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Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

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Related in: MedlinePlus

MEF-2 inhibition perturbs HTLV-1-mediated T-cell transformation in correlation with a heightened MEF-2 expression in ATL patients. (A) PBMCs were transduced with scrambled or shMEF-2 expressing lentivirus by spinoculation. Viable cell proliferation of PBMCs was determined by trypan blue exclusion assay, after co-culture with lethally irradiated MT-2 cells for the indicated times. Error bars represent standard deviation of triplicate samples with high significance (***p < 0.001) between shMEF-2 versus shControl samples. (B) MT-2 and Jurkat cells were collected at 24 hr post a 3-day transfection followed by staining with propidium iodide (PI-25 μg/ml, RNAase- 40 μg/ml, sodium citrate-0.1% and Triton-100 × −0.03%). Cell cycle progression of MT-2 and Jurkat cells were observed via flow cytometry with no transfection (upper panel), mock transfected (middle panel) and shMEF2 plasmid (2.5 μg/1×106 cells) transfection (lower panel). The percentage of sub-G1 cells, G0/G1, S and G2-M cells was analyzed using the FLowjo software. (C) MEF-2A mRNA levels was determined by the quantitative real-time PCR as described in Methods. At least two replicates per donor were processed and MEF-2 levels were compared between seronegative controls and ATL patients (n = 3, each). Each point represents average mRNA expression in individual donors. Bars represent mean with Standard Error (SEM) derived by a two-tailed, unpaired nonparametric t-test (Mann–Whitney).
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Fig2: MEF-2 inhibition perturbs HTLV-1-mediated T-cell transformation in correlation with a heightened MEF-2 expression in ATL patients. (A) PBMCs were transduced with scrambled or shMEF-2 expressing lentivirus by spinoculation. Viable cell proliferation of PBMCs was determined by trypan blue exclusion assay, after co-culture with lethally irradiated MT-2 cells for the indicated times. Error bars represent standard deviation of triplicate samples with high significance (***p < 0.001) between shMEF-2 versus shControl samples. (B) MT-2 and Jurkat cells were collected at 24 hr post a 3-day transfection followed by staining with propidium iodide (PI-25 μg/ml, RNAase- 40 μg/ml, sodium citrate-0.1% and Triton-100 × −0.03%). Cell cycle progression of MT-2 and Jurkat cells were observed via flow cytometry with no transfection (upper panel), mock transfected (middle panel) and shMEF2 plasmid (2.5 μg/1×106 cells) transfection (lower panel). The percentage of sub-G1 cells, G0/G1, S and G2-M cells was analyzed using the FLowjo software. (C) MEF-2A mRNA levels was determined by the quantitative real-time PCR as described in Methods. At least two replicates per donor were processed and MEF-2 levels were compared between seronegative controls and ATL patients (n = 3, each). Each point represents average mRNA expression in individual donors. Bars represent mean with Standard Error (SEM) derived by a two-tailed, unpaired nonparametric t-test (Mann–Whitney).

Mentions: In order to investigate whether MEF-2A is required for HTLV-1-induced transformation of T cells, we used a well-established co-culture model wherein HTLV-1 immortalizes primary T lymphocytes [64]. PBMCs were transduced with lentivirus expressing control scrambled or ShMEF-2A and subjected to a co-culture assay with MT-2 cells as the source of virus. Transduced cells were selected with puromycin after 3 weeks. As seen in Figure 2A, PBMCs transduced with shMEF-2A ceased proliferating at about 3 weeks whereas PBMCs expressing control shRNA continued cell growth and became immortalized. Control untransduced PBMCs were rapidly killed upon adding puromycin (Figure 2A). As expected, PBMCs by themselves without MT-2 did not survive long enough, thus it is difficult to determine from this assay whether MEF-2 is required for survival of cells in the absence of HTLV-1. Therefore, to further investigate this phenomenon, we determined changes in the cell cycle of Jurkat and MT-2 cells in the presence or absence of MEF-2 (shMEF-2A) by the PI-based assessment of DNA content in G0/G1, S, and G2/M phase. Early steps in cell death are characterized by internucleosomal DNA fragmentation and chromatin condensation, thus the apoptotic stage sub-G1 was also deduced. Figure 2B (left panel) displays a dramatic decrease of G0/G1 (13%) phase in shMEF-2A-transfected MT-2 cells compared to control- (56%) or mock transfected (43%). Concurrently, the cell population of the G2/M and M phases also decreased from 12% to 5% and those of S phase decreased from 12% to 9% in mock versus shMEF-2A conditions, respectively. This population reduction was concomitant with the emergence of a characteristic hypodiploid (<2 N DNA) sub-G1 peak, which indicates apoptotic cells. shMEF-2A containing cells showed a dramatic increase in the sub-G1 peak with 73% cells showing signs of undergoing apoptosis compared to 28% in the mock-transfected cells. Interestingly, this phenomenon of increased sub-G1 events was not observed in Jurkat cells (Figure 2B, right panel). For Jurkat, control, mock or shMEF-2A samples showed fairly similar percentages of cells in the G0/G1, S and G2/M phases. This data suggest that observed MEF-2 effects are specific to HTLV-1 and more so to Tax since lack of MEF-2A reduces LTR transactivation and subsequent Tax expression (Figure 1), consequently leading to cell cycle arrest and dispensing MT-2 cells of G0/G1 and G2/M phases to apoptosis.Figure 2


Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

MEF-2 inhibition perturbs HTLV-1-mediated T-cell transformation in correlation with a heightened MEF-2 expression in ATL patients. (A) PBMCs were transduced with scrambled or shMEF-2 expressing lentivirus by spinoculation. Viable cell proliferation of PBMCs was determined by trypan blue exclusion assay, after co-culture with lethally irradiated MT-2 cells for the indicated times. Error bars represent standard deviation of triplicate samples with high significance (***p < 0.001) between shMEF-2 versus shControl samples. (B) MT-2 and Jurkat cells were collected at 24 hr post a 3-day transfection followed by staining with propidium iodide (PI-25 μg/ml, RNAase- 40 μg/ml, sodium citrate-0.1% and Triton-100 × −0.03%). Cell cycle progression of MT-2 and Jurkat cells were observed via flow cytometry with no transfection (upper panel), mock transfected (middle panel) and shMEF2 plasmid (2.5 μg/1×106 cells) transfection (lower panel). The percentage of sub-G1 cells, G0/G1, S and G2-M cells was analyzed using the FLowjo software. (C) MEF-2A mRNA levels was determined by the quantitative real-time PCR as described in Methods. At least two replicates per donor were processed and MEF-2 levels were compared between seronegative controls and ATL patients (n = 3, each). Each point represents average mRNA expression in individual donors. Bars represent mean with Standard Error (SEM) derived by a two-tailed, unpaired nonparametric t-test (Mann–Whitney).
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Related In: Results  -  Collection

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Fig2: MEF-2 inhibition perturbs HTLV-1-mediated T-cell transformation in correlation with a heightened MEF-2 expression in ATL patients. (A) PBMCs were transduced with scrambled or shMEF-2 expressing lentivirus by spinoculation. Viable cell proliferation of PBMCs was determined by trypan blue exclusion assay, after co-culture with lethally irradiated MT-2 cells for the indicated times. Error bars represent standard deviation of triplicate samples with high significance (***p < 0.001) between shMEF-2 versus shControl samples. (B) MT-2 and Jurkat cells were collected at 24 hr post a 3-day transfection followed by staining with propidium iodide (PI-25 μg/ml, RNAase- 40 μg/ml, sodium citrate-0.1% and Triton-100 × −0.03%). Cell cycle progression of MT-2 and Jurkat cells were observed via flow cytometry with no transfection (upper panel), mock transfected (middle panel) and shMEF2 plasmid (2.5 μg/1×106 cells) transfection (lower panel). The percentage of sub-G1 cells, G0/G1, S and G2-M cells was analyzed using the FLowjo software. (C) MEF-2A mRNA levels was determined by the quantitative real-time PCR as described in Methods. At least two replicates per donor were processed and MEF-2 levels were compared between seronegative controls and ATL patients (n = 3, each). Each point represents average mRNA expression in individual donors. Bars represent mean with Standard Error (SEM) derived by a two-tailed, unpaired nonparametric t-test (Mann–Whitney).
Mentions: In order to investigate whether MEF-2A is required for HTLV-1-induced transformation of T cells, we used a well-established co-culture model wherein HTLV-1 immortalizes primary T lymphocytes [64]. PBMCs were transduced with lentivirus expressing control scrambled or ShMEF-2A and subjected to a co-culture assay with MT-2 cells as the source of virus. Transduced cells were selected with puromycin after 3 weeks. As seen in Figure 2A, PBMCs transduced with shMEF-2A ceased proliferating at about 3 weeks whereas PBMCs expressing control shRNA continued cell growth and became immortalized. Control untransduced PBMCs were rapidly killed upon adding puromycin (Figure 2A). As expected, PBMCs by themselves without MT-2 did not survive long enough, thus it is difficult to determine from this assay whether MEF-2 is required for survival of cells in the absence of HTLV-1. Therefore, to further investigate this phenomenon, we determined changes in the cell cycle of Jurkat and MT-2 cells in the presence or absence of MEF-2 (shMEF-2A) by the PI-based assessment of DNA content in G0/G1, S, and G2/M phase. Early steps in cell death are characterized by internucleosomal DNA fragmentation and chromatin condensation, thus the apoptotic stage sub-G1 was also deduced. Figure 2B (left panel) displays a dramatic decrease of G0/G1 (13%) phase in shMEF-2A-transfected MT-2 cells compared to control- (56%) or mock transfected (43%). Concurrently, the cell population of the G2/M and M phases also decreased from 12% to 5% and those of S phase decreased from 12% to 9% in mock versus shMEF-2A conditions, respectively. This population reduction was concomitant with the emergence of a characteristic hypodiploid (<2 N DNA) sub-G1 peak, which indicates apoptotic cells. shMEF-2A containing cells showed a dramatic increase in the sub-G1 peak with 73% cells showing signs of undergoing apoptosis compared to 28% in the mock-transfected cells. Interestingly, this phenomenon of increased sub-G1 events was not observed in Jurkat cells (Figure 2B, right panel). For Jurkat, control, mock or shMEF-2A samples showed fairly similar percentages of cells in the G0/G1, S and G2/M phases. This data suggest that observed MEF-2 effects are specific to HTLV-1 and more so to Tax since lack of MEF-2A reduces LTR transactivation and subsequent Tax expression (Figure 1), consequently leading to cell cycle arrest and dispensing MT-2 cells of G0/G1 and G2/M phases to apoptosis.Figure 2

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Show MeSH
Related in: MedlinePlus