Lennart Zabeau

The ins and outs of leptin receptor activation

The adipocyte‐derived hormone leptin signals the status of body energy stores by activating its receptor in hypothalamic nuclei. In contrast to the initial expectations, leptin treatment of human obesity was largely unsuccessful. One explanation for this is the marked leptin resistance, which likely operates in part at the receptor level. The leptin receptor is a member of the class I cytokine receptor family, which uses the Janus kinase/signal transducer and activator of transcription pathway as a major signaling route. In this review, we focus on the molecular mechanisms underlying leptin receptor activation. Different modes of leptin‐induced clustering of the ectodomains and the subsequent signaling events will be discussed.

Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex.

The leptin·leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine·cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I–III). We demonstrated previously that leptin has similar binding sites I–III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038–41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the β-sheet formed by β-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin·LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A–B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 α-receptor, and forms the center of binding site I. The 2:4 hexameric leptin·LR complex offers a rational explanation for mutagenesis studies and residue conservation.

Mapping of the interface between leptin and the leptin receptor CRH2 domain

Despite the impact of the leptin system on body weight and other physiologic processes, little is known about the binding of leptin to its receptor. The extracellular domain of the leptin receptor consists of two cytokine receptor homology (CRH) domains separated by an immunoglobulin-like domain, and followed by two juxtamembrane fibronectin type III modules. The CRH2 domain functions as a high-affinity binding site for leptin, and we previously demonstrated interaction with helices A and C of leptin. In this work, we constructed a homology model for the leptin/CRH2 complex and performed a detailed mutation analysis of the CRH2/leptin interface. Using both cell-based and in vitro binding assays using the isolated CRH2 domain, we show the critical role of hydrophobic interactions between Leu 13 and Leu 86 of leptin and Leu 504 in CRH2 in leptin binding and signalling. This binding pattern closely resembles the interaction of other four-helix bundle long chain cytokines with the CRH domain of their cognate receptors.

A complex interaction pattern of CIS and SOCS2 with the leptin receptor

Hypothalamic leptin receptor signalling plays a central role in weight regulation by controlling fat storage and energy expenditure. In addition, leptin also has direct effects on peripheral cell types involved in regulation of diverse body functions including immune response, bone formation and reproduction. Previous studies have demonstrated the important role of SOCS3 (suppressor of cytokine signalling 3) in leptin physiology. Here, we show that CIS (cytokine-inducible SH2 protein) and SOCS2 can also interact with the leptin receptor. Using MAPPIT (mammalian protein-protein interaction trap), a cytokine receptor-based two-hybrid method operating in intact cells, we show specific binding of CIS with the conserved Y985 and Y1077 motifs in the cytosolic domain of the leptin receptor. SOCS2 only interacts with the Y1077 motif, but with higher binding affinity and can interfere with CIS and STAT5a prey recruitment at this site. Furthermore, although SOCS2 does not associate with Y985 of the leptin receptor, we find that SOCS2 can block interaction of CIS with this position. This unexpected interference can be explained by the direct binding of SOCS2 on the CIS SOCS box, whereby elongin B/C recruitment is crucial to suppress CIS activity.

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.

Leptin Modulates Innate and Adaptive Immune Cell Recruitment after Cigarette Smoke Exposure in Mice

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p < 0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p < 0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.

The role of leptin in the development of pulmonary neutrophilia in infection and Acute Lung Injury

Objectives:One of the hallmarks of severe pneumonia and associated acute lung injury is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and acute lung injury. Design:Controlled human and murine in vivo and ex vivo experimental studies. Setting:Research laboratory of a university hospital. Subjects:Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. Interventions:Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated acute lung injury were immunostained for leptin. Human bronchoalveolar lavage samples obtained after lipopolysaccharide-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with Escherichia coli- or Klebsiella pneumoniae-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E. coli model. Bronchoalveolar lavage neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. Measurements and Main Results:Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human bronchoalveolar lavage following lipopolysaccharide instillation. Bronchoalveolar lavage neutrophilia in uninjured and infected mice was increased and lung bacterial load decreased by airway leptin administration, whereas bronchoalveolar lavage neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by lipopolysaccharide, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated toward leptin in vitro, and this required intact signaling through the Janus Kinase 2/phosphatidylinositol-4,5-bisphosphate 3-kinase pathway. Conclusions:We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.

Leptin induces inflammation-related genes in RINm5F insulinoma cells

BackgroundLeptin acts not only on hypothalamic centers to control food intake but has additional functions in peripheral tissues, e.g. inhibition of insulin secretion from pancreatic islets. The leptin receptor (LEPRb) is a class I cytokine receptor that mediates activation of STAT transcription factors. In this study, we characterise the regulation of inflammation-related genes by leptin in insulinoma cells and compare the effect of transcriptional regulation by leptin with that of other cytokines.ResultsWe have used RINm5F insulinoma cells as a model system for a peripheral target cell of leptin. Six transcripts encoding inflammation-related proteins were found to be upregulated by activation of LEPRb, namely lipocalin-2, pancreatitis-associated protein, preprotachykinin-1, fibrinogen-β, tissue-type plasminogen activator (tPA) and manganese-dependent superoxide dismutase (MnSOD). Four of these transcripts (fibrinogen-β, lipocalin-2, tPA, MnSOD) were also induced by the proinflammatory cytokine interleukin-1β (IL-1β). Interferon-γ alone had no effect on the leptin-induced transcripts but enhanced the upregulation by IL-1β of lipocalin-2, tPA and MnSOD mRNA levels. Experiments with LEPRb point mutants revealed that the upregulation of the inflammation-related genes depended on the presence of tyrosine-1138 which mediates the activation of the transcription factors STAT1 and STAT3. Reporter gene assays showed that leptin induced the expression of preprotachykinin-1 and lipocalin-2 on the level of promoter regulation. Finally, leptin treatment increased caspase 3-like proteolytic activity in RINm5F cells.ConclusionThe present data show that leptin induces a cytokine-like transcriptional response in RINm5F cells, consistent with the proposed function of leptin as a modulator of immune and inflammatory responses.

Neutralizing monoclonal antibodies can potentiate IL-5 signaling.

IL‐5 is a major determinant in the survival, differentiation and effector‐functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ligand‐specific α‐chain and a β‐chain, shared with the receptors for IL‐3 and granulocyte‐macrophage colony‐stimulating factor. We have generated and mapped the epitopes of three monoclonal antibodies (mAb) directed against this cytokine: the strong neutralizing mAb 5A5 and 1E1, and the very weak neutralizing mAb H30. We found that H30 as well as 5A5 can increase proliferation above the level induced by human (h)IL‐5 alone, in a JAK‐2‐dependent manner, and at every sub‐optimal hIL‐5 concentration analyzed. This effect is dependent on mAb‐mediated cross‐linking of IL‐5R complexes, and is only observed on cell lines expressing a hybrid human/mouse IL‐5Rα‐chain. We discuss these findings in view of the stoichiometric and topological requirements for an activated IL‐5R. Since humanized anti‐IL‐5 mAb are currently in clinical testing, our findings imply that such mAb should be carefully evaluated for their potentiating effects.