Leptin Signaling Maintains B-Cell Homeostasis Via Induction of Bcl-2 and Cyclin D1

Leptin signaling maintains B-cell homeostasis via induction of Bcl-2 and Cyclin D1

Queenie Lai Kwan Lama,1, Shengjun Wangb, Otis King Hung Koa, Paul W. Kincadec, and Liwei Lua,1

aDepartment of Pathology and Centre of Infection and Immunology, University of Hong Kong, Hong Kong, China; bDepartment of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China; and cImmunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Edited by Tak Wah Mak, Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved June 22, 2010 (received for review April 2, 2010) Regulation of apoptosis and cell cycle progression plays an essential of lymphocytes with impaired humoral responses, but many ques- role in the maintenance of B-cell homeostasis, because a fine tions remain about the molecular mechanisms (13, 14). A variety of balance of survival and expansion is critical for preventing lympho- human cancer cells express the leptin receptor and show enhanced cytic disorders. Although remarkable progress in understanding proliferation in response to leptin stimulation (11). Common B-cell development has been achieved, much less is known concern- polymorphisms in the leptin gene or its receptor are linked to the ing niches that are critical to the maintenance of B-cell homeostasis. pathogenesis of various hematological malignancies including non- Leptin has recently been recognized to be important for modulating Hodgkin’s lymphoma (15). Serum leptin level was significantly the immune responses, but it has remained unclear how leptin sig- elevated in patients with multiple myeloma and chronic lympho- naling influences B-cell physiology. A variety of lymphocytic malig- cytic leukemia (16). It is therefore important to understand how nancies have been reported to be linked to leptin, and therefore it is leptin signaling is involved in such malignancies (17). In this study necessary to elucidate the mechanisms involved. Here we demon- we present direct evidence that leptin maintains B-cell homeostasis strate that leptin promotes B-cell homeostasis by inhibiting apopto- by protecting them from apoptosis and inducing cell-cycle entry sis and by inducing cell cycle entry through the activation of via the induction of Bcl-2 and cyclin D1. Leptin elevates Bcl-2 and expressions of B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1. We fur- cyclin D1 levels through at least two mechanisms, by activating ther show that leptin can induce Bcl-2 and cyclin D1 expression by their promoters and suppressing miRNAs that target the putative two pathways, including the direct activation of their promoters 3′untranslated regions (UTR) of Bcl-2 and cyclin D1 mRNAs. and suppression of microRNAs (miRNAs) that target their putative Amplification of these leptin-modulated miRNAs led to sup- ′ fi 3 untranslated regions. Ampli cation of these leptin-modulated pression of Bcl-2 and/or cyclin D1 expression and inhibition of fi miRNAs inhibited B lymphoma cell growth. These ndings provide B lymphoma cell growth. These results demonstrate critical roles insights into mechanisms for leptin regulation of the humoral im- for leptin in B cell survival as well as proliferation, and suggest mune system and suggest new therapeutic strategies for leptin re- new targets for cancer therapy. ceptor expressing malignancies. Results B cell survival | apoptosis | proliferation | microRNA Functional Leptin Receptors Are Expressed on B Lymphocytes. We began our study by confirming that the leptin receptor is expressed poptosis plays an essential role in the maintenance of B-cell on various B lymphocyte subsets (Fig. S1 A and B). Because STAT3 Ahomeostasis, a process that is regulated by a fine balance is a major regulator of B-cell development, we examined its activity between the continual generation of new B lymphocytes and their upon leptin treatment in B cells (18). We found that phosphoryla- elimination through multiple checkpoints in both bone marrow tion of STAT3 occurred when leptin was added to spleen B cells in (BM) and secondary lymphoid organs (1). The balance between culture, whereas a significantly reduced level of phosphorylated B cell survival and death is critical because excessive apoptosis STAT3 was found in db/db mouse B cells (Fig. S1 C and D). How- may lead to immunodeficiencies whereas insufficient apoptosis ever, these data do not exclude the possibility that leptin acts in part may result in autoimmunity and even lymphoid malignancies (2). B by an influence on components of lymphocyte niches. Therefore, we − − cells that are optimally activated escape apoptosis and transit transferred equal numbers of db/db or WT CD19+CD43 sIgM through different cell cycle checkpoints. B-cell CLL/lymphoma pre-B cells to irradiated CD45.1 C57B/6 mice along with B cell- 2 (Bcl-2) family proteins, which function to preserve or disturb depleted WT BM cells. At 6 wk after transfer, reduced frequencies − mitochondrial integrity, are required for the maintenance of im- of total splenic B220+ B cells, B220+IgM+IgD B cells, B220+ − mune homeostasis (2, 3). Targeted loss of the antiapoptotic IgM+IgD+ B cells, B220+IgM IgD+ B cells, B220+CD23+CD21+ − member gene Bcl-2 has led to massive apoptosis of lymphocytes, follicular (FO) B cells, B220+CD23 CD21+ MZ B cells, B220+ whereas overexpression of Bcl-2 has caused the accumulation of GL-7+ GC B cells as well as B220-tolowCD138+ plasma cells from B cells (4, 5). D-type cyclins are key players in the G1 checkpoint the CD45.2+ donors were observed in the spleens of db/db chimera control mechanism and are critical for cell proliferation (6). Both (Fig. 1A). As control, we detected very low frequency (3.6% ± 1.3%) Bcl-2 and D-type cyclins are prime targets for novel therapeutics of reconstituted CD45.2+ B cells in the recipients’ spleens at 6 wk because their overexpression is common in many hematological post transfer with only the B cell-depleted WT BM cells (Fig. S2A). − diseases and many types of cancers (7, 8). MicroRNAs (miRNAs) B220+AA4.1+ Tr B cells, B220+AA4.1+CD23 IgM+ Transitional are increasingly recognized for their prominent role in immune homeostasis, while the accumulating evidence for aberrant miRNA expression in B-cell–derived tumors underscores miRNAs as po- Author contributions: Q.L.K.L., P.W.K., and L.L. designed research; Q.L.K.L., S.W., and O.K. tential targets for cancer therapeutics (9). Therefore, elucidation H.K. performed research; Q.L.K.L.., S.W., P.W.K., and L.L. analyzed data; and Q.L.K.L.. and of the regulatory mechanisms of miRNAs in peripheral B cells L.L. wrote the paper. will contribute to a fuller understanding of B cell biology. The authors declare no conflict of interest. Leptin, originally discovered as an endocrine hormone, has 1To whom correspondence may be addressed. E-mail: [email protected] or qlam@ been shown to play a crucial role in modulating immune responses pathology.hku.hk. – fi ob/ob (10 12). Earlier studies on leptin de cient and leptin- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. receptor–deficient db/db mice showed markedly reduced numbers 1073/pnas.1004185107/-/DCSupplemental.

13812–13817 | PNAS | August 3, 2010 | vol. 107 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1004185107 Downloaded by guest on September 27, 2021 were found in db/db T1, T2, and T3 B cell subsets (Fig. S2F). These results suggest that in addition to attenuation of B-cell apoptosis, leptin induces cell-cycle entry. While in the presence of costimulation, leptin activates cell cycle progression in B cells.

Leptin Signaling Induces Bcl-2 and Cyclin D1 Expression in B Cells. We found markedly reduced Bcl-2 transcripts in all of the db/db Tr, FO, and MZ B cell subsets (Fig. 3A). Bcl-xL expression was de- creased only in the db/db MZ B cell subset, whereas the expression of Bax and Bim was increased in all db/db B cell subsets. Bad expression was only increased in db/db MZ B cells. Incubation of splenic WT B cells with leptin significantly induced Bcl-2 expression, but inhibited the expression of the proapoptotic Bcl-2 family proteins, Bax, Bim, and Bad (Fig. 3B). Next, we used real- time PCR to analyze the expression of a panel of cell cycle reg- ulators in the db/db Tr, FO and MZ subsets (Fig. 3C). Cyclin D1 is the first cyclin produced in cell cycle in response to extracellular signals and allows quiescent cells to enter the G1 phase. Consis- Fig. 1. Functional leptin receptors are expressed on B lymphocytes. (A) tent with a role of leptin in regulating genes relating to early G1, Phenotypic FACS analysis of FSC, B220, IgM, IgD, CD23, CD21, GL-7, and we detected marked reductions in cyclin D1 transcripts in db/db B CD138 expressions on splenic B cells from chimeras 6 wk after reconstitution cells. We also found significantly increased expression of p27 Kip1 with either WT or db/db BM pre-B cells as described in Materials and in all of the db/db B cell subsets. These results were confirmed by Methods. Numbers indicate percentages of gated populations within D CD45.2+ cells. (B) Total live cell numbers of various B-cell populations from incubating B cells with leptin (Fig. 3 ). Taken together, these the chimeras as described in E. Transitional type 1 (T1), transitional type 2 data point to a positive effect of leptin on cell cycle regulation in B (T2), transitional type 3 (T3), FO, MZ, GC, and plasma cells (PC) are as shown. cells. Because leptin alone did not induce cell cycle progression, Data in A, B, E, and F are representative of four independent experiments, we further analyzed the gene expression profiles of leptin-treated whereas data in C and D are representative of three independent experi- B cells in the absence or presence of costimulation (Fig. 3E). ments. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Consistently, cyclin E1, cyclin A2, and CDK2 were unchanged in B cells treated with leptin alone, but were markedly up-regulated 1 (T1), B220+AA4.1+CD23+ IgM+ T2 and B220+AA4.1+ on costimulation (Fig. 3E). With BCR ligation or LPS stimula- − CD23+IgM T3 B cells from the donors were also found to be re- tion, leptin could even up-regulate the expression of cyclin B1, an duced in the db/db chimera (Fig. S2B). Also, B cells from the db/db important activator of G2/ M progression. These data indicate − − donors with the B220+AA4.1 CD23 IgMHigh MZ phenotype were that leptin differentially regulates Bcl-2 family members and reduced in frequency (Fig. S2B). We also used an alternative promotes cell cycle progression in B lymphocytes that are simul- staining scheme to confirm the reduced GC B cells in db/db chi- taneously receiving activation signals. meras (Fig. S2C). Accordingly, the cell numbers of various B cell subsets from the donors were significantly reduced in db/db chi- Leptin Signaling Activates Bcl-2 and Cyclin D1 Promoters Mediating meras (Fig. 1B). Immunization of the chimeras with TNP-Ficoll Prosurvival Responses in B Cells. We next focused on determining resulted in significantly reduced serum levels of TNP-specific IgM, how leptin modulates Bcl-2 and cyclin D1 levels. STAT-3, Akt, κ IgA, IgG1 and IgG2a in db/db chimeras (Fig. S2D). We conclude and NF- B are known inducers of Bcl-2 and cyclin D1. As noted that leptin acts directly on a spectrum of B lymphocyte types, above, STAT3 was phosphorylated in B cells exposed to leptin in regulating their numbers under normal, steady-state conditions. culture. We also observed significantly enhanced phosphorylation of Akt and PDK-1 upon leptin treatment (Fig. 4A). In addition, Leptin Signaling Promotes B-Cell Survival and Is Important for leptin caused phosphorylation of IκB, the inhibitor of NF-κB, and Proliferation. We observed increased numbers of early apoptotic stimulated the nuclear translocation of p65 subunit in the ca- − Annexin V+7AAD cells in Tr, FO, MZ and GC subsets of db/db nonical pathway (Fig. 4 B and C). We also found that leptin ac- mice compared with WT controls (Fig. 2A). Similar observations tivated the noncanonical pathway by stimulating Rel B and p52 to were found in db/db T1, T2 and T3 B-cell subsets (Fig. S2E). We enter the nucleus, indicating its potent activation of the NF-κB then treated B cells with leptin in the absence or presence of anti- pathway (Fig. 4D). IgM F(ab′)2 fragments (anti-IgM), CD40L, or LPS for 24 h and Leptin has been shown to activate CD40 promoter directly (10). However, addition of leptin to CD40 promoter luciferase found that leptin protected B cells from apoptosis in all conditions IMMUNOLOGY (Fig. 2B). We next performed a Mitotracker Red uptake assay and construct-transfected 293T cells could not stimulate firefly lucif- confirmed that leptin exerted antiapoptotic function via the mi- erase activity, so we next generated a 293T cell line stably express- tochondrial pathway in the absence or presence of anti-IgM, ing the long functional isoform of the leptin receptor OB-Rb (293T- CD40L, or LPS (Fig. 2C). Leptin alone had limited influence but OB-Rb cells) (Figs. 4E and S3 G and H). We transfected 293T-OB- markedly enhanced the proliferation of B cells in the presence of Rb cells with a construct containing either STAT3 enhancers or costimulation as assessed by carboxyfluorescein succinimidyl ester an NK-κB element fused to the 5′ end of the firefly luciferase, (CFSE) assay (Fig. 2C). Consistently, we observed significant pSTAT3-TA-luc, or pTAL-NF-kB, respectively (Fig. 4F). Leptin increases in DNA replication as assessed by BrdU incorporation treatment significantly activated both the STAT3 enhancer and NK- in the presence of leptin and costimulators (Fig. 2C). Leptin κB element (Fig. 4F). We also validated a direct role of leptin in treatment led to an increase in viable cell numbers (Fig. 2D). We activating the promoters of Bcl-2 and cyclin D1 by transfecting detected an increase in B cell size, enhanced surface expression of 293T-OB-Rb cells with constructs containing the promoter of either CD23, CD86, CD40, and MHC II, as well as migratory capacity of Bcl-2 or cyclin D1 fused to the 5′ end of the firefly luciferase B cells when incubated with leptin either alone or in the presence (Fig. 4G). Leptin treatment enhanced the normalized firefly lucif- of the stimuli, consistent with an activated state of B cells for entry erase activity in both transfections (Fig. 4G). Finally, we sought to into cell cycle (Fig. 2 E and F). Finally, we found significantly analyze whether leptin promotes B cells survival via induction of reduced uptake of BrdU in Tr, FO, MZ, and GC subsets of db/db Bcl-2 and/or cyclin D1 by using nonreplicating lentiviruses that mice compared with WT controls (Fig. 2G). Similar observations express siRNAs for Bcl-2 (LV-siBcl-2) and cyclin D1 (LV-sicyclin

Lam et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13813 Downloaded by guest on September 27, 2021 Fig. 2. Leptin signaling promotes B cell survival. (A) Percentage of early − apoptosis (annexin V+7AAD ) cells analyzed by FACS in the B-cell populations, transitional (Tr), FO, MZ, and GC B cells of WT and db/db mice that had been incubated in complete culture medium for 24 h. (B) Percentage of early apoptosis of WT splenic B cells treated with or without leptin (10 ng/ mL) in the absence or presence of anti- IgM (10 μg/mL), CD40L (10 μg/mL), or LPS (10 μg/mL) for 24 h. Db/db splenic B cells treated with or without leptin were shown as control. (C) FACS analysis of Mitotracker Red ﬂuores- cence intensity, CFSE dilution and BrdU uptake of WT splenic B cells treated as in B. Numbers indicate percentage of cells. Reduction of CFSE by successive cell division (gray line) is indicated. Representative histograms from three sets of similar results are shown. (D) WT splenic B cells treated as in B and numbers of live cells − (7AAD ) were determined in triplicate cultures on days 0, 1, 2, and 3. (E) FACS analysis of FSC, CD23, CD86, CD40, and MHC II of WT B cells treated as in B for 1 d. Untreated (black line), leptin- treated (gray line), and isotypic control mAb (shaded line) are as shown. (F) WT splenic B cells were treated as in B, and percentage of migrated B cells in a transwell at 6, 12, or 24 h, as analyzed by FACS and calculated based on original input cell numbers. (G) Percentage of BrdU-positive (BrdU+) cells analyzed by FACS of Tr, FO, MZ, and GC B cells of WT and db/db mice that had been fed with BrdU for 2, 3, or 4 d. Data are mean ± SD. All data are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

D1), respectively (Figs. 4H and S3D). As expected, B cells completely abolished with knockdown of both Bcl-2 and cyclin infected with a lentivirus expressing the ORF of leptin (LV-lep- D1 (Fig. 4 K and L). Similar results were obtained with malignant tin) showed up-regulated expression of Bcl-2 and cyclin D1 (Fig. A20 cells (Fig. S3 A–F). 4I). We then infected LPS-stimulated splenic B cells of both WT and db/db mice with the lentiviruses and found that the knock- Leptin Signaling Promotes B Cell Survival by Inhibiting Expression of down of Bcl-2 or cyclin D1 in WT and db/db B cells led to a smaller miRNAs That Target the Putative 3′UTR of Bcl-2 and Cyclin D1. The difference in live cell numbers between them, indicating that each 3′UTR of mRNA can be important for its stability (19). To test factor partially mediates the prosurvival effects of leptin (Fig. 4J). whether leptin regulates Bcl-2 and/or cyclin D1 expression via However, when B cells were infected with both LV-siBcl-2 and their 3′UTRs, we performed a reporter assay by using a firefly LV-sicyclin D1, the difference between WT and db/db B cell luciferase chimeric plasmid containing the 3′UTR region of either numbers was abolished, suggesting that Bcl-2 and cyclin D1 to- Bcl-2 (pEZX-Bcl-2–3′UTR) or cyclin D1 (pEZX-Cyclin D1-3′ gether contribute significantly to leptin-induced prosurvival re- UTR) downstream of the firefly luciferase coding sequence and sponses. These findings were further confirmed in WT B cells with transfected that into 293T-OB-Rb cells (Fig. S4A). Treatment coinfection of LV-leptin or leptin treatment, and the effect was with leptin enhanced the normalized firefly luciferase activity,

13814 | www.pnas.org/cgi/doi/10.1073/pnas.1004185107 Lam et al. Downloaded by guest on September 27, 2021 mediates leptin-induced expression of Bcl-2 and cyclin D1 in B cells, we selected a panel of miRNAs that are predicted to target the 3′ UTR of Bcl-2 and cyclin D1 mRNAs (Fig. 5A). We examined expression patterns for miRNA candidates in splenic B cells and shortlisted those that were expressed at high or medium levels (cycle threshold ≤30) (Fig. S4B). We then examined the physiological effect of leptin on their expression levels by analyzing splenic B cells of WT mice versus db/db littermates (Fig. S4C), and confirmed the direct effect of leptin by incubating WT B cells with recombinant leptin in vitro (Fig. S4D). Together, five and 14 miRNA candidates that can potentially target Bcl-2 and cyclin D1 respectively were also regulated by leptin in B cells (Fig. S4E). Transfection of A20 cells with these miRNA inhibitors up- regulated d expression of Bcl-2 or cyclin D1 proteins (Fig. 5B). To directly show whether these miRNAs can target the 3′UTR of Bcl-2 or cyclin D1 mRNA, we transfected 293T cells with pEZX- Bcl-2–3′UTR or pEZX-cyclin D1-3′UTR along with a mimic for each of the miRNAs (Fig. 5C). Each transfection significantly inhibited the normalized firefly luciferase activity. We also found leptin overexpression in A20 cells inhibited expression of these miRNAs (Fig. S4F). Moreover, transfection of A20 cells with the miRNA mimics impaired cell survival, suggesting that leptin can promote the survival of B cells with a possible impact on proliferation via the miRNA dependent mechanisms (Fig. 5D). Discussion We have described two target molecules and two mechanisms through which leptin regulates B lymphocyte numbers. Experi- ments conducted with db/db leptin receptor defective mice and chimeras suggest that leptin promotes cell survival as well as cell cycle progression in the presence of costimulatory signals. Levels of the antiapoptotic protein Bcl-2 and the cell cycle regulator cyclin D1 are critical and function synergistically in these responses. Leptin controls expression of these proteins through at least two pathways: direct activation of their promoters and indirect stabilization of their 3′UTR via suppression of miRNAs. These findings further our understanding of homeostasis in the humoral immune system. The capability of leptin to induce cyclin D1 expression is cor- roborative with its function in triggering cell cycle entry of B cells. Leptin alone was unable to induce DNA replication but pro- foundly enhanced G1/S transition in the presence of costimulation with anti-IgM, CD40L or LPS. Bcl-2 protein is an important regulator of B-cell homeostasis by regulating an intrinsic apoptosis pathway. Our findings that leptin activates Akt, STAT3, and NF- KB pathways are consistent with the link of these signaling molecules to Bcl-2 and cyclin D1 expression. In addition, our data that Fig. 3. Leptin signaling induces Bcl-2 and cyclin D1 expression in B cells. (A) identify leptin as a B cell survival factor that protects B cells from Quantitative RT-PCR analysis of relative mRNA levels of Bcl-2, Bcl- xL, Bax, Bim, apoptosis and induces cell cycle entry similar to the function of and Bad in WT and db/db Tr, FO and MZ B-cell populations normalized to β-actin. (B) Western blot analysis of Bcl-2, Bcl- xL, Bax, Bim, and Bad from WT BAFF (20). B cell development depends critically on BAFF, mouse splenic B cells that were untreated on day 0 that have been cultured which initiates signaling and transcriptional programs including

for 3 h before lysis, or treated with leptin (50 ng/mL) or LPS (5 μg/mL) for 1 or activation of Akt and NF-KB pathways and induction of anti- IMMUNOLOGY 2d.(C) Quantitative RT-PCR analysis of relative mRNA levels of cyclin D1, apoptotic members of the Bcl-2 family as well as cyclin D1 and D2 cyclin D2, CDK4, CDK6, p27 Kip1, p21 Waf/Cip1, and p15 INK4B in WT and db/ expression (20, 21). Although there are similar properties be- db Tr, FO, and MZ B cell populations normalized to β-actin. (D) Western blot tween leptin and BAFF signaling, whereas db/db B cells express analysis of cyclin D1, cyclin D2, CDK4, CDK6, p27 Kip1, p21 Waf/Cip1, and p15 normal level of BAFF receptor, they are not as responsive to INK4B in WT mouse splenic B cells treated as in B. GAPDH was used as loading BAFF stimulation as WT control, suggesting the possibility of the control for all Western blot analyses. (E) Quantitative RT-PCR analysis of rel- two cytokines sharing both common as well as nonredundant ative mRNA levels of cyclin D1, cyclin D2, cyclin E1, cyclin A2, cyclin B1, CDK2, G H CDK4, and CDK6 in WT splenic total B cells treated with or without leptin (10 pathways (Fig. S2 and ). Notably, NF-KB, STAT3, and Akt ng/mL) in the absence or presence of anti-IgM (10 μg/mL), CD40L (10 μg/mL), signaling pathways are frequently activated in cancer cells in- or LPS (10 μg/mL) for 6 h. Data are mean ± SD. All data are representative of cluding B lymphomas, suggesting that leptin signaling is likely to three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. play a role in B-cell neoplasias (22, 23). In addition, the high similarity of leptin’s structural and signaling capabilities to those of IL-6 warrants further studies to determine its function in the suggesting that leptin regulates expression of Bcl-2 and cyclin D1 regulation of B cell differentiation into plasma cells (24). through its effect on their 3′UTRs. Notably, both Bcl-2 and cyclin In recent years, accumulating evidence of aberrant miRNA ex- D1 possess a long 3′-UTR (∼5.1 kb and ∼2.7 kb, respectively), pression in B-cell–derived tumors underscores the importance of which are indicative of an expressional control mechanism via miRNA as potential targets for cancer therapeutics (9). Recent these regions (Fig. 5A). To analyze whether the miRNA pathway studies have identiﬁed signiﬁcant correlation between the expres-

Lam et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13815 Downloaded by guest on September 27, 2021 Fig. 4. Leptin signaling activates Bcl-2 and cyclin D1 promoters mediating prosurvival responses in B cells. (A) Western blot analysis of PDK-1 P(S241), Akt P (S473) or Akt of splenic B cells treated with leptin (50 ng/mL), LPS (5 μg/mL) or both for 0, 30, 60, or 120 min. (B) Western blot analysis of I-κB P(S32/36)and I-κB of splenic B cells treated as in A. GAPDH was used as loading control for A and B.(C and D) Western blot analysis of (C) p65 and (D) Rel B, p100, p52 in cytoplasmic and nuclear extracts of splenic B cells treated with leptin (50 ng/mL), LPS (5 μg/mL) or both for 0, 2, or 4 h. (E) Western blot analysis of leptin receptor of 293T cells and 293T cells stably transfected with pCIneo-OB-Rb construct (293T-OB-Rb). (F) Relative luciferase activity (RLA) of transfection of 293T- OB-Rb cells with constructs of STAT3 or NF-κB binding elements inserted upstream of firefly luciferase, or empty control construct, untreated or untreated with leptin. (G) RLA of transfection of 293T-OB-Rb cells with constructs comprising promoter sequence of Bcl-2 or cyclin D1 inserted upstream of firefly luciferase, or empty control construct, untreated or untreated with leptin. (H) Western blot analysis of Bcl-2 and cyclin D1 expressions in A20 cells infected with lentivirus expressing si-luciferase (LV-siCTL), 2 siRNAs for Bcl-2 (LV-siBcl-2), or 2 siRNAs for cyclin D1 (LV-sicyclin D1) at a final MOI of 15. (I) Western blot analysis of Bcl-2, cyclin D1 and leptin expressions in LPS-stimulated splenic B cells infected with control lentivirus (LV-CTL) or lentivirus expressing leptin (LV- Leptin) at a final MOI of 15. β-Actin was used as loading control for C–E, H, and I.(J) LPS-stimulated B cells from WT or db/db mice were infected with LV-siCTL, − LV-siBcl-2, or LV-sicyclin D1, or both LV-siBcl-2 and LV-sicyclin D1 at a final MOI of 15. Numbers of live GFP+ cells (7AAD GFP+) were determined in triplicate cultures at 2 d posttransduction. (K and L) WT splenic B cells were infected as in J with (K) coinfection of LV-CTL or LV-Leptin, or (L) absence (CTL) or presence of 20 ng/mL leptin. Numbers of 7AAD−GFP+ cells were determined as in J. Data are mean ± SD. All data are representative of three independent experiments. *P < 0.01; **P < 0.001.

sion of certain miRNAs and circulating leptin (25). In this study, we benefit our understanding of not only B-cell physiology but also show that leptin could suppress the expressions of a panel of the development of potential targets for cancer therapy. miRNAs to mediate induction of gene expressions in B cells. We have also found that all of the leptin-modulated miRNAs that Materials and Methods target the 3′UTR of Bcl-2 or cyclin D1 mRNAs are expressed at 3- Mice and Cell Lines. Male C57BL/6 db/db, C57BL/6 wild-type (WT) control and to 160-fold higher in normal primary B cells than those in their C57BL/6 CD45.1 mice were obtained from the Jackson Laboratory. For gen- × 6 fi malignant counterparts (Table S1). Although both Bcl-2 and cyclin eration of chimeric mice, 5 10 BM pre-B cells puri ed from 4-wk-old db/db or WT control littermates were injected with 3 × 106 B cell-depleted BM cells D1 are frequently over-expressed in many B cell lymphoprolifer- into the tail veins of C57BL/6 CD45.1 mice that had been irradiated at 9 Gy ative disorders, our observations suggest that leptin might play 1 d before transfer. All mice were maintained in a specific pathogen-free a role in B-cell malignancies via suppression of miRNAs leading to animal facility at the University of Hong Kong under the institutional amplification of Bcl-2 and cyclin D1 during cancer development. guidelines for usage of experimental animals. All db/db and control mice Nevertheless, although attempts to analyze Bcl-2 status associated had free access to food and water and were used at age of 4–5 wk before with human B cell lymphoproliferative disorders have been suc- the onset of diabetes as revealed by normal blood glucose levels (112 ± 5 cessful, the initial attempts to establish cyclin D1 as a dominant B- mg/dL) (27). A20 and 293T cells were purchased from ATCC. cell oncogene were not, although it is speculated that the CDKs might play a role in the action of cyclin D1 (26). Cyclin D1 is nor- Flow Analysis and Cell Sorting. Immunostained cells were examined using fl mally expressed at a very low level by B cells in the absence of a FACSCalibur ow cytometer and analyzed by CellQuest software (Becton Dickinson) (10, 27). Cell sorting was performed with an EPICS-Altra flow growth factors, and our data show that knockdown of cyclin D1 fi cytometer (Beckman Coulter) and the purity of collected cells was routinely alone has not impeded B cell survival as signi cantly as that of Bcl-2. >96% (27). Total B cells were also isolated using negative selection with mag- It is important to understand how leptin signaling is involved netic microbeads according to manufacturer’s instructions (Miltenyi Biotec). in such malignancies (17). Further elucidation of the miRNA layer of the regulatory mechanisms for the function of leptin Statistical Analysis. Data are presented as mean ± SD. Means were compared signaling in normal B cells and their malignant counterparts will with the Student t test where appropriate for statistical analysis. Values of P

13816 | www.pnas.org/cgi/doi/10.1073/pnas.1004185107 Lam et al. Downloaded by guest on September 27, 2021 Fig. 5. Leptin signaling promotes B cell survival by inhibiting expression of miRNAs that target the putative 3′UTR of Bcl-2 and cyclin D1 in B cells. (A) Schematic diagram showing approximate locations of predicted highly conserved target sites for the miRNAs on the 3′ untranslated region (UTR) of Bcl-2 and cyclin D1 mRNAs. Bcl-2 mRNA 3′UTR (filled bar); Cyclin D1 mRNA 3′UTR (open bar); miRNA target sites (triangle). (B) Western blot analysis of Bcl-2 and cyclin D1 expressions in A20 cells on days 2 and 3 after transfection with 2’O-methylated inhibitor for each of the miRNAs. β-Actin was used as loading control. (C) RLA of transfection of 293T cells with constructs comprised of 3′UTR of Bcl-2 or cyclin D1 inserted downstream of firefly luciferase along with different miRNA mimics. miR-control (CTL). (D) A20 cells were transfected with mimic for each of the miRNAs as shown, and the numbers of live cells (7AAD−) were determined in triplicate cultures at 24 h. Data are mean ± SD. All data are representative of four independent experiments. *P < 0.01; **P < 0.001.

< 0.05 are considered statistically signiﬁcant. Unless otherwise speciﬁed, all ACKNOWLEDGMENTS. We thank Dr. Lin Zheng for excellent technical studies are representative of at least three independent experiments. assistance. This work was supported by Research Grants Council of Hong Additional details including primer sequences for Tables S2–S5 are pro- Kong Grants HKU 771809M (to L.L.) and HKU 200909159002 and HKU vided in SI Materials and Methods. 20087176006 (to Q.L.K.L.).

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