovery Division our results suggest that BAFF may be a new adipokine, Korea Research Institute of Chemical Technology representing a link between obesity and inflammation. Daejeon 305-343, Korea 3Corresponding author: Tel, 82-54-260-1301; Keywords: adipokine; B-cell maturation antigen; obe- Fax, 82-54-260-1319; E-mail, [email protected] sity; rosiglitazone; TNFRSF13C protein, human; DOI 10.3858/emm.2009.41.3.024 TNFSF13B protein, human; transmembrane activator and CAML Interactor protein Accepted 18 November 2008 Abbreviations: BAFF, B cell activation factor; BAFF-R, BAFF Introduction receptor; BCMA, B cell maturation antigen; BCS, bovine calf serum; BLyS, B lymphocyte stimulator; CCL, CC chemokine ligand; COX, B cell activation factor (BAFF) was recently intro- cyclooxygenase; CRP, C-reactive protein; EAT, epididymal adipose duced as a novel member of the TNF ligand tissue; ECL, enhanced chemiluminescence; IBMX, methylisobutyl- superfamily and is also named BLyS, THANK, xanthine; MCP-1, monocyte chemotatic protein-1; PAI-1, plas- TALL-1, and TNFSF13B (Moore et al., 1999; minogen activator inhibitor-1; SAA, serum amyloid A; TACI, trans- Nardelli et al., 2001). BAFF is a type II transmem- membrane activator and CAML interactor; TALL-1, TNF- and ApoL- brane protein, containing 285 amino acids. The related leukocyte-expressed ligand 1; THANK, TNF homologue that soluble form of BAFF, cleaved by "furin-like" pro- activates apoptosis, NF-kB, and JNK; TNFSF13B, TNF superfamily tease, is a 152 amino acid protein, with a mass of 13B; VAT, visceral adipose tissue 17,038 daltons (Moore et al., 1999). BAFF is produced by myeloid lineage cells, malignant B cells, activated T cells, and bone marrow stromal Abstract cells (Nardelli et al., 2001; Novak et al., 2002; Scapini et al., 2003; Lavie et al., 2004). The B cell activation factor (BAFF) is a novel member of the recombinant soluble BAFF (aa 134-285) binds spe- TNF ligand superfamily, mainly produced by myeloid cifically to human primary B cells and B cell tumor cells. BAFF has been shown to participate in B-cell sur- lines, but not to T cells, monocytes, NK cells, or vival and B- and T-cell maturation. BAFF expression in granulocytes (Moore et al., 1999). adipocytes has been recently demonstrated. In the cur- BAFF has three receptors that belong to the TNF rent study, we verified that BAFF expression is in- receptor superfamily: B cell maturation antigen creased during adipocyte differentiation. BAFF ex- (BCMA), transmembrane activator and CAML pression was augmented by TNF-α treatment and was interactor (TACI), and BAFF receptor (BAFF-R) decreased by rosiglitazone treatment. BAFF secretion (Gross et al., 2000; Thompson et al., 2001). These in lean and in ob/ob mice sera were compared and receptors are primarily expressed in B cells, but TACI and BAFF-R are known to be expressed by T smaller amount of BAFF was secreted in ob/ob mice. cell subsets as well (Huard et al., 2001; Ng et al., mRNA and protein expression were different between 2004). Because of the receptor distribution, the epididymal and visceral adipose tissue. BAFF ex- studies on BAFF have been focused on B cells pression was also increased in ob/ob mouse adipose and T cells. It has been shown that BAFF is res- tissue. We sought to identify known BAFF receptors ponsible for B cell survival and maturation (Mackey (BAFF-R, BCMA, and TACI) in adipocytes, and de- et al., 1999; Craxton et al., 2003), and also func- termined that all three were present and upregulated tions as a T-cell co-stimulatory molecule (Huard et during adipocyte differentiation. However, the ex- al., 2001; Ng et al., 2004). Furthermore, overexpre- BAFF linking obesity and inflammation 209 ssion of BAFF results in autoimmune-like manifes- obesity and related diseases. tations (Mackey et al., 1999), and BAFF levels are Previously, we have investigated the expression elevated in human autoimmune diseases such as profiles of inflammatory genes in human SGBS systemic lupus erythematosus, rheumatoid arthritis, adipocytes using DNA microarrays, and found that and multiple myeloma (Zhang et al., 2001; Novak TNFSF13B (another term for BAFF) is also et al., 2004; Seyler et al., 2005). The expression expressed by human adipocytes. We reported that and functional effects of BAFF on other cell linea- TNFST13B expression is increased during adipo- ges have not been elucidated. cyte differentiation and TNF-α treatment (Do et al., Adipose tissue has long been known to provide 2006). Recently, Pelekanou and his coworkers energy storage for the body, but recent studies have reported the expression of BAFF in breast have resulted in redefinition of this tissue as an tumor lesions and also in adipocytes surrounding endocrine organ (Mohamed-Ali et al., 1998). Adi- the tumor lesions (Pelekanou et al., 2008). We pose tissue causes inflammation by secreting were therefore interested in further examination of cytokines (such as TNF-α and IL-6), chemokines BAFF expression in adipocytes. (CCL2; MCP-1), and acute-phase molecules (CRP, In adipocytes, secreted TNF acts on cells via an haptoglobin, SAA, PAI-1) (Fantuzzi et al., 2005; autocrine/paracrine pathway through TNFR1 acti- Kim et al., 2006). Moreover, the adipokines, speci- vation (Nguyen et al., 2005). Additionally, it has fic cytokines that are mainly produced by adipo- been reported that BAFF and BAFF receptors are cytes e.g. leptin, adiponectin, and resistin, are co-expressed by B cells, and that BAFF may signal increasingly being shown to have important roles through an autocrine pathway as a member of the as mediators linking the adipose tissue and infla- TNF ligand superfamily (Haiat et al., 2006). There- mmation (Fantuzzi et al., 2005; Kim et al., 2006; fore, verification of the existence of BAFF recep- Tilg et al., 2006). Obesity, now considered to be a tors on adipocytes may lead to a greater unders- low-grade chronic inflammation state, increases tanding of the function of BAFF in adipocytes. the risk of many metabolic diseases such as type 2 Here we provide the first report of the expression diabetes, atherosclerosis, certain cancers, and au- of BAFF and BAFF receptors in 3T3-L1 adipocytes. toimmune diseases (Wisse et al., 2004; Fantuzzi et We examined mRNA and protein expression levels al., 2005; Kim et al., 2006; Tilg et al., 2006). The of BAFF during adipocyte differentiation, and their dysregulation of adipokine secretion is often changes under pro-inflammatory and anti-inflam- observed in patients with obesity; therefore, adipo- matory conditions. We also examined the existence kines may provide a link between obesity and of BAFF receptors on 3T3-L1 adipocytes and metabolic disorders. In this regard, further research observed their changes in response to induction or into the identification and characterization of novel inhibition of inflammation. BAFF and BAFF receptor adipokines will improve our understanding of expression in adipose tissue of ob/ob transgenic Figure 1. BAFF expression during adipocyte differentiation. (A) The relative amount of BAFF gene expression was evaluated by real time RT-PCR on ev- ery other day of 3T3-L1 adipocyte differentiation (*P ＜ 0.05). (B) The relative amount of BAFF protein was evaluated by western blot analysis on every other day of 3T3-L1 adipocyte differentiation. Data are expressed relative to untreated control cells and represent means ± S.E. Experiments were con- ducted twice, each included triplication. 210 Exp. Mol. Med. Vol. 41(3), 208-216, 2009 mice was examined as well. We further investigated 3T3-L1 adipocytes on every other day during the the autocrine effects on 3T3-L1 adipocytes by progression of differentiation. We examined mRNA treatment with soluble BAFF protein and examined and protein expression levels of BAFF using real the changes of BAFF receptors expression. time RT-PCR and western blot analysis, respec- tively. BAFF mRNA expression levels were increa- sed as differentiation progressed, and were aug- Results mented 1.7-fold after six days of differentiation (Figure 1A). Similarly, protein expression levels BAFF expression is increased during adipocyte were increased in accordance with differentiation, differentiation and on day six of differentiation BAFF protein level In order to determine whether BAFF expression is was 1.5-fold higher than before the induction of affected by adipocyte differentiation, we collected differentiation (Figure 1B). Figure 2. Changes of BAFF expression by TNF-α and Rosiglitazone treatment. (A) The relative amount of BAFF gene expression was evaluated by real time RT-PCR at different time points after simulation with 10 ng/ml TNF-α (*P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001). (B) The relative amount of BAFF pro- tein was evaluated by western blot analysis at different time points after simulation with 10 ng/ml TNF-α. (C) The relative amount BAFF gene expression was evaluated by real time RT-PCR at different time points after simulation with 1.5 μM Rosiglitazone (*P ＜ 0.05, **P ＜ 0.01). (D) The relative amount of BAFF protein was evaluated by western blot analysis at different time points after simulation with 1.5 μM Rosiglitazone. Data are expressed relative to untreated control cells and represent means ± S.E.