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Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor

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ABSTRACT

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line.

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Inactivation of MAPKs does not prevent CSC-induced membrane damage. (a and b). Spermatocytes were exposed to DMSO (0.1%) or CSC (40 μg/ml) for 17 h. In the antagonists treatment groups, the cells were first pretreated with AHR or MAPK inhibitors or together for 1 h followed by exposure to CSC for 17 h (a). The percentage of cells segregated following co-staining with annexin V alexa fluor 488 and PI was determined by FACS analysis using FlowJo software (v9.7.5, FLOWJO, LLC.) as described under materials and methods. The representative histogram demonstrates the distribution of spermatocytes at 17 h. The percent difference in annexin V+ cells is shown within the quadrant. (b) Histograms represent the mean flow cytometric data from DMSO-, CSC- or antagonists pretreated samples at 17 h of more than three independent experiments, each assayed in triplicate±S.E.M. n=4.
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fig7: Inactivation of MAPKs does not prevent CSC-induced membrane damage. (a and b). Spermatocytes were exposed to DMSO (0.1%) or CSC (40 μg/ml) for 17 h. In the antagonists treatment groups, the cells were first pretreated with AHR or MAPK inhibitors or together for 1 h followed by exposure to CSC for 17 h (a). The percentage of cells segregated following co-staining with annexin V alexa fluor 488 and PI was determined by FACS analysis using FlowJo software (v9.7.5, FLOWJO, LLC.) as described under materials and methods. The representative histogram demonstrates the distribution of spermatocytes at 17 h. The percent difference in annexin V+ cells is shown within the quadrant. (b) Histograms represent the mean flow cytometric data from DMSO-, CSC- or antagonists pretreated samples at 17 h of more than three independent experiments, each assayed in triplicate±S.E.M. n=4.

Mentions: Given our previous study demonstrating crosstalk between p38-MAPK, ERK-44/42 and AHR,19 we asked whether pharmacological inhibition of MAPKs could prevent CSC-induced membrane damage. We first confirmed that neither the p38 inhibitor SB203580 nor the ERK inhibitor PD98059 could, on their own, increase the percentage of annexin-V-positive spermatocytes (Supplementary Figure 3). Pretreatment with MAPK inhibitors alone or together with AHR-inh could not prevent the externalization of phosphatidylserine in spermatocytes and thus the membrane damage (Figures 7a and b). These data ruled out the possibility of direct participation of MAPKs in membrane damage while implicating the specificity of AHR pathway in apoptosis.


Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor
Inactivation of MAPKs does not prevent CSC-induced membrane damage. (a and b). Spermatocytes were exposed to DMSO (0.1%) or CSC (40 μg/ml) for 17 h. In the antagonists treatment groups, the cells were first pretreated with AHR or MAPK inhibitors or together for 1 h followed by exposure to CSC for 17 h (a). The percentage of cells segregated following co-staining with annexin V alexa fluor 488 and PI was determined by FACS analysis using FlowJo software (v9.7.5, FLOWJO, LLC.) as described under materials and methods. The representative histogram demonstrates the distribution of spermatocytes at 17 h. The percent difference in annexin V+ cells is shown within the quadrant. (b) Histograms represent the mean flow cytometric data from DMSO-, CSC- or antagonists pretreated samples at 17 h of more than three independent experiments, each assayed in triplicate±S.E.M. n=4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979462&req=5

fig7: Inactivation of MAPKs does not prevent CSC-induced membrane damage. (a and b). Spermatocytes were exposed to DMSO (0.1%) or CSC (40 μg/ml) for 17 h. In the antagonists treatment groups, the cells were first pretreated with AHR or MAPK inhibitors or together for 1 h followed by exposure to CSC for 17 h (a). The percentage of cells segregated following co-staining with annexin V alexa fluor 488 and PI was determined by FACS analysis using FlowJo software (v9.7.5, FLOWJO, LLC.) as described under materials and methods. The representative histogram demonstrates the distribution of spermatocytes at 17 h. The percent difference in annexin V+ cells is shown within the quadrant. (b) Histograms represent the mean flow cytometric data from DMSO-, CSC- or antagonists pretreated samples at 17 h of more than three independent experiments, each assayed in triplicate±S.E.M. n=4.
Mentions: Given our previous study demonstrating crosstalk between p38-MAPK, ERK-44/42 and AHR,19 we asked whether pharmacological inhibition of MAPKs could prevent CSC-induced membrane damage. We first confirmed that neither the p38 inhibitor SB203580 nor the ERK inhibitor PD98059 could, on their own, increase the percentage of annexin-V-positive spermatocytes (Supplementary Figure 3). Pretreatment with MAPK inhibitors alone or together with AHR-inh could not prevent the externalization of phosphatidylserine in spermatocytes and thus the membrane damage (Figures 7a and b). These data ruled out the possibility of direct participation of MAPKs in membrane damage while implicating the specificity of AHR pathway in apoptosis.

View Article: PubMed Central - PubMed

ABSTRACT

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line.

No MeSH data available.


Related in: MedlinePlus