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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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Effect of SOCS3 knockdown on reporter gene induction by LEPRb point mutants. A, SOCS3 expression levels. - HEPG2 cells were transfected with the expression plasmids for the indicated LEPRb constructs, pSUPER-SOCS3 (300 ng/well) or pEF-SOCS3 (10 ng/well). Levels of endogenous SOCS3 in leptin-treated cells (16 h, 100 ng/ml) or overexpressed FLAG-SOCS3 were determined by immunoprecipitation and Western blot analysis with SOCS3-specific antibody. B and C, Knockdown of SOCS1 or SOCS3. The reporter gene assays were performed as in Figure 3 except that shRNA vectors specific for SOCS1 or SOCS3 were co-transfected (300 ng/well). Columns reflect luciferase activities relative to the activity in cells transfected with empty knockdown vector (means ± S.D., n = 3 independent experiments in B and n = 5 in C). The difference of the responses of FYY and FFY to the knockdown of SOCS3 was significant (Student's t-test, P < 0.05).
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Figure 5: Effect of SOCS3 knockdown on reporter gene induction by LEPRb point mutants. A, SOCS3 expression levels. - HEPG2 cells were transfected with the expression plasmids for the indicated LEPRb constructs, pSUPER-SOCS3 (300 ng/well) or pEF-SOCS3 (10 ng/well). Levels of endogenous SOCS3 in leptin-treated cells (16 h, 100 ng/ml) or overexpressed FLAG-SOCS3 were determined by immunoprecipitation and Western blot analysis with SOCS3-specific antibody. B and C, Knockdown of SOCS1 or SOCS3. The reporter gene assays were performed as in Figure 3 except that shRNA vectors specific for SOCS1 or SOCS3 were co-transfected (300 ng/well). Columns reflect luciferase activities relative to the activity in cells transfected with empty knockdown vector (means ± S.D., n = 3 independent experiments in B and n = 5 in C). The difference of the responses of FYY and FFY to the knockdown of SOCS3 was significant (Student's t-test, P < 0.05).

Mentions: We performed knockdown experiments to address whether endogenous levels of SOCS3 suffice to inhibit LEPRb signaling via binding to pTyr1077. Endogenous SOCS3 accumulated to higher levels after stimulation of LEPRb-FFY than LEPRb-FYY, confirming that Tyr1077 contributes to the negative regulation of the effects of leptin on gene expression. Expression of a SOCS3-directed shRNA reduced the expression of leptin-induced SOCS3 below the limits of detection (Figure 5A). Promoter activation by wild type LEPRb was enhanced 5-fold by knockdown of SOCS3 expression (Figure 5B), indicating that the negative feedback through SOCS3 plays a critical role in the modulation of the leptin response in HepG2 cells. Promoter activation by LEPRb-FYY was also significantly enhanced by knockdown of SOCS3, indicating that Tyr985 is not essential for the inhibitory effect of SOCS3. In contrast, the activity of LEPRb-FFY was not altered by the knockdown plasmid, suggesting that Tyr1077 must be responsible for the inhibitory effect of SOCS3 on LEPRb-FYY. Knockdown of SOCS1 expression increased LEPRb-induced promoter activity only by approximately 50% (Figure 5C) since SOCS1 is not induced by leptin (see above, Figure 3). Consistent with the above observation that the effect of SOCS1 was partially dependent on receptor tyrosine motifs, knockdown of SOCS1 did not further enhance the activity of LEPRb-FFY. However, the difference between the mutant receptor construct did not reach statistical significance.


Mechanism of attenuation of leptin signaling under chronic ligand stimulation.

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

Effect of SOCS3 knockdown on reporter gene induction by LEPRb point mutants. A, SOCS3 expression levels. - HEPG2 cells were transfected with the expression plasmids for the indicated LEPRb constructs, pSUPER-SOCS3 (300 ng/well) or pEF-SOCS3 (10 ng/well). Levels of endogenous SOCS3 in leptin-treated cells (16 h, 100 ng/ml) or overexpressed FLAG-SOCS3 were determined by immunoprecipitation and Western blot analysis with SOCS3-specific antibody. B and C, Knockdown of SOCS1 or SOCS3. The reporter gene assays were performed as in Figure 3 except that shRNA vectors specific for SOCS1 or SOCS3 were co-transfected (300 ng/well). Columns reflect luciferase activities relative to the activity in cells transfected with empty knockdown vector (means ± S.D., n = 3 independent experiments in B and n = 5 in C). The difference of the responses of FYY and FFY to the knockdown of SOCS3 was significant (Student's t-test, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Effect of SOCS3 knockdown on reporter gene induction by LEPRb point mutants. A, SOCS3 expression levels. - HEPG2 cells were transfected with the expression plasmids for the indicated LEPRb constructs, pSUPER-SOCS3 (300 ng/well) or pEF-SOCS3 (10 ng/well). Levels of endogenous SOCS3 in leptin-treated cells (16 h, 100 ng/ml) or overexpressed FLAG-SOCS3 were determined by immunoprecipitation and Western blot analysis with SOCS3-specific antibody. B and C, Knockdown of SOCS1 or SOCS3. The reporter gene assays were performed as in Figure 3 except that shRNA vectors specific for SOCS1 or SOCS3 were co-transfected (300 ng/well). Columns reflect luciferase activities relative to the activity in cells transfected with empty knockdown vector (means ± S.D., n = 3 independent experiments in B and n = 5 in C). The difference of the responses of FYY and FFY to the knockdown of SOCS3 was significant (Student's t-test, P < 0.05).
Mentions: We performed knockdown experiments to address whether endogenous levels of SOCS3 suffice to inhibit LEPRb signaling via binding to pTyr1077. Endogenous SOCS3 accumulated to higher levels after stimulation of LEPRb-FFY than LEPRb-FYY, confirming that Tyr1077 contributes to the negative regulation of the effects of leptin on gene expression. Expression of a SOCS3-directed shRNA reduced the expression of leptin-induced SOCS3 below the limits of detection (Figure 5A). Promoter activation by wild type LEPRb was enhanced 5-fold by knockdown of SOCS3 expression (Figure 5B), indicating that the negative feedback through SOCS3 plays a critical role in the modulation of the leptin response in HepG2 cells. Promoter activation by LEPRb-FYY was also significantly enhanced by knockdown of SOCS3, indicating that Tyr985 is not essential for the inhibitory effect of SOCS3. In contrast, the activity of LEPRb-FFY was not altered by the knockdown plasmid, suggesting that Tyr1077 must be responsible for the inhibitory effect of SOCS3 on LEPRb-FYY. Knockdown of SOCS1 expression increased LEPRb-induced promoter activity only by approximately 50% (Figure 5C) since SOCS1 is not induced by leptin (see above, Figure 3). Consistent with the above observation that the effect of SOCS1 was partially dependent on receptor tyrosine motifs, knockdown of SOCS1 did not further enhance the activity of LEPRb-FFY. However, the difference between the mutant receptor construct did not reach statistical significance.

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