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 ho- meostasis, 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 ex- pression, 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.