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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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Inhibition of leptin or Akt induces apoptosis. (A–D) TUNEL staining (red) after treatment with 5 μg/ml α-leptin antibody (B) or 5 μM Akt inhibitor XI (D) compared with the control (A and C, respectively). DAPI, blue. (E) Percentage of total cells that are TUNEL positive (controls on left, treatment on right): IgG versus α-leptin antibody (gray bars) and DMSO versus Akt inhibitor XI (black bars). (F) Representative Western blot of PCNA after treatment with either 5 μg/ml IgG or α-leptin antibody and either DMSO or 5 μM Akt inhibitor XI. β-Tubulin was used as a loading control. (G–I) Percentage of total cells that are TUNEL positive (G), total cells present in and on the gel (H), and percentage of total cells undergoing EMT (black bars) or migrating into the collagen gel (gray bars; I) after treatment with IgG, α-leptin antibody, or α-leptin antibody plus 40 mM z-VAD. Error bars represent SD. Bar, 40 μm. *, P < 0.05.
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fig7: Inhibition of leptin or Akt induces apoptosis. (A–D) TUNEL staining (red) after treatment with 5 μg/ml α-leptin antibody (B) or 5 μM Akt inhibitor XI (D) compared with the control (A and C, respectively). DAPI, blue. (E) Percentage of total cells that are TUNEL positive (controls on left, treatment on right): IgG versus α-leptin antibody (gray bars) and DMSO versus Akt inhibitor XI (black bars). (F) Representative Western blot of PCNA after treatment with either 5 μg/ml IgG or α-leptin antibody and either DMSO or 5 μM Akt inhibitor XI. β-Tubulin was used as a loading control. (G–I) Percentage of total cells that are TUNEL positive (G), total cells present in and on the gel (H), and percentage of total cells undergoing EMT (black bars) or migrating into the collagen gel (gray bars; I) after treatment with IgG, α-leptin antibody, or α-leptin antibody plus 40 mM z-VAD. Error bars represent SD. Bar, 40 μm. *, P < 0.05.

Mentions: The cell survival properties of leptin have been described in multiple cell types in the vascular, bone, and nervous systems (Artwohl et al., 2002; Najib and Sanchez-Margalet, 2002; Gordeladze and Reseland, 2003; Guo et al., 2007). Therefore, an alternative explanation for changes in total cell number may be leptin's effects on proliferation and apoptosis. To evaluate this hypothesis, TUNEL staining was performed on cultures treated with leptin antibody (Fig. 7 B) or Akt inhibitor XI (Fig. 7 D). Inhibition of leptin resulted in 49.8 ± 4.3% of total cells displaying TUNEL reactivity compared with 17 ± 3.4% in the control group (n = 3; P < 0.0001; Fig. 7 E). Similarly, inhibition of Akt resulted in 34.4 ± 5.3% of total cells displaying TUNEL reactivity compared with 10 ± 2.8% in the control group (n = 3; P < 0.0009; Fig. 7 E). A 2.8- or 3.4-fold induction in cell death was detected after treatment with α-leptin antibody or Akt inhibitor XI, respectively, suggesting that leptin/Akt signaling plays a role in cell survival during cardiac EMT or that the failure of normal differentiation results in cell death.


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)

Inhibition of leptin or Akt induces apoptosis. (A–D) TUNEL staining (red) after treatment with 5 μg/ml α-leptin antibody (B) or 5 μM Akt inhibitor XI (D) compared with the control (A and C, respectively). DAPI, blue. (E) Percentage of total cells that are TUNEL positive (controls on left, treatment on right): IgG versus α-leptin antibody (gray bars) and DMSO versus Akt inhibitor XI (black bars). (F) Representative Western blot of PCNA after treatment with either 5 μg/ml IgG or α-leptin antibody and either DMSO or 5 μM Akt inhibitor XI. β-Tubulin was used as a loading control. (G–I) Percentage of total cells that are TUNEL positive (G), total cells present in and on the gel (H), and percentage of total cells undergoing EMT (black bars) or migrating into the collagen gel (gray bars; I) after treatment with IgG, α-leptin antibody, or α-leptin antibody plus 40 mM z-VAD. Error bars represent SD. Bar, 40 μm. *, P < 0.05.
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Related In: Results  -  Collection

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fig7: Inhibition of leptin or Akt induces apoptosis. (A–D) TUNEL staining (red) after treatment with 5 μg/ml α-leptin antibody (B) or 5 μM Akt inhibitor XI (D) compared with the control (A and C, respectively). DAPI, blue. (E) Percentage of total cells that are TUNEL positive (controls on left, treatment on right): IgG versus α-leptin antibody (gray bars) and DMSO versus Akt inhibitor XI (black bars). (F) Representative Western blot of PCNA after treatment with either 5 μg/ml IgG or α-leptin antibody and either DMSO or 5 μM Akt inhibitor XI. β-Tubulin was used as a loading control. (G–I) Percentage of total cells that are TUNEL positive (G), total cells present in and on the gel (H), and percentage of total cells undergoing EMT (black bars) or migrating into the collagen gel (gray bars; I) after treatment with IgG, α-leptin antibody, or α-leptin antibody plus 40 mM z-VAD. Error bars represent SD. Bar, 40 μm. *, P < 0.05.
Mentions: The cell survival properties of leptin have been described in multiple cell types in the vascular, bone, and nervous systems (Artwohl et al., 2002; Najib and Sanchez-Margalet, 2002; Gordeladze and Reseland, 2003; Guo et al., 2007). Therefore, an alternative explanation for changes in total cell number may be leptin's effects on proliferation and apoptosis. To evaluate this hypothesis, TUNEL staining was performed on cultures treated with leptin antibody (Fig. 7 B) or Akt inhibitor XI (Fig. 7 D). Inhibition of leptin resulted in 49.8 ± 4.3% of total cells displaying TUNEL reactivity compared with 17 ± 3.4% in the control group (n = 3; P < 0.0001; Fig. 7 E). Similarly, inhibition of Akt resulted in 34.4 ± 5.3% of total cells displaying TUNEL reactivity compared with 10 ± 2.8% in the control group (n = 3; P < 0.0009; Fig. 7 E). A 2.8- or 3.4-fold induction in cell death was detected after treatment with α-leptin antibody or Akt inhibitor XI, respectively, suggesting that leptin/Akt signaling plays a role in cell survival during cardiac EMT or that the failure of normal differentiation results in cell death.

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