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Mechanism of attenuation of leptin signaling under chronic ligand stimulation.

Knobelspies H, Zeidler J, Hekerman P, Bamberg-Lemper S, Becker W - BMC Biochem. (2010)

Bottom Line: Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance).In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077.The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.

ABSTRACT

Background: Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.

Results: Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.

Conclusions: Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

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Attenuation of LEPRb signaling is not due to receptor internalization and depends on active gene expression. A, Binding of leptin-SEAP to RINm5F cells stably expressing LEPRb was determined after cells were treated with leptin (20 ng/nl) for 2 h or were not treated (Ctrl). Nonspecific binding was determined by incubating parallel cultures with leptin-SEAP in the presence of excess unlabeled leptin (2 μg/ml) and was subtracted. Bound phosphatase activity was determined using a chemoluminescence-based assay. Bars represent means and S.D. values of six independent experiments. Reduction of LEPRb surface expression after treatment with leptin was not significant (paired t-test). B, RINm5F cells stably expressing wild type LEPRb were treated with actinomycin D (5 μg/ml) for 30 min before stimulation with leptin (20 ng/ml). The time course of leptin-induced STAT3 phosphorylation was analyzed by Western blots of total cellular lysates with the indicated antibodies. This results are representative of two independent experiments.
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Figure 2: Attenuation of LEPRb signaling is not due to receptor internalization and depends on active gene expression. A, Binding of leptin-SEAP to RINm5F cells stably expressing LEPRb was determined after cells were treated with leptin (20 ng/nl) for 2 h or were not treated (Ctrl). Nonspecific binding was determined by incubating parallel cultures with leptin-SEAP in the presence of excess unlabeled leptin (2 μg/ml) and was subtracted. Bound phosphatase activity was determined using a chemoluminescence-based assay. Bars represent means and S.D. values of six independent experiments. Reduction of LEPRb surface expression after treatment with leptin was not significant (paired t-test). B, RINm5F cells stably expressing wild type LEPRb were treated with actinomycin D (5 μg/ml) for 30 min before stimulation with leptin (20 ng/ml). The time course of leptin-induced STAT3 phosphorylation was analyzed by Western blots of total cellular lysates with the indicated antibodies. This results are representative of two independent experiments.

Mentions: We determined the surface expression of LEPRb in untreated and leptin-treated RINm5F cells to examine whether leptin signaling was downregulated by receptor internalization and/or degradation. After 2 h of leptin treatment, binding assays showed only a weakly reduced leptin binding to the cell surface, which could not account for the reduced STAT3 phosphorylation (Figure 2A). Next we used the transcriptional inhibitor, actinomycin D, to reveal whether active gene expression was required for feedback inhibition. As shown in Figure 2B, treatment with actinomycin D prevented the decrease of leptin-induced STAT3 phosphorylation over time. A similar result was obtained by treatment with cycloheximide (10 μg/ml; data not shown). These observations are consistent with the hypothesis that LEPRb signaling in RINm5F cells is attenuated by the upregulation of SOCS family proteins. Therefore, we examined whether leptin induced the expression of SOCS1 and SOCS3 in RINm5F cells. As previously observed in other cell lines (CHO, PC12, and 32D, rat skeletal muscle cells [39-41,45]), leptin induced a rapid upregulation of SOCS3 mRNA levels within 1 h but did not affect the expression of SOCS1 (Figure 3A). However, SOCS1 mRNA was upregulated in response to IL-1β or the simultaneous application of leptin and IL-1β. We tested this combination of cytokines because we had previously shown that IL-1β enhanced the expression of several leptin-induced genes in RINm5F cells [44]. In contrast to a previous study in RINm5F cells [46], we observed no upregulation of SOCS3 mRNA by IL-1β. Consistent with previous reports that the transcription of the SOCS3 gene is regulated by STAT3, Tyr1138 was sufficient for the induction of SOCS3 expression in RINm5F cells (Figure 3B). Thus, the LEPRb constructs used throughout this study were fully capable of inducing the expression of SOCS3.


Mechanism of attenuation of leptin signaling under chronic ligand stimulation.

Knobelspies H, Zeidler J, Hekerman P, Bamberg-Lemper S, Becker W - BMC Biochem. (2010)

Attenuation of LEPRb signaling is not due to receptor internalization and depends on active gene expression. A, Binding of leptin-SEAP to RINm5F cells stably expressing LEPRb was determined after cells were treated with leptin (20 ng/nl) for 2 h or were not treated (Ctrl). Nonspecific binding was determined by incubating parallel cultures with leptin-SEAP in the presence of excess unlabeled leptin (2 μg/ml) and was subtracted. Bound phosphatase activity was determined using a chemoluminescence-based assay. Bars represent means and S.D. values of six independent experiments. Reduction of LEPRb surface expression after treatment with leptin was not significant (paired t-test). B, RINm5F cells stably expressing wild type LEPRb were treated with actinomycin D (5 μg/ml) for 30 min before stimulation with leptin (20 ng/ml). The time course of leptin-induced STAT3 phosphorylation was analyzed by Western blots of total cellular lysates with the indicated antibodies. This results are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821298&req=5

Figure 2: Attenuation of LEPRb signaling is not due to receptor internalization and depends on active gene expression. A, Binding of leptin-SEAP to RINm5F cells stably expressing LEPRb was determined after cells were treated with leptin (20 ng/nl) for 2 h or were not treated (Ctrl). Nonspecific binding was determined by incubating parallel cultures with leptin-SEAP in the presence of excess unlabeled leptin (2 μg/ml) and was subtracted. Bound phosphatase activity was determined using a chemoluminescence-based assay. Bars represent means and S.D. values of six independent experiments. Reduction of LEPRb surface expression after treatment with leptin was not significant (paired t-test). B, RINm5F cells stably expressing wild type LEPRb were treated with actinomycin D (5 μg/ml) for 30 min before stimulation with leptin (20 ng/ml). The time course of leptin-induced STAT3 phosphorylation was analyzed by Western blots of total cellular lysates with the indicated antibodies. This results are representative of two independent experiments.
Mentions: We determined the surface expression of LEPRb in untreated and leptin-treated RINm5F cells to examine whether leptin signaling was downregulated by receptor internalization and/or degradation. After 2 h of leptin treatment, binding assays showed only a weakly reduced leptin binding to the cell surface, which could not account for the reduced STAT3 phosphorylation (Figure 2A). Next we used the transcriptional inhibitor, actinomycin D, to reveal whether active gene expression was required for feedback inhibition. As shown in Figure 2B, treatment with actinomycin D prevented the decrease of leptin-induced STAT3 phosphorylation over time. A similar result was obtained by treatment with cycloheximide (10 μg/ml; data not shown). These observations are consistent with the hypothesis that LEPRb signaling in RINm5F cells is attenuated by the upregulation of SOCS family proteins. Therefore, we examined whether leptin induced the expression of SOCS1 and SOCS3 in RINm5F cells. As previously observed in other cell lines (CHO, PC12, and 32D, rat skeletal muscle cells [39-41,45]), leptin induced a rapid upregulation of SOCS3 mRNA levels within 1 h but did not affect the expression of SOCS1 (Figure 3A). However, SOCS1 mRNA was upregulated in response to IL-1β or the simultaneous application of leptin and IL-1β. We tested this combination of cytokines because we had previously shown that IL-1β enhanced the expression of several leptin-induced genes in RINm5F cells [44]. In contrast to a previous study in RINm5F cells [46], we observed no upregulation of SOCS3 mRNA by IL-1β. Consistent with previous reports that the transcription of the SOCS3 gene is regulated by STAT3, Tyr1138 was sufficient for the induction of SOCS3 expression in RINm5F cells (Figure 3B). Thus, the LEPRb constructs used throughout this study were fully capable of inducing the expression of SOCS3.

Bottom Line: Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance).In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077.The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.

ABSTRACT

Background: Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.

Results: Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.

Conclusions: Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.

Show MeSH
Related in: MedlinePlus