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Leptin affects endocardial cushion formation by modulating EMT and migration via Akt signaling cascades.

Nath AK, Brown RM, Michaud M, Sierra-Honigmann MR, Snyder M, Madri JA - J. Cell Biol. (2008)

Bottom Line: Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels.Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects.Our data suggest that an Akt signaling pathway underlies the observed phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT 06510, USA.

ABSTRACT
Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin-independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of alphavbeta3 integrin receptor and resulted in decreased alphavbeta3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT.

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Akt localization in the endocardial cushion and phosphorylation by exogenous leptin. (A and B) Akt immunofluorescence staining (red) was performed on embryonic hearts excised just before EMT (A) and during EMT (B). (C and D) Representative Western blots for pAkt (serine 473) and Akt in AVCs treated with 5 μg/ml α-leptin antibody (C) or recombinant leptin (50, 100, and 500 pM for 30 min; D). DAPI, blue. Myo, myocardium; En, endocardium; Mes, mesenchyme; CJ, cardiac jelly.
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fig4: Akt localization in the endocardial cushion and phosphorylation by exogenous leptin. (A and B) Akt immunofluorescence staining (red) was performed on embryonic hearts excised just before EMT (A) and during EMT (B). (C and D) Representative Western blots for pAkt (serine 473) and Akt in AVCs treated with 5 μg/ml α-leptin antibody (C) or recombinant leptin (50, 100, and 500 pM for 30 min; D). DAPI, blue. Myo, myocardium; En, endocardium; Mes, mesenchyme; CJ, cardiac jelly.

Mentions: To determine whether the Akt pathway may be present in the embryonic heart, immunofluorescent localization of Akt (antibody detects Akt1, 2, and 3) was performed on embryonic hearts excised from timed pregnant matings at stages of endocardial cushion morphogenesis before EMT (E8.5) and during EMT (E9.5). The presence of Akt in the endocardial cells before EMT (Fig. 4 A) suggests recruitment of Akt signaling in the endocardial response to EMT inductive stimuli. The expression of Akt in mesenchymal cells (Fig. 4 B) signifies a potential role in maintenance of the mesenchymal phenotype after EMT or participating in invasion-associated cellular activities. The expression patterns of leptin receptor and Akt prompted the investigation of the Akt pathway as a downstream mediator of leptin action. Inhibition of leptin in the AVC explants resulted in decreased Akt phosphorylation at serine 473 after 2 h (Fig. 4 C). The intensity of the bands was measured using Quantity One software to determine relative phosphorylation levels. In the displayed blot, 89% of total Akt was phosphorylated in control hearts compared with 62% after leptin antibody treatment for 2 h (Fig. 4 C), resulting in an ∼30% decrease in pAkt. The potential role of Akt was further supported by the observation that dose-dependent induction of Akt phosphorylation at serine 473 occurs in AVCs treated with exogenous recombinant leptin (50, 100, and 500 pM) for 30 min (Fig. 4 D). In Fig. 4 D, initial relative phosphorylation levels in control hearts was 16%. After the addition of 50, 100, and 500 pM of recombinant leptin for 30 min, the percent total Akt that was phosphorylated increased to 26, 57, and 80%, respectively. At the highest dose of recombinant leptin, there was a fivefold increase in relative pAkt levels compared with the control. These doses did not induce the phosphorylation of STAT3 at serine 727 and tyrosine 705 (unpublished data).


Leptin affects endocardial cushion formation by modulating EMT and migration via Akt signaling cascades.

Nath AK, Brown RM, Michaud M, Sierra-Honigmann MR, Snyder M, Madri JA - J. Cell Biol. (2008)

Akt localization in the endocardial cushion and phosphorylation by exogenous leptin. (A and B) Akt immunofluorescence staining (red) was performed on embryonic hearts excised just before EMT (A) and during EMT (B). (C and D) Representative Western blots for pAkt (serine 473) and Akt in AVCs treated with 5 μg/ml α-leptin antibody (C) or recombinant leptin (50, 100, and 500 pM for 30 min; D). DAPI, blue. Myo, myocardium; En, endocardium; Mes, mesenchyme; CJ, cardiac jelly.
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Related In: Results  -  Collection

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fig4: Akt localization in the endocardial cushion and phosphorylation by exogenous leptin. (A and B) Akt immunofluorescence staining (red) was performed on embryonic hearts excised just before EMT (A) and during EMT (B). (C and D) Representative Western blots for pAkt (serine 473) and Akt in AVCs treated with 5 μg/ml α-leptin antibody (C) or recombinant leptin (50, 100, and 500 pM for 30 min; D). DAPI, blue. Myo, myocardium; En, endocardium; Mes, mesenchyme; CJ, cardiac jelly.
Mentions: To determine whether the Akt pathway may be present in the embryonic heart, immunofluorescent localization of Akt (antibody detects Akt1, 2, and 3) was performed on embryonic hearts excised from timed pregnant matings at stages of endocardial cushion morphogenesis before EMT (E8.5) and during EMT (E9.5). The presence of Akt in the endocardial cells before EMT (Fig. 4 A) suggests recruitment of Akt signaling in the endocardial response to EMT inductive stimuli. The expression of Akt in mesenchymal cells (Fig. 4 B) signifies a potential role in maintenance of the mesenchymal phenotype after EMT or participating in invasion-associated cellular activities. The expression patterns of leptin receptor and Akt prompted the investigation of the Akt pathway as a downstream mediator of leptin action. Inhibition of leptin in the AVC explants resulted in decreased Akt phosphorylation at serine 473 after 2 h (Fig. 4 C). The intensity of the bands was measured using Quantity One software to determine relative phosphorylation levels. In the displayed blot, 89% of total Akt was phosphorylated in control hearts compared with 62% after leptin antibody treatment for 2 h (Fig. 4 C), resulting in an ∼30% decrease in pAkt. The potential role of Akt was further supported by the observation that dose-dependent induction of Akt phosphorylation at serine 473 occurs in AVCs treated with exogenous recombinant leptin (50, 100, and 500 pM) for 30 min (Fig. 4 D). In Fig. 4 D, initial relative phosphorylation levels in control hearts was 16%. After the addition of 50, 100, and 500 pM of recombinant leptin for 30 min, the percent total Akt that was phosphorylated increased to 26, 57, and 80%, respectively. At the highest dose of recombinant leptin, there was a fivefold increase in relative pAkt levels compared with the control. These doses did not induce the phosphorylation of STAT3 at serine 727 and tyrosine 705 (unpublished data).

Bottom Line: Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels.Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects.Our data suggest that an Akt signaling pathway underlies the observed phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT 06510, USA.

ABSTRACT
Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin-independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of alphavbeta3 integrin receptor and resulted in decreased alphavbeta3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT.

Show MeSH
Related in: MedlinePlus