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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 on potential intrinsic mitochondrial pathways of apoptosis.A. The macrophages were cultured under serum free conditions and were lysed and immunoblotted for BAX and Bcl-2. There was no change in band intensities for BAX and Bcl-2 relative to beta-actin in α-MSH treated and untreated (SFM) cells, nor was there any change in the BAX to Bcl-2 ratio. These results are representative of 3 independent experiments. B. The macrophages were cultured as before, and in the last hour of incubation they were treated with Mito-Flow dye. The cells were assayed by flow cytometry for expression of retained mitochondrial dye in treated and untreated (SFM) macrophages. In all the cultures less than half the cells retained high levels of the dye (*) indicating mitochondrial membrane integrity, with greater than half the cells with low levels of dye indicating loss in mitochondrial membrane integrity. Treating the cells with α-MSH had no effect on this pattern of dye retention. The flow cytometry histogram presented is representative of 3 experiments with the same result of no measurable change seen related to α-MSH treatment. There is no effect on potential intrinsic apoptotic activity by α-MSH.
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pone-0074488-g005: Effects of α-MSH on potential intrinsic mitochondrial pathways of apoptosis.A. The macrophages were cultured under serum free conditions and were lysed and immunoblotted for BAX and Bcl-2. There was no change in band intensities for BAX and Bcl-2 relative to beta-actin in α-MSH treated and untreated (SFM) cells, nor was there any change in the BAX to Bcl-2 ratio. These results are representative of 3 independent experiments. B. The macrophages were cultured as before, and in the last hour of incubation they were treated with Mito-Flow dye. The cells were assayed by flow cytometry for expression of retained mitochondrial dye in treated and untreated (SFM) macrophages. In all the cultures less than half the cells retained high levels of the dye (*) indicating mitochondrial membrane integrity, with greater than half the cells with low levels of dye indicating loss in mitochondrial membrane integrity. Treating the cells with α-MSH had no effect on this pattern of dye retention. The flow cytometry histogram presented is representative of 3 experiments with the same result of no measurable change seen related to α-MSH treatment. There is no effect on potential intrinsic apoptotic activity by α-MSH.

Mentions: There is a possibility that α-MSH protects the cells from the stress of serum-free induced death by reducing mitochondrial stress. The cells were treated as before and lysed. The lysates were assayed for BAX and Bcl-2 by immunoblotting. There was no observable or measurable change in the expression of BAX or Bcl-2 or the ratio of the two with α-MSH treatment (Figure 5A). Mitochondrial stress was further assayed by loading the cells with Mito-Flow stain to assay for the integrity of the mitochondrial membrane potential. The cells were incubated under serum-free conditions, treated with α-MSH, and assayed by flow cytometry. There was no change in dye retention by the mitochondria in macrophages treated with α-MSH (Figure 5B). The results show that there was a loss in mitochondrial membrane potential in more than 50% of the macrophages under serum-free conditions treated or not treated with α-MSH. Therefore, α-MSH is not providing survival signals through changes in mitochondrial activity; moreover, it may allow for apoptotic signals to propagate from the stressed mitochondria.


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 on potential intrinsic mitochondrial pathways of apoptosis.A. The macrophages were cultured under serum free conditions and were lysed and immunoblotted for BAX and Bcl-2. There was no change in band intensities for BAX and Bcl-2 relative to beta-actin in α-MSH treated and untreated (SFM) cells, nor was there any change in the BAX to Bcl-2 ratio. These results are representative of 3 independent experiments. B. The macrophages were cultured as before, and in the last hour of incubation they were treated with Mito-Flow dye. The cells were assayed by flow cytometry for expression of retained mitochondrial dye in treated and untreated (SFM) macrophages. In all the cultures less than half the cells retained high levels of the dye (*) indicating mitochondrial membrane integrity, with greater than half the cells with low levels of dye indicating loss in mitochondrial membrane integrity. Treating the cells with α-MSH had no effect on this pattern of dye retention. The flow cytometry histogram presented is representative of 3 experiments with the same result of no measurable change seen related to α-MSH treatment. There is no effect on potential intrinsic apoptotic activity by α-MSH.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757010&req=5

pone-0074488-g005: Effects of α-MSH on potential intrinsic mitochondrial pathways of apoptosis.A. The macrophages were cultured under serum free conditions and were lysed and immunoblotted for BAX and Bcl-2. There was no change in band intensities for BAX and Bcl-2 relative to beta-actin in α-MSH treated and untreated (SFM) cells, nor was there any change in the BAX to Bcl-2 ratio. These results are representative of 3 independent experiments. B. The macrophages were cultured as before, and in the last hour of incubation they were treated with Mito-Flow dye. The cells were assayed by flow cytometry for expression of retained mitochondrial dye in treated and untreated (SFM) macrophages. In all the cultures less than half the cells retained high levels of the dye (*) indicating mitochondrial membrane integrity, with greater than half the cells with low levels of dye indicating loss in mitochondrial membrane integrity. Treating the cells with α-MSH had no effect on this pattern of dye retention. The flow cytometry histogram presented is representative of 3 experiments with the same result of no measurable change seen related to α-MSH treatment. There is no effect on potential intrinsic apoptotic activity by α-MSH.
Mentions: There is a possibility that α-MSH protects the cells from the stress of serum-free induced death by reducing mitochondrial stress. The cells were treated as before and lysed. The lysates were assayed for BAX and Bcl-2 by immunoblotting. There was no observable or measurable change in the expression of BAX or Bcl-2 or the ratio of the two with α-MSH treatment (Figure 5A). Mitochondrial stress was further assayed by loading the cells with Mito-Flow stain to assay for the integrity of the mitochondrial membrane potential. The cells were incubated under serum-free conditions, treated with α-MSH, and assayed by flow cytometry. There was no change in dye retention by the mitochondria in macrophages treated with α-MSH (Figure 5B). The results show that there was a loss in mitochondrial membrane potential in more than 50% of the macrophages under serum-free conditions treated or not treated with α-MSH. Therefore, α-MSH is not providing survival signals through changes in mitochondrial activity; moreover, it may allow for apoptotic signals to propagate from the stressed mitochondria.

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