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Alpha-melanocyte stimulating hormone (α-MSH) is a post-caspase suppressor of apoptosis in RAW 264.7 macrophages.

Taylor AW - PLoS ONE (2013)

Bottom Line: There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity.In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding.These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA. awtaylor@bu.edu

ABSTRACT
The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

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Effects of α-MSH treatment on Caspase 9 activity.The macrophages were cultured and treated as described in Figure 1 and the lysate was assayed for protein concentration, Caspase 9 activity, and immunoblotted for Caspase 9 protein. A. There was no significant difference seen between α-MSH treated and untreated (SFM) cells. Results presented are the mean ± standard deviation of 3 independent experiments. B. The lysates were immunoblotted with antibody that detected precursor and the activation fragments of Caspase 9. The band intensities were measured and made relative to beta-actin band intensity. C. The ratio of Caspase 9 activation fragments to precursor protein did not change between the untreated SFM cells and the α-MSH treated cells. These results are representative of 3 independent experiments showing that α-MSH has no effect on Caspase 9 activation.
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pone-0074488-g002: Effects of α-MSH treatment on Caspase 9 activity.The macrophages were cultured and treated as described in Figure 1 and the lysate was assayed for protein concentration, Caspase 9 activity, and immunoblotted for Caspase 9 protein. A. There was no significant difference seen between α-MSH treated and untreated (SFM) cells. Results presented are the mean ± standard deviation of 3 independent experiments. B. The lysates were immunoblotted with antibody that detected precursor and the activation fragments of Caspase 9. The band intensities were measured and made relative to beta-actin band intensity. C. The ratio of Caspase 9 activation fragments to precursor protein did not change between the untreated SFM cells and the α-MSH treated cells. These results are representative of 3 independent experiments showing that α-MSH has no effect on Caspase 9 activation.

Mentions: To see if α-MSH had any effect on caspase activities within the macrophages under serum-free conditions, the α-MSH treated cells were lysed and assayed for Caspase 9 and Caspase 8 activity. The activity of Caspase 9 was detectible in the macrophages cultured without serum, and α-MSH had no effect on Caspase 9 activity (Figure 2A). In addition, immunoblotting for the precursor protein and activation fragments of Caspase 9 from cell lysates were unchanged with α-MSH treatment (Figure 2B). This suggests that intrinsic signals of apoptosis are most likely not suppressed by α-MSH treatment. In contrast there was significant suppression of Caspase 8 activity (Figure 3A). Also, α-MSH treatment reduced the expression of Caspase 8 p18 activation-fragments in the macrophages (Figure 3B, C). In addition, the ratio of Caspase 8 activation p18 fragments to Caspase 8 precursor proteins was suppressed by α-MSH at the concentrations tested (Figure 3D) These results demonstrate a suppression of Caspase 8 activity in the α-MSH treated macrophages mostly at lower concentration of α-MSH. It is possible that the changes in Caspase 8 activity with the α-MSH concentration is more related to increased cell numbers in the 10 ng/ml α-MSH treated cell cultures.


Alpha-melanocyte stimulating hormone (α-MSH) is a post-caspase suppressor of apoptosis in RAW 264.7 macrophages.

Taylor AW - PLoS ONE (2013)

Effects of α-MSH treatment on Caspase 9 activity.The macrophages were cultured and treated as described in Figure 1 and the lysate was assayed for protein concentration, Caspase 9 activity, and immunoblotted for Caspase 9 protein. A. There was no significant difference seen between α-MSH treated and untreated (SFM) cells. Results presented are the mean ± standard deviation of 3 independent experiments. B. The lysates were immunoblotted with antibody that detected precursor and the activation fragments of Caspase 9. The band intensities were measured and made relative to beta-actin band intensity. C. The ratio of Caspase 9 activation fragments to precursor protein did not change between the untreated SFM cells and the α-MSH treated cells. These results are representative of 3 independent experiments showing that α-MSH has no effect on Caspase 9 activation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757010&req=5

pone-0074488-g002: Effects of α-MSH treatment on Caspase 9 activity.The macrophages were cultured and treated as described in Figure 1 and the lysate was assayed for protein concentration, Caspase 9 activity, and immunoblotted for Caspase 9 protein. A. There was no significant difference seen between α-MSH treated and untreated (SFM) cells. Results presented are the mean ± standard deviation of 3 independent experiments. B. The lysates were immunoblotted with antibody that detected precursor and the activation fragments of Caspase 9. The band intensities were measured and made relative to beta-actin band intensity. C. The ratio of Caspase 9 activation fragments to precursor protein did not change between the untreated SFM cells and the α-MSH treated cells. These results are representative of 3 independent experiments showing that α-MSH has no effect on Caspase 9 activation.
Mentions: To see if α-MSH had any effect on caspase activities within the macrophages under serum-free conditions, the α-MSH treated cells were lysed and assayed for Caspase 9 and Caspase 8 activity. The activity of Caspase 9 was detectible in the macrophages cultured without serum, and α-MSH had no effect on Caspase 9 activity (Figure 2A). In addition, immunoblotting for the precursor protein and activation fragments of Caspase 9 from cell lysates were unchanged with α-MSH treatment (Figure 2B). This suggests that intrinsic signals of apoptosis are most likely not suppressed by α-MSH treatment. In contrast there was significant suppression of Caspase 8 activity (Figure 3A). Also, α-MSH treatment reduced the expression of Caspase 8 p18 activation-fragments in the macrophages (Figure 3B, C). In addition, the ratio of Caspase 8 activation p18 fragments to Caspase 8 precursor proteins was suppressed by α-MSH at the concentrations tested (Figure 3D) These results demonstrate a suppression of Caspase 8 activity in the α-MSH treated macrophages mostly at lower concentration of α-MSH. It is possible that the changes in Caspase 8 activity with the α-MSH concentration is more related to increased cell numbers in the 10 ng/ml α-MSH treated cell cultures.

Bottom Line: There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity.In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding.These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA. awtaylor@bu.edu

ABSTRACT
The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

Show MeSH
Related in: MedlinePlus