The gene for the lysosomal protein LAMP3 is a direct target of the transcription factor ATF4

Abstract

Autophagy and lysosomal activities play a key role in the cell by initiating and carrying out the degradation of misfolded proteins. Transcription factor EB (TFEB) functions as a master controller of lysosomal biogenesis and function during lysosomal stress, controlling most but, importantly, not all lysosomal genes. Here, we sought to better understand the regulation of lysosomal genes whose expression does not appear to be controlled by TFEB. Sixteen of these genes were screened for transactivation in response to diverse cellular insults. mRNA levels for lysosomal-associated membrane protein 3 (LAMP3), a gene that is highly up-regulated in many forms of cancer, including breast and cervical cancers, were significantly increased during the integrated stress response, which occurs in eukaryotic cells in response to accumulation of unfolded and misfolded proteins. Of note, results from siRNA-mediated knockdown of activating transcription factor 4 (ATF4) and overexpression of exogenous ATF4 cDNA indicated that ATF4 up-regulates LAMP3 mRNA levels. Finally, ChIP assays verified an ATF4-binding site in the LAMP3 gene promoter, and a dual-luciferase assay confirmed that this ATF4-binding site is indeed required for transcriptional up-regulation of LAMP3 These results reveal that ATF4 directly regulates LAMP3, representing the first identification of a gene for a lysosomal component whose expression is directly controlled by ATF4. This finding may provide a key link between stresses such as accumulation of unfolded proteins and modulation of autophagy, which removes them.

Keywords: activating transcription factor 4 (ATF4); autophagy; cell stress; eukaryotic initiation factor 2 (eIF2); lysosomal-associated membrane protein 3 (LAMP3); lysosome; mammalian target of rapamycin complex 1 (mTORC1); protein misfolding; transcription factor EB (TFEB); unfolded protein response (UPR).

Figure 1.

Effects of ER stress, mTORC1/2 inhibition, and lysosomal stress on the expression of selected genes in HeLa and A549 cell lines. Total RNA was extracted from HeLa or A549 cells that had been treated with 1:1000 DMSO, 5 μm BFA (6 h), 200 nm rapamycin (6 h), 1 μm AZD8055 (6 h), or 100 mm sucrose (24 h) and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin. Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D. For clarity, not all significant differences are indicated.

Figure 2.

LAMP3 transcript levels increase with time in HeLa and A549 cells treated with BFA. Total RNA was extracted from HeLa or A549 cells that had been treated with 1:1000 DMSO for 24 h or 5 μm BFA for 2, 4, 8, and 24 h and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify the part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (DMSO). Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D.

Figure 3.

Increased TFEB activity does not increase LAMP3 mRNA levels. A, HeLa cells were transfected with pTFEB–3xFLAG–CMV-10 or empty vector (CONTROL). Six h later, cells were treated with 1:1000 DMSO or 1 μm AZD8055. Twenty four h later, cells were lysed, and samples were analyzed via immunoblotting with the indicated antibodies (n = 3). B, total RNA was extracted from similarly-treated cells and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (CONTROL + DMSO). Significance was calculated using Student's t test with means for n = 3. Error bars represent ± S.D.

Figure 4.

LAMP3 transcript levels are enhanced by additional chemical inducers of the ISR. A, HeLa and A549 cells were treated with 1:1000 DMSO, 5 μm BFA, 1 μm TPG, or 2 mm histidinol for 24 h. Extracts were then immunoblotted with antibodies to ATF4 and β-actin (n = 3). B, total RNA was extracted from similarly-treated cells and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (DMSO). Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D.

Figure 5.

BFA-induced increases in LAMP3 are attenuated by ISRIB. A, HeLa and A549 cells were treated with 1:1000 DMSO, 5 μm BFA (6 h), 5 μm BFA +200 nm ISRIB (6 h), or 200 nm ISRIB (6 h). Extracts were then immunoblotted with antibodies to ATF4 and β-actin (n = 3). B, total RNA was extracted from similarly-treated cells and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (DMSO). Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D. For clarity, not all the significant differences are indicated.

Figure 6.

BFA-induced LAMP3 expression is impaired upon siRNA-mediated ATF4 knockdown. A, HeLa cells were transfected with 5 nm scrambled siRNA (CONTROL) or one directed toward ATF4 (siATF4). After 48 h, cells were treated with 1:1000 DMSO or 5 μm BFA. 24 h later, cells were lysed, and samples were analyzed via immunoblotting with antibodies to ATF4 and β-actin (n = 3). B, ATF4 was quantified using densitometric analysis and normalized against β-actin, represented as the mean of the three biological replicates. C, total RNA was extracted from similarly-treated cells and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (CONTROL + DMSO). Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D.

Figure 7.

LAMP3 expression is up-regulated upon ATF4 overexpression under basal conditions. A, HeLa cells were transfected with pRK-ATF4 (ATF4) or empty vector (CONTROL). 24 h later, cells were lysed, and samples were analyzed via immunoblotting with antibodies to ATF4 and β-actin (n = 3). B, total RNA was extracted from similarly-treated cells and then used as a template for preparation of cDNA. cDNA samples were amplified and analyzed by qPCR using primers designed to amplify part of the coding sequence of the specified genes. Transcript levels were normalized to β-actin and presented as a fold-enrichment compared with the control (DMSO). Significance was calculated using Student's t test with mean for n = 3. Error bars represent ± S.D.

Figure 8.

Verification of an ATF4-binding site near the TSS of LAMP3. HeLa cells were treated with 1:1000 DMSO and 5 μm BFA for 6 h, and then formaldehyde was used to cross-link DNA and protein. Samples were sonicated to shear chromatin, then immunoprecipitated with 1:200 ATF4 antibody or 1:500 normal rabbit IgG antibody. Chromatin was purified, then analyzed by qPCR in technical triplicates using primers designed to analyze known or potential ATF4-binding sites near the specified gene, as well as a region ∼1000 bp upstream of each site (negative). Results are presented as a fold-enrichment of ATF4 versus normal rabbit IgG. DNA from the normal rabbit IgG was normalized to 1 for comparison with the ATF4 pulldown. Significance was calculated using two-way ANOVA with mean for n = 3. Error bars represent ± S.D.

Figure 9.

ATF4-binding site near the TSS of LAMP3 mediates BFA-induced expression of a reporter. A, illustration of oligonucleotides designed for insertion into the pGL3-pro plasmid. Boldface nucleotides represent the putative ATF4-binding sequence (WT L3). Underlined nucleotides represent mutations introduced into the mutant variants (MT1 L3 and MT2 L3). Lowercase represents nucleotides chosen to allow for insertion into the KpnI/NheI-digested plasmid. B, HeLa cells were transfected with 50 ng of pRL-TK DNA and 50 ng of one of pGL3-pro, pGL3-pro + WT L3, pGL3-pro + MT1 L3, and pGL3-pro + MT2 L3, and then, 8 h later, were treated for 16 h with DMSO or BFA, as indicated. The Promega Dual-Glo luciferase assay system was then used to measure firefly and Renilla luciferase activity in each well. Results are presented as relative luciferase units (RLU) of firefly to Renilla luciferase activity. Significance was calculated using a Student's t test with mean for n = 3. Error bars represent ± S.D. For clarity, not all the significant differences are indicated.