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. 2016 Dec;39(12):877-887.
doi: 10.14348/molcells.2016.0161. Epub 2016 Dec 13.

Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy Through Transmembrane Protein 192

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Free PMC article

Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy Through Transmembrane Protein 192

Rong-Yaun Shyu et al. Mol Cells. .
Free PMC article

Abstract

Tazarotene-induced gene 1 (TIG1) is a retinoic acid-inducible protein that is considered a putative tumor suppressor. The expression of TIG1 is decreased in malignant prostate carcinoma or poorly differentiated colorectal adenocarcinoma, but TIG1 is present in benign or well-differentiated tumors. Ectopic TIG1 expression led to suppression of growth in cancer cells. However, the function of TIG1 in cell differentiation is still unknown. Using a yeast two-hybrid system, we found that transmembrane protein 192 (TMEM192) interacted with TIG1. We also found that both TIG1A and TIG1B isoforms interacted and co-localized with TMEM192 in HtTA cervical cancer cells. The expression of TIG1 induced the expression of autophagy-related proteins, including Beclin-1 and LC-3B. The silencing of TMEM192 reduced the TIG1-mediated upregulation of autophagic activity. Furthermore, silencing of either TIG1 or TMEM192 led to alleviation of the upregulation of autophagy induced by all-trans retinoic acid. Our results demonstrate that the expression of TIG1 leads to cell autophagy through TMEM192. Our study also suggests that TIG1 and TMEM192 play an important role in the all-trans retinoic acid-mediated upregulation of autophagic activity.

Keywords: Beclin-1; LC3B; all-trans retinoic acid; autophagy; tazarotene-induced gene 1; transmembrane protein 192.

Figures

Fig. 1
Fig. 1
TIG1 interacts and co-localizes with TMEM192. HtTA cells plated in a 10-cm dish were transfected with 3 μg of TMEM192-Flag expression vector along with the TIG1A-myc or TIG1B-myc expression vector for 24 h. Cell lysates were prepared, and the interaction between TIG1 and TMEM192 was analyzed by immunoprecipitation followed by Western blot analysis. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the anti-MYC antibody (A) or anti- FLAG antibody (B). Total cellular extracts from HtTA cells were subjected to Western blot analysis for TIG1, TMEM192, and actin. HtTA cells were co-transfected with EGFP-TMEM192 along with the TIG1A-myc or TIG1B-myc expression vectors for 18 h. The cells were fixed and then incubated with anti-MYC and anti-LAMP1 (lysosomal marker) antibodies followed by Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 405 goat anti-rabbit IgG antibodies. The cells were then analyzed with a laser scanning confocal microscope. Scale bar: 10 μm. The localization of TIG1 (red), TMEM192 (green), and lysosomes (blue) was analyzed using a laser scanning confocal microscope. Arrows indicate co-localization of TIG1 and TMEM192 (C). Scale bar, 10 μm.
Fig. 2
Fig. 2
Effects of TIG1 on expression of TMEM192 in HtTA cells. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc (A) or TMEM192-flag (B) expression vector for 24 h. Total RNA was extracted and relative levels of the indicated mRNAs were measured by real-time RT-PCR. HtTA cells plated in a 6-cm dish were transfected with 0.5 μg of TMEM192-Flag expression vector along with 0.5–1.5 μg of TIG1A-myc (C) or TIG1B-myc (D) expression vector for 12 h. Alternatively, cells were transfected with 0.5–1.5 μg of TMEM192-Flag expression vector along with 0.5 μg of TIG1A-myc (E) or TIG1B-myc (F) expression vector for 12 h. Cell lysates were prepared, and the levels of TIG1A, TIG1B, and actin were determined by immunoblotting.
Fig. 3
Fig. 3
TIG1A induced the expression of autophagy-related proteins. HtTA cells plated in a 6-cm dish were transfected with 1.5 μg of TMEM192-Flag expression vector along with 1.5 μg of TIG1A-myc expression vector for 24 h. Cells were refreshed in medium without serum and treated with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the levels of LC-3B, Beclin-1, SQSTM1/p62, TIG1A, and actin were determined by immunoblotting (A). Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (B, C). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of the indicated vectors or the control vector for 24 h and were then cultured in serum-free medium with 200 nM rapamycin for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown (D). HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 75 ng of TMEM192-Flag expression vector and 150 ng of pGFP-LC3 expression vector for 24 h. Cells were refreshed in medium without serum and treated with 400 nM rapamycin for 6 h. Representative images with GFP-LC3 puncta formation (E). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (F). Scale bar, 10 μm. *Indicates p value < 0.05.
Fig. 4
Fig. 4
TMEM192 siRNAs alleviated TIG1A-induced expression of autophagy-related proteins. HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1B-myc (A) or TMEM192-Flag (B) expression vector along with 30 nM TIG1, TMEM192, or NC siRNA for 48 h. Alternatively, cells were transfected with 0.5 μg of TIG1A-myc (C–E) expression vector along with the indicated siRNA (30 nM) for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the expression of TIG1, TMEM192, Beclin-1, or LC3B was determined using anti-MYC, anti-FLAG, anti-Beclin-1, or anti-LC3B antibodies, respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (D, E). HtTA cells plated in 6-well dishes were transfected with 0.5 μg of TIG1A-myc expression vectors along with the indicated siRNA for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the level of Beclin-1 was detected using an enzyme immunoassay. Representative results of three independent experiments are shown. HtTA cells plated in triplicate in 24-well plates were transfected with 75 ng of TIG1A-myc expression vector, 150 ng pGFP-LC3 expression vector and with the indicated 30 nM siRNA for 48 h and were then cultured in serum-free medium for 6 h. Representative images with GFP-LC3 puncta formation (G). Bar chart indicating the percentage of cells with GFP-LC3 puncta formation from three independent experiments (H). Scale bar, 10 μm. *Indicates p value < 0.05. #Indicates p value < 0.05 when cells were co-transfected with TIG1A expression vector and indicated siRNA compared to cells that were co-transfected with TIG1A expression vector and NC siRNA.
Fig. 5
Fig. 5
TIG1 siRNAs suppressed the expression of autophagy-related proteins. HtTA cells plated in 6-well dishes were transfected with the indicated siRNA (30 nM) for 48 h and were then cultured in serum-free medium for 6 h. Cell lysates were prepared, and the expression of TIG1, TMEM192, or LC3B was determined using anti-TIG1 (A), anti-TMEM192 (B), or anti-LC3B and Beclin-1 (A, B) antibodies, respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (C–F). The level of Beclin-1 was detected using an enzyme immunoassay (G, H). Representative results of three independent experiments are shown. *Indicates p value < 0.05.
Fig. 6
Fig. 6
TIG1 and TMEM192 siRNAs decrease ATRA-induced upregulation of autophagy. HtTA cells plated in 6-well dishes were treated daily with the indicated concentration of ATRA for 48 h (A). Alternatively, cells were transfected with the indicated siRNA (30 nM) and then treated daily with 10−6 M ATRA for 48 h. Cells were then cultured in serum-free medium for 6 h (B–D). Cell lysates were prepared, and the expression of TIG1, TMEM192, Beclin-1, or LC3B was determined using anti-TIG1, anti-TMEM192, Beclin-1, or anti-LC3B antibodies (B), respectively. Experimental results are summarized as the mean percentage (± SD) of the ratio of LC3B-II to LC3B-I and the level of Beclin-1 with each sample normalized to the level of actin protein in two independent experiments (C, D). The level of Beclin-1 was detected using an enzyme immunoassay (E). Representative results of three independent experiments are shown. *Indicates p value < 0.05. #Indicates p value < 0.05 when cells were transfected with the indicated siRNA and then treated with ATRA compared to cells transfected with NC siRNA and then treated with ATRA.

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