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Effects of hypoxia inducible factor-1α on apoptotic inhibition and glucocorticoid receptor downregulation by dexamethasone in AtT-20 cells.

Zhang C, Qiang Q, Jiang Y, Hu L, Ding X, Lu Y, Hu G - BMC Endocr Disord (2015)

Bottom Line: BrdU was used to determine the effects of hypoxia on cell viability.However, the viability of AtT-20 cells decreased greatly when they were first transfected with HIF-1α-siRNA and then exposed to hypoxia.Downregulation of GR by dexamethasone was greatly prevented in cells that were transfected with HIF-1α siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Shanghai Changzheng Hospital, Second Military Medical University, No. 415, Feng-Yang Road, Shanghai, 200003, China. christopher0708@163.com.

ABSTRACT

Background: Hypoxia inducible factor-1α (HIF-1α) is the central transcriptional regulator of hypoxic responses during the progression of pituitary adenomas. Although previous immunohistochemical studies revealed that HIF-1α is expressed in adreno-cortico-tropic-hormone (ACTH) pituitary adenomas, the role of HIF-1α remains unclear.

Methods: AtT-20 cells were incubated under hypoxic conditions (1 % O2) for 12 h. HIF-1α mRNA and protein expression levels were measured by real-time PCR and western blotting, respectively. BrdU was used to determine the effects of hypoxia on cell viability. AtT-20 cells were transfected with siRNA targeting HIF-1α, followed by hypoxia (1 % O2) for 12 h. Apoptosis was determined by annexin V-FITC flow cytometry and Tdt-mediated dUTP nick end-labelling (TUNEL) assay. In addition, we examined interactions between HIF-1α, glucocorticoid receptor (GR), and dexamethasone under both normoxic and hypoxic conditions.

Results: Hypoxia triggered the time-dependent proliferation of AtT-20 cells in association with increased HIF-1α mRNA and protein levels. However, the viability of AtT-20 cells decreased greatly when they were first transfected with HIF-1α-siRNA and then exposed to hypoxia. According to flow cytometry (annexin V-FITC and PI staining) and TUNEL analyses, a greater percentage of cells were apoptotic when transfected with HIF-1α siRNA and subsequently cultured under hypoxic conditions compared to those in the normoxia and mock groups. After AtT-20 cells were cultured in 1 % O2 and then treated with dexamethasone, HIF-1α levels significantly increased or decreased in normoxic or hypoxic conditions, respectively. Dexamethasone suppressed GR expression to a higher degree in hypoxic than normoxic conditions. Downregulation of GR by dexamethasone was greatly prevented in cells that were transfected with HIF-1α siRNA.

Conclusions: These findings strongly suggest that HIF-1α exerts an antiapoptotic role and participates in the downregulation of GR by dexamethasone in hypoxic AtT-20 cells.

No MeSH data available.


Related in: MedlinePlus

Role of HIF-1α in protecting AtT-20 cells from hypoxia-induced apoptosis. AtT-20 cells were transfected with 10 μM HIF-1α siRNA or mock transfected, and subsequently cultured in hypoxic or normoxic conditions for 12h. (a) Annexin V-FITC and (b) TUNEL assay showed increased apoptosis when cells were transfected with HIF-1α siRNA and subsequently cultured in 1% O2 for 12 h compared with normoxia and mock groups (*P < 0.05 vs.the other groups)
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Fig2: Role of HIF-1α in protecting AtT-20 cells from hypoxia-induced apoptosis. AtT-20 cells were transfected with 10 μM HIF-1α siRNA or mock transfected, and subsequently cultured in hypoxic or normoxic conditions for 12h. (a) Annexin V-FITC and (b) TUNEL assay showed increased apoptosis when cells were transfected with HIF-1α siRNA and subsequently cultured in 1% O2 for 12 h compared with normoxia and mock groups (*P < 0.05 vs.the other groups)

Mentions: RNA was prepared using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA, 1 μL oligo(dT) 15 primer (Promega, USA) and DEPC water were added to a total volume of 10 μL, heated to 70 °C for 5 min, and placed on ice for 5 min. Then a mixture of M-MLV RT 5× reaction buffer (5 μL), 100 mM dNTPs (0.5 μL), 1 μL M-MLV RT H(−) point mutant, and DEPC water in a final volume of 15 μL (all from Promega, Madison, USA) was added to each sample, followed by incubation at 40 °C for 60 min and 70 °C for 15 min. Real-time PCR was performed using the SYBR® Premix Ex Taq™ PCR kit (Takara, Japan) on the Applied Biosystems 7300 Real-Time PCR System (Foster, CA, USA). The 20-μL reaction of the SYBR Green assay contained 10 μL of 2× SYBR Premix Ex Taq, 0.4 μL PCR forward primers and 0.4 μL reverse primers, 0.4 μL ROX reference dye (50×), 2 μL cDNA, and 6.8 μL double-distilled H2O. PCR was carried out as follows: one cycle of 95 °C for 10 s (pre-denature) and 40 cycles of two steps (95 °C for 5 s and 60 °C for 31 s). At the end of the amplification, a dissociation curve (melting curve) was plotted in the temperature range 65–95 °C. All amplifications and detections were carried out in a MicroAmp optical 96-well reaction plate with optical adhesive covers (Applied Biosystems). PCRs were performed in triplicate, and a reliable internal control under hypoxia, 28S rRNA, was co-amplified to normalize the amount of RNA added to the reaction [14]. Normoxia group in Fig. 1b, 1 % O2-0 h without dexamethasone in Fig. 2a and b, 1 % O2-0 h in Fig. 3a and b were used as calibrator for relative quantitative PCR. All data were analysed using the Applied Biosystems 7300 SDS Software (Applied Biosystems, CA, USA). -2-△△ct method was used to analyze the real time PCR results.Fig. 1


Effects of hypoxia inducible factor-1α on apoptotic inhibition and glucocorticoid receptor downregulation by dexamethasone in AtT-20 cells.

Zhang C, Qiang Q, Jiang Y, Hu L, Ding X, Lu Y, Hu G - BMC Endocr Disord (2015)

Role of HIF-1α in protecting AtT-20 cells from hypoxia-induced apoptosis. AtT-20 cells were transfected with 10 μM HIF-1α siRNA or mock transfected, and subsequently cultured in hypoxic or normoxic conditions for 12h. (a) Annexin V-FITC and (b) TUNEL assay showed increased apoptosis when cells were transfected with HIF-1α siRNA and subsequently cultured in 1% O2 for 12 h compared with normoxia and mock groups (*P < 0.05 vs.the other groups)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464719&req=5

Fig2: Role of HIF-1α in protecting AtT-20 cells from hypoxia-induced apoptosis. AtT-20 cells were transfected with 10 μM HIF-1α siRNA or mock transfected, and subsequently cultured in hypoxic or normoxic conditions for 12h. (a) Annexin V-FITC and (b) TUNEL assay showed increased apoptosis when cells were transfected with HIF-1α siRNA and subsequently cultured in 1% O2 for 12 h compared with normoxia and mock groups (*P < 0.05 vs.the other groups)
Mentions: RNA was prepared using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA, 1 μL oligo(dT) 15 primer (Promega, USA) and DEPC water were added to a total volume of 10 μL, heated to 70 °C for 5 min, and placed on ice for 5 min. Then a mixture of M-MLV RT 5× reaction buffer (5 μL), 100 mM dNTPs (0.5 μL), 1 μL M-MLV RT H(−) point mutant, and DEPC water in a final volume of 15 μL (all from Promega, Madison, USA) was added to each sample, followed by incubation at 40 °C for 60 min and 70 °C for 15 min. Real-time PCR was performed using the SYBR® Premix Ex Taq™ PCR kit (Takara, Japan) on the Applied Biosystems 7300 Real-Time PCR System (Foster, CA, USA). The 20-μL reaction of the SYBR Green assay contained 10 μL of 2× SYBR Premix Ex Taq, 0.4 μL PCR forward primers and 0.4 μL reverse primers, 0.4 μL ROX reference dye (50×), 2 μL cDNA, and 6.8 μL double-distilled H2O. PCR was carried out as follows: one cycle of 95 °C for 10 s (pre-denature) and 40 cycles of two steps (95 °C for 5 s and 60 °C for 31 s). At the end of the amplification, a dissociation curve (melting curve) was plotted in the temperature range 65–95 °C. All amplifications and detections were carried out in a MicroAmp optical 96-well reaction plate with optical adhesive covers (Applied Biosystems). PCRs were performed in triplicate, and a reliable internal control under hypoxia, 28S rRNA, was co-amplified to normalize the amount of RNA added to the reaction [14]. Normoxia group in Fig. 1b, 1 % O2-0 h without dexamethasone in Fig. 2a and b, 1 % O2-0 h in Fig. 3a and b were used as calibrator for relative quantitative PCR. All data were analysed using the Applied Biosystems 7300 SDS Software (Applied Biosystems, CA, USA). -2-△△ct method was used to analyze the real time PCR results.Fig. 1

Bottom Line: BrdU was used to determine the effects of hypoxia on cell viability.However, the viability of AtT-20 cells decreased greatly when they were first transfected with HIF-1α-siRNA and then exposed to hypoxia.Downregulation of GR by dexamethasone was greatly prevented in cells that were transfected with HIF-1α siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Shanghai Changzheng Hospital, Second Military Medical University, No. 415, Feng-Yang Road, Shanghai, 200003, China. christopher0708@163.com.

ABSTRACT

Background: Hypoxia inducible factor-1α (HIF-1α) is the central transcriptional regulator of hypoxic responses during the progression of pituitary adenomas. Although previous immunohistochemical studies revealed that HIF-1α is expressed in adreno-cortico-tropic-hormone (ACTH) pituitary adenomas, the role of HIF-1α remains unclear.

Methods: AtT-20 cells were incubated under hypoxic conditions (1 % O2) for 12 h. HIF-1α mRNA and protein expression levels were measured by real-time PCR and western blotting, respectively. BrdU was used to determine the effects of hypoxia on cell viability. AtT-20 cells were transfected with siRNA targeting HIF-1α, followed by hypoxia (1 % O2) for 12 h. Apoptosis was determined by annexin V-FITC flow cytometry and Tdt-mediated dUTP nick end-labelling (TUNEL) assay. In addition, we examined interactions between HIF-1α, glucocorticoid receptor (GR), and dexamethasone under both normoxic and hypoxic conditions.

Results: Hypoxia triggered the time-dependent proliferation of AtT-20 cells in association with increased HIF-1α mRNA and protein levels. However, the viability of AtT-20 cells decreased greatly when they were first transfected with HIF-1α-siRNA and then exposed to hypoxia. According to flow cytometry (annexin V-FITC and PI staining) and TUNEL analyses, a greater percentage of cells were apoptotic when transfected with HIF-1α siRNA and subsequently cultured under hypoxic conditions compared to those in the normoxia and mock groups. After AtT-20 cells were cultured in 1 % O2 and then treated with dexamethasone, HIF-1α levels significantly increased or decreased in normoxic or hypoxic conditions, respectively. Dexamethasone suppressed GR expression to a higher degree in hypoxic than normoxic conditions. Downregulation of GR by dexamethasone was greatly prevented in cells that were transfected with HIF-1α siRNA.

Conclusions: These findings strongly suggest that HIF-1α exerts an antiapoptotic role and participates in the downregulation of GR by dexamethasone in hypoxic AtT-20 cells.

No MeSH data available.


Related in: MedlinePlus