Regulation of the Immune Response by Leptin
4. MECHANISMS OF LEPTIN ACTION IN IMMUNE CELLS
Leptin receptor (Ob-R) belongs to the family of class I cytokine receptors, which include receptors for IL-2, IL-3, IL-4, IL-6, IL-7, LIF, G-CSF, growth hormone-releasing hormone, prolactin, and erythropoietin (12). As mentioned in the previous section, Ob-R expression is present in hematopoietic cells as well as the cells that participate in the innate and adaptive immune response. Leptin signaling has recently been reviewed (47,48), and we have previously reviewed leptin signaling in mononuclear cells (10). Most members of the cytokine family of receptors stimulate tyrosine phosphorylation of signal transduc- ers and activators of transcription (STAT) proteins by activating JAK kinases, which are associated with the intracellular part of the transmembrane receptor (49,50).
Tyrosine phosphorylation of the activated leptin receptor has already been reported in other systems (51,52). In human blood mononuclear cells, we have also found that human leptin stimulates tyrosine phosphorylation of the long form of the leptin recep- tor (53), as assessed by immunoprecipitation with an antibody against the C-terminal of the protein and immunoblotting with antibodies against phosphotyrosine. This effect is dependent on the dose at 5 min incubation. Maximal phosphorylation can be observed with 10 nM leptin.
The leptin receptor lacks intrinsic tyrosine kinase activity, but requires the activation of receptor-associated kinases of the Janus family (JAKs) (54), which initiate down- stream signalling including members of the STAT family of transcription factors (13,51). After ligand binding, JAKs autophosphorylate and tyrosine phosphorylates var- ious STATs. Activated STATs by leptin stimulation in the hypothalamus dimerize and translocate to the nucleus, where specific gene responses are elicited (55,56). In this context, we have studied the JAK–STAT signaling pathway triggered by leptin stimula- tion in human peripheral blood mononuclear cells (10,57). To investigate the activation of JAK kinases by leptin receptor in human peripheral blood mononuclear cells, we studied the effect of leptin on tyrosine phosphorylation of immunprecipitated JAK pro- teins by immunoblot. We found that both JAK-2 and JAK-3 are transiently activated, with maximal response at 5 min after leptin stimulation. Moreover, both isoforms have been found physically associated with the leptin receptor, as assessed by coimmunopre- cipitation studies. This association turned out to be constitutive, and it occurs both in the absence and presence of leptin in peripheral blood mononuclear cells. Preassociation of
Fig. 2. Model of leptin signaling in immune cells.
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JAK proteins with cytokine receptors has been described for other members of the fam- ily (58), and for the leptin receptor itself with JAK-2 (51). The relative contribution of each JAK isoform in leptin receptor signaling in human peripheral blood mononuclear cells, however, remains to be assessed.
The possible activation of STAT-3 by human leptin in mononuclear cells has also been studied at different time-points in peripheral blood mononuclear cells (57) by antiphosphotyrosine immunoblotting of anti-STAT-3 immunoprecipitates. The effect of leptin promoting STAT-3 tyrosine phosphorylation is maximal at 10 min, and the effect is dose-dependent, reaching maximal effect at 10 nM leptin. Leptin stimulation of mononuclear cells not only activates and phosphorylates STAT-3 but also promotes the tyrosine phosphorylation of the RNA binding protein Sam68, which has been previ- ously found to be recruited upon TCR, and insulin receptor activation (59,60) associates with STAT-3 in the same signaling complex (57). Tyrosine phosphorylation of Sam68 by the Src family kinase p59fyn has been shown previously to regulate negatively its association with RNA (61). In mononuclear cells we have found that leptin not only promotes the tyrosine phosphorylation of Sam68 (57), but also inhibits the RNA bind- ing efficiency of this protein (53). This effect of leptin was also found to be dependent on the dose. Even though we do not know the role of Sam68 in leptin signaling and lep- tin action, the effect of leptin regulating the RNA binding capacity of Sam68 may be involved in the post-transcriptional modulation of RNA, regulating either the meta- bolism, the splicing, or the localization of RNA. In this way, Sam68 has been proposed to provide the means for a rapid pathway to regulate protein expression by modifying the mRNA stability and/or mRNA translation.
Leptin has been shown to promote the translocation of STAT-3 to the nucleus in the rat hypothalamus (55,56). Some STAT-3 can be detected in nuclear lysates in basal con- ditions, but a significant increase in the amount of STAT-3 is found in the nuclear extract from cells stimulated with human leptin (10 nM) for 15 min, suggesting the transloca- tion of STAT-3 to the nucleus upon leptin stimulation. Other STAT isoforms (STAT-1, STAT-5, STAT-6) have also been shown to be activated by the leptin receptor, although only in transfected systems (62,63). However, other STAT forms have not been assayed in lymphocytes and therefore we cannot rule out the possible implication of STATs other than STAT-3. Nevertheless, STAT-3 is the only STAT that has been shown to be activated by leptin in the hypothalamus (64).
In this context, the tyrosine phosphorylation of leptin receptor and the activation of JAK-2 and STAT-3 by leptin stimulation has been confirmed in a murine macrophage cell line (65).
Different pathways in addition to STATs are known to be involved in leptin receptor signaling in a similar way to other members of the cytokine family. Thus, leptin has been shown to activate mitogen-activated protein kinase (MAPK) (52,66,67) and phos- phatidylinositol 3-kinase (PI3K) (68,69). More precisely, leptin receptor signaling in mononuclear cells has been shown to activate MAPK and p70 S6K pathways (70). We have also found that leptin stimulates tyrosine/threonine phosphorylation of MAPK in blood mononuclear cells, as assessed by immunoblot (53). Both Erk-1 and Erk-2 were phosphorylated after 10 min incubation with leptin. This effect was dose-dependent and maximal response was obtained at 10 nM leptin. The activation of MAPK was transient and decreased after 15 to 30 min incubation (53). More important, we have found that
MAPK activation by leptin is necessary for the antiapoptotic effect in human mono- nuclear cells (25).
Evidence that leptin initiates a signaling cascade involving MAPK-dependent path- way has been also found in neutrophils (31), whereby leptin also inhibits apoptotic path- ways. Neutrophils seem to express only the short isoform of leptin receptor, Ob-Ra (32), which does not signal via the JAK–STAT pathway, but may be sufficient to stimulate the MAPK pathway.
We have also explored the PI3K pathway in peripheral blood mononuclear cells (PBMC) in response to human leptin. Thus, PI3K activity associated with tyrosine phosphorylated proteins is found to be increased more than threefold after 10 nM lep- tin stimulation (53). PI3K activation is regulated by the association of tyrosine phospho- rylated proteins with the SH2 domains of p85. Thus, in response to leptin, a band corresponding to the molecular mass of the insulin receptor substrate (IRS)-1 and sev- eral bands of 60 to 70 kDa (including Sam68) are phosphorylated and associated with p85 in a dose–response manner, similar to our previous data in response to insulin (53,60,71). Maximal response is observed at 10 nM leptin and 5 min incubation time.
The activation of PI3K in a macrophage cell line has also been demonstrated (65).
Leptin also initiates the activation of PI3K pathway in neutrophils, even though they express only the short isoform of leptin receptor (31). This signaling pathway is involved in transducing leptin-mediated antiapoptotic signals into neutrophils.
Finally, new mechanisms by which leptin can promote inflammatory responses have been recently provided, at least in alveolar macrophages—i.e., the upregulation of phospholipase A2 activity and phospholipase A2Lprotein levels (24).
5. LEPTIN AND PATHOPHYSIOLOGY OF THE IMMUNE SYSTEM