Abstract
Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus–pituitary–thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, that is the Ctsk−/−/Mct8−/y/Mct10−/− genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk−/−/Mct8−/y/Mct10−/− thyroid glands. Our data revealed that while mRNA amounts and subcellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk−/−/Mct8−/y/Mct10−/− mice in comparison to WT controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk−/−/Mct8−/y/Mct10−/− mice. Indeed, thyroid tissue of Lat2−/− mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.
Introduction
The main function of the thyroid gland is the production of the thyroid hormones (TH) 3,3’,5-triiodo-L-thyronine (T3) and 3,3’,5,5’-tetraiodo-L-thyronine (T4), which are involved in fundamental regulatory processes in the body. To do so, the thyroid gland synthesizes TH on the backbone of thyroglobulin (Tg), liberates these upon sequential Tg proteolysis, and releases them into the bloodstream (Brix et al. 2019, 2020, Coscia & Taler-Verčič 2021). Tg solubilization and utilization is initiated in the thyroid follicle lumen and continues within endocytic compartments, mediated by proteases such as endo-lysosomal cathepsins (Friedrichs et al. 2003, Jordans et al. 2009). TH translocation across membranes is enabled by transmembrane transporters such as monocarboxylate transporters Mct8 (Slc16a2) or Mct10 (Slc16a10) and L-type amino acid transporter 2 (Lat2, Slc7a8) (Groeneweg et al. 2020). The thyroid gland is canonically regulated by the hypothalamus–pituitary–thyroid (HPT) axis, whereby low circulatory concentrations of TH result in the release of thyroid-stimulating hormone (TSH) from pituitary thyrotropes, which activates the thyroid gland (Fliers et al. 2014, Ortiga-Carvalho et al. 2016).
It is worth mentioning that distinct cathepsins play specific roles in Tg processing and degradation. For instance, while cathepsins B and L mainly enable Tg solubilization from its covalently cross-linked storage forms, TH liberation is mediated by cathepsins K and L (Friedrichs et al. 2003). Importantly, only cathepsin K is capable of directly liberating T4 from intact Tg (Tepel et al. 2000). Previously, we have shown that besides normal serum TSH concentrations, mice lacking cathepsin K (Ctsk−/−) feature normal thyroid morphological and functional phenotypes (Dauth et al. 2020, Venugopalan et al. 2021a,b). This may be partly attributed to the functional compensation through TSH-independent cathepsin L upregulation in Ctsk−/− mice (Friedrichs et al. 2003, Weber et al. 2017, Venugopalan et al. 2021a), implying the existence of intrathyroidal regulatory mechanisms (Brix et al. 2020). Since cathepsin K deficiency is also associated with increased Mct8 protein amounts (Weber et al. 2017), we have previously examined whether TH transporters are part of such thyroid auto-regulatory mechanisms by assessing thyroid phenotypes in Ctsk−/−/Mct10−/−, Ctsk−/−/Mct8−/y, and Ctsk−/−/Mct8−/y/Mct10−/− mice (Venugopalan et al. 2021a). The data showed that despite impaired TH export, male mice lacking Mct8 (Mct8−/y) display persistent cathepsin-mediated Tg proteolysis due to enhanced lysosomal biogenesis, which is triggered by autophagy induction (Venugopalan et al. 2021a). The observation that Ctsk−/−/Mct8−/y/Mct10−/− mice exhibited a prominent induction of autophagy motivated us to elucidate the molecular mechanism of autophagy induction in the triple-deficient mice in the present study.
In tissues such as kidney, pancreas, and skeletal muscle, Lat2-mediated amino acid transport has been implicated to stimulate signaling via mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy (Kurayama et al. 2011, Suryawan et al. 2013, Feng et al. 2018). Since Lat2 is also synthesized in the thyroid gland and involved in TH besides large neutral amino acid transport (Di Cosmo et al. 2010, Braun et al. 2011b, Kinne et al. 2011), we proposed that Lat2 might be involved in regulating autophagy in thyroid tissue as well. Therefore, we asked whether protein abundance or localization patterns of Lat2 are changed in the triple-deficient mice, that is in the Ctsk−/−/Mct8−/y/Mct10 genotype.
The results of this study indeed demonstrate that the protein levels of Lat2 are diminished in thyroid tissue of the triple-deficient murine model. We propose that the lack of Lat2 possibly triggers the observed autophagy and lysosomal biogenesis resulting in enhanced cathepsin expression and activities, which were previously observed in the Ctsk−/−/Mct8−/y/Mct10−/− genotype (Venugopalan et al. 2021a). Additionally, reduced TH export due to the lack of Mct8 and Mct10 might contribute to intrathyroidal TH accumulation, which possibly activates autophagy through thyrotoxic stress in the thyroid tissue of these mice. Consistent with this notion, induced autophagy-mediated increases in cathepsin amounts and activities were observed in thyroid tissue of Lat2-deficient mice. This observation further supports the interpretation that reduced protein amounts of thyroidal Lat2 result in autophagy-triggered lysosomal biogenesis in triple-deficient mice.
Materials and methods
Animals
Animals analyzed in the present study were 5–8 months old male Ctsk−/−/Mct8−/y/Mct10−/−, Lat2−/−, and WT C57Bl/6J mice. The generation of triple-deficient animals and the Lat2−/− mice and the respective genotyping protocols have been described previously (Braun et al. 2011a, b, Venugopalan et al. 2021a, b). Mice were housed under standardized conditions of light and darkness cycles (12 h/12 h) with water and food ad libitum.
Tissue sampling and cryo-sectioning
Thyroid glands were removed from mice after sacrificing them by CO2 inhalation and following perfusion with 0.9% NaCl containing 200 IU heparin (Braun Melsungen AG, Melsungen, Germany) as described previously (Venugopalan et al. 2021a, b). While still attached to the trachea, the thyroid gland was fixed overnight at 4°C in 4% paraformaldehyde in 200 mM HEPES, pH 7.4, cryo-preserved by embedding overnight at 4°C in tissue freezing medium (Jung, through Leica Microsystems), frozen in the gas phase of liquid nitrogen, and stored at −80°C. For immunohistochemistry analyses, thyroid tissue was cryo-sectioned into 5 µm thick transverse sections using a cryostat (Leica CM1900, Leica Microsystems), thaw-mounted onto microscopic slides, and stored at −20°C until subsequent use. For protein biochemistry studies, individual thyroid lobes were snap-frozen in liquid nitrogen and stored at −80°C until further use for the preparation of whole tissue lysates.
Indirect immunofluorescence, image acquisition, and automated image analysis
Thyroid cryo-sections were immunostained as previously described (Venugopalan et al. 2021a, b) using the antibodies listed in Table 1. Draq5TM (10 µM; DR05500, BioStatus Limited, Shepshed, Leicestershire, UK) was used to counter-stain nuclear DNA. Immunolabeled tissue sections on microscopic slides were embedded in mounting medium comprising 33% glycerol and 14% Mowiol (Hoechst AG, Frankfurt, Germany) in 200 mM Tris-HCl, pH 8.5, and stored at 4°C until subsequent analysis by confocal laser scanning microscopy. Thyroid cryo-sections were imaged by confocal laser scanning microscopy using Argon and Helium-Neon or diode lasers (LSM 510 Meta, Carl Zeiss Jena GmbH and LSM 980 with Airyscan 2 and Multiplex, Carl Zeiss Microscopy GmbH). Images were taken with a pinhole opening of 1 Airy unit and at a resolution of 1024 × 1024 pixels or using high-resolution Airyscan modes. Micrographs were analyzed using the LSM 510 software (version 3.2; Carl Zeiss Jena GmbH) and with the LSM 980 ZEN 3.2 software (Carl Zeiss Microscopy GmbH). Immunofluorescence intensities were quantified using Cell Profiler (version 3.1.9) (McQuin et al. 2018), an open-source automated image analysis software available from the Broad Institute at www.cellprofiler.org (last accessed on September 17, 2021). Cathepsin B, D, L, and Lat2 fluorescence intensity measurements were determined and normalized to the numbers of cells (i.e. Draq5™-positive nuclei) as described previously (Venugopalan et al. 2021a).
List of antibodies used in this study.
Antigen | Biological source | Species reactivity | Company/provider | Catalog no. | Dilution in IF | Dilution in IB |
---|---|---|---|---|---|---|
Primary antibodies | ||||||
Cath B | Goat | Mouse | Neuromics (through Acris, Herford, Germany) | GT15047 | 1:100 | 1:1000 |
Cath D | Rabbit | Human | Calbiochem (through Merck Millipore) | IM-16 | 1:10 | 1:80 |
Cath L | Goat | Mouse | Neuromics | GT15049 | 1:100 | 1:1000 |
Lat2 | Rabbit | Mouse | Immunoglobe (Himmelstadt, Germany) | 0142-10 | 1:30 | 1:200 |
LC3 | Rabbit | Mouse, human, rat | Novus Biologicals (through Bio-Techne, Wiesbaden-Nordenstadt, Germany) | NB100-2331 | 1:100 | 1:500 |
p62 | Mouse | Human | Abcam | ab56146 | – | 1:500 |
Secondary antibodies | ||||||
Rabbit anti-goat Alexa 488 | Molecular Probes (through Invitrogen, Darmstadt, Germany) | A-21222 | 1:200 | – | ||
Goat anti-rabbit Alexa 488 | Molecular Probes | A-11070 | 1:200 | – | ||
Rabbit-anti-goat IgG (H+L)-HRP | Jackson Immunoresearch | 305-035-003 | – | 1:5000 | ||
Goat-anti-rabbit IgG (H+L)-HRP | Jackson Immunoresearch | 111-035-003 | – | 1:5000 | ||
Goat anti-mouse IgG (H+L)-HRP | Southern Biotech (through Biozol Diagnostica, Eching, Germany) | 1036-05 | – | 1:3000 |
IB, immunoblotting; IF, immunofluorescence; HRP, horseradish peroxidase.
Preparation of whole tissue lysates, SDS-PAGE and immunoblotting
Tissue lysates were prepared as described previously (Venugopalan et al. 2021a,b) by homogenization of snap-frozen thyroid glands in PBS, pH 7.4, containing 0.5% Triton X-100 and protease inhibitors (0.02 M EDTA, 10 µM E64, 1 µM pepstatin A, and 2 ng/mL aprotinin). Active thyroidal cysteine cathepsins were analyzed by excluding the addition of protease inhibitors and adding 5 µM biotin-conjugated activity-based probe DCG-04 (Greenbaum et al. 2000) to the lysis buffer. Tissue lysates were cleared by centrifugation at 16,000 g for 10 min at 4°C and stored at −20°C. Neuhoff assay was performed to determine protein concentrations (Neuhoff et al. 1979) with BSA as standard protein. Samples were prepared by normalizing tissue lysates to equal protein amounts, that is 0.6 µg/µL protein per sample was used and 15 µg were loaded per lane. Samples were denatured in sample buffer (50 mM Tris–HCl (pH 7.6), 2.5% sodium dodecyl sulfate (SDS), 125 mM dithiothreitol, and 4 μM bromophenol blue) for 5 min at 95°C and separated on commercially available horizontal SDS Gradient 8–18 ExcelGel gels (GE Healthcare) along with PageRuler prestained protein ladder (#26616, Thermo Fisher Scientific) at 300 V and 50 mA. Subsequently, proteins were transferred onto nitrocellulose membranes by semi-dry blotting in a Novoblot Western Blotting chamber (GE Healthcare) for 1 h at 30 V and 216 mA. Total protein per lane was determined by incubating membranes for 10 min with Ponceau S solution (#A2395, AppliChem, Darmstadt, Germany). Immunostaining of the membranes was performed as described previously (Venugopalan et al. 2021a,b) by blocking membranes using 5% blotting-grade milk powder in PBS containing 0.3% Tween-20 (PBS-T) for 1 h at room temperature and, subsequently, incubating with antibodies detailed in Table 1. Next, membranes were incubated with ECL Western blotting substrate (#34580, Thermo Fisher Scientific) for 3 min, and immuno-recognized bands were visualized by exposure onto CL-XPosureTM films (Pierce via Thermo Fisher Scientific). Exposed films were scanned using a transmitted-light scanner (Desk Scan II version 2.9, Hewlett-Packard Co., Palo Alto, California, USA). Densitometry analyses were done using Image Studio Lite version 5.2 (LI-COR Biosciences GmbH, Bad Homburg, Germany).
Transcriptome and proteome analysis data
The generation of global gene expression data by comparative analyses of thyroidal transcriptomes and proteomes from Ctsk−/−/Mct8−/y/Mct10−/− and WT control mice was described earlier (Venugopalan et al. 2021a). Microarray-based transcriptome analyses (four to five animals per genotype) as well as LC-MS/MS-based proteome analyses (five animals per genotype) were performed using total RNA and protein extracted from snap-frozen thyroid tissue as detailed previously (Lietzow et al. 2016, Venugopalan et al. 2021a). The Rosetta Resolver software (Rosetta Biosoftware, Seattle, WA, USA) was used for analysis of the transcriptome data set, while for relative protein quantification, normalized label-free quantification (LFQ) intensities were imported in GeneData Analyst software (version 10.0.3) (Genedata, Basel, Switzerland) and log10-transformed. Data from transcriptome and proteome analyses were statistically analyzed by performing one-way ANOVA and Welch’s t-test, respectively, where P-values were corrected for multiple testing using the Benjamini–Hochberg adjustment. Finally, P- and q-values below 0.05 in combination with fold changes ≥|1.5| were considered statistically significant. The transcriptome and proteome data sets are available in the GEO and the MassIVE data repositories, respectively, with the reference numbers GSE163168 and MSV000086595.
Statistical analysis
Densitometry and fluorescence intensity measurements are shown as means ± standard deviations and as fold changes over WT controls to interpret differences between genotypes. Levels of significance were determined by performing two-tailed unpaired t-test using GraphPad PrismTM (version 5.01; GraphPad Software Inc.), and P‑values below 0.05 were considered statistically significant.
Results
Transcript amounts of Lat2 and its heavy chain CD98 in thyroid glands of Ctsk−/−/Mct8−/y/Mct10−/− mice based on omics analyses
We have recently analyzed the thyroid functional phenotype of murine models with combined cathepsin K and TH transporter deficiencies and reported that Ctsk−/−/Mct8−/y/Mct10−/− mice show enhanced cathepsin-mediated Tg degradation due to autophagy induction (Venugopalan et al. 2021a). Because the Lat2 protein has been suggested to be involved in the regulation of autophagy in other tissues, we now compared the thyroidal transcriptome and proteome data from Ctsk−/−/Mct8−/y/Mct10−/− mice with WT control animals (Venugopalan et al. 2021a) for changes in Lat2 (Slc7a8) expression. We observed a non-significant trend toward lower Lat2 mRNA abundance in the triple-deficient murine model compared to WT controls (Fig. 1A), while the Lat2 protein was among those proteins that were not detectable in the corresponding proteome analysis (see the MassIVE data repository entry MSV000086595). The related transporter Lat1 (Slc7a5) is also expressed in the mouse thyroid gland and could possibly compensate for altered Lat2 function. Therefore, we examined Lat1 mRNA levels in Ctsk−/−/Mct8−/y/Mct10−/− mice in comparison to WT controls, which remained unchanged (Fig. 1B).
Because it is known that the covalent binding of the heavy chain CD98 (4F2hc, Slc3a2) to Lat2 enables cell-surface trafficking and proper function of the latter by acting as its chaperone (Nakamura et al. 1999, Rosell et al. 2014), we next sought to address whether this was affected in the triple-deficient genotype in comparison to WT controls. The transcriptome as well as the proteome data demonstrated no significant differences in either the CD98 transcript or the CD98 protein amounts (Fig. 1C and D, respectively).
While these data revealed that the abundances of Lat2 and CD98 transcripts as well as those of CD98 protein remain unchanged in triple-deficient mice, no statement could be made for Lat2 protein amounts.
Lat2 protein in the thyroid gland of Ctsk−/−/Mct8−/y/Mct10−/− mice
To assess whether Lat2 protein is affected in the triple-deficient murine model using an alternative methodological approach, protein lysates of whole thyroid tissue from Ctsk−/−/Mct8−/y/Mct10−/− mice and WT controls were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunolabeled using antibodies specific for Lat2 (Fig. 2A). Lat2 was detected at the expected molecular mass of approximately 58 kDa in the investigated genotypes, and additionally, a potential degradation fragment of 38 kDa was immuno-recognized, while no immunoreaction was observed in Lat2-deficient thyroid tissue lysates, as reported previously (Wirth et al. 2009, Braun et al. 2011b, Badziong et al. 2017). Densitometry analysis was performed by normalizing the signal of immunopositive bands at 58 kDa to the amount of total Ponceau-stained proteins per lane. Results revealed a significant decrease in Lat2-representative band intensities in thyroid tissue lysates of Ctsk−/−/Mct8−/y/Mct10−/− animals compared to WT controls (Fig. 2B).
These data indicate that reduced Lat2 protein amounts represent a characteristic feature of thyroid gland tissue of the triple-deficient Ctsk−/−/Mct8−/y/Mct10−/− murine model.
Subcellular Lat2 localization in the thyroid gland of Ctsk−/−/Mct8−/y/Mct10−/− mice
Thyroid epithelial cells express different amino acid transporters, and molecules with dual functionality, thus, also involved in TH translocation across cellular membranes. Presumably, specific transporter molecules are each strategically positioned at individual subcellular locations for translocation of amino acids or TH across endo-lysosomal membranes or the plasma membrane of the apical and basolateral poles of thyroid epithelial cells, with the latter mainly involved in T4 export from thyroid follicles (Weber et al. 2017, Brix et al. 2020). Hence any alterations in the subcellular localization of such transporters could affect thyroid function.
Therefore, tissue cryo-sections from the thyroid glands of Ctsk−/−/Mct8−/y/Mct10−/− mice and WT controls were immunostained using Lat2-specific antibodies and analyzed by immunofluorescence microscopy. Results showed that Lat2 predominantly localizes to vesicles in both WT and triple-deficient mice (Fig. 3A, A′ and B, B′, respectively, arrows), while no immunostaining was observed in Lat2-deficient tissue (Fig. 3C), indicating a possible role of Lat2 as amino acid translocator across endo-lysosomal membranes of thyrocytes. Furthermore, intensity measurements of Lat2 immunofluorescence signals using a cell profiler-based pipeline with normalization to cell counts revealed a significant reduction of vesicular Lat2 signal in Ctsk−/−/Mct8−/y/Mct10−/− thyroid cryo-sections compared to WT thyroid tissue (Fig. 3D).
Thus, although Lat2 localization remained unaltered in Ctsk−/−/Mct8−/y/Mct10−/− thyrocytes, immunopositive fluorescence signals were diminished in these mice compared to WT controls (Fig. 3). This result suggests that the absence of Mct8 and Mct10 in the triple-deficient mice does not result in functional compensation through upregulation of the Lat2 protein but is similarly associated with reduced Lat2 amounts, which in turn may affect amino acid transport processes within thyroid epithelial cells and/or may result in altered autophagy.
Autophagy is induced in the thyroid gland of Lat2−/− mice
Phenotyping of Lat2−/− mice has previously shown that these animals exhibit normal serum TH and TSH concentrations (Braun et al. 2011b). In order to clarify whether the detected reduced amounts of Lat2 may be mechanistically involved in activation of the autophagic pathway in Ctsk−/−/Mct8−/y/Mct10−/− thyroid glands, we set out to assess autophagy also in thyroid glands of Lat2-deficient mice. For this purpose, thyroid cryo-sections from Lat2−/− mice and WT controls were incubated with antibodies specific for the autophagosomal marker microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3A/LC3) and assessed by confocal laser scanning microscopy. While LC3 localized mainly to the cytosol in WT controls (Fig. 4A, left panel), Lat2-deficient thyroid cryo-sections demonstrated vesicle-associated LC3 localization (Fig. 4A, right panel, arrows). The predominance of LC3-positive puncta in Lat2−/− thyroid tissue suggested an increase in autophagosome numbers as a result of autophagy induction upon Lat2 deficiency.
This assumption was confirmed by separating proteins from thyroid tissue lysates of WT and Lat2−/− mice on SDS-gels and subsequently performing immunoblotting using LC3-specific antibodies (Fig. 4B, left panel). Two LC3-representative bands, LC3-I at ~19 kDa and LC3-II at ~17 kDa, were detected. It is observed that upon autophagy induction, LC3-I represents processed cytosolic LC3, while LC3-II is the lipidated form that associates with autophagosomal membranes (Yoshii & Mizushima 2017, Venugopalan et al. 2021a). Analysis by densitometry was performed by normalizing band intensities to total Ponceau-stained protein per lane. Results showed a significant three-fold increase in LC3-II intensity in Lat2−/− thyroid tissue lysates in comparison to WT controls (Fig. 4B, right panel).
These results indicated that lack of Lat2 protein in the thyroid gland indeed correlates with induced autophagy.
Autophagic flux is enhanced in Lat2 deficiency
Increased LC3-II levels could either be a consequence of enhanced autophagosome formation due to the initiation of autophagy or caused by diminished autophagosomal turnover due to impaired autophagosome-lysosome fusion and reduced autophagic activity that results in the accumulation of autophagosomes (Yoshii & Mizushima 2017). To differentiate between these two possibilities, we evaluated the levels of p62, a cargo protein that is degraded during autophagy and is hence used to investigate autophagic flux. Immunoblotting of whole thyroid tissue lysates from WT and Lat2-deficient mice with anti-p62 antibodies (Fig. 5A) revealed a three-fold reduction in p62 protein amounts in thyroid tissue of Lat2−/− mice, indicating that autophagic flux is indeed enhanced in Lat2 deficiency (Fig. 5B).
These results demonstrated that the absence of Lat2 protein does not only correlate with initiation of autophagy but also with induction of autophagic flux in murine thyroid glands.
Lysosomal biogenesis is induced in Lat2-deficient thyroid glands
The degradation of cellular components by the process of autophagy depends on the availability of hydrolases in the endo-lysosomes. Since the Tg-processing cathepsins B, D, and L are endo-lysosomal enzymes, we examined whether the amounts of these cathepsins are altered in Lat2−/− thyroid glands as a result of autophagy induction. Whole thyroid tissue lysates from Lat2−/− mice and WT controls were immunoblotted using cathepsin B-, D-, or L-specific antibodies (Fig. 6). It is known that these cathepsins are synthesized as proforms (pro) in the endoplasmic reticulum (ER) and later reach the endo-lysosomes wherein they are proteolytically activated to either mature single-chain (SC) or two-chain forms consisting of disulfide-linked heavy (HC) and light chains (LC). In both genotypes, anti-cathepsin B and L antibodies recognized the proform, SC, and HC forms (Fig. 6A and B, upper left panels). In contrast, cathepsin D immunoblotting detected only the proform due to the epitope specificity of the anti-cathepsin D antibody used in this study (Fig. 6C, left panel). Densitometric analysis of immunoblots showed that the signals representing all immuno-recognized forms of the investigated cathepsins were significantly enhanced in Lat2−/− thyroid lysates compared to WT controls (Fig. 6A, B and C, upper right and bottom panels). While the derived protein amounts of both proform and mature forms of cathepsins B and L were approximately two-fold elevated (Fig. 6A and B, bottom panels), pro-cathepsin D displayed a three-fold increase in Lat2-deficient thyroid glands in comparison to WT controls (Fig. 6C, right panel). Thus, the data confirmed enhanced biogenesis of endo-lysosomal cathepsins B, D, and L in Lat2−/− thyroid tissue, comparable to the Ctsk−/−/Mct8−/y/Mct10−/− animals (Venugopalan et al. 2021a).
Next, we investigated whether Lat2 deficiency affects the subcellular localization of cathepsins. Immunofluorescence analysis of thyroid cryo-sections from Lat2−/− and WT mice revealed comparable subcellular localization of cathepsins B, D, and L mainly to endo-lysosomal compartments (Fig. 7A, B, C, D, E and F, arrows) and within the lumen of some follicles (asterisks). However, the fluorescence signal intensities of the immunostainings were enhanced in Lat2−/− mice, thereby confirming the immunoblotting results. Furthermore, cathepsin-harboring vesicles appeared enlarged in Lat2−/− thyroid sections in comparison to WT controls (Fig. 7A, B, C, D, E and F, right panels), as expected under conditions of induced autophagy and as reported before for the Ctsk−/−/Mct8−/y/Mct10−/− animals (Venugopalan et al. 2021a).
The upregulation of cathepsin protein amounts is indicative of autophagy-triggered lysosomal biogenesis in Lat2 deficiency. Since an increase in the sizes of cathepsin-positive vesicles could also be explained by an impaired lysosomal function that consequently leads to an accumulation of lysosomal substrates as observed in lysosomal storage disorders, it became necessary to determine whether lysosomal activity is increased in Lat2-deficient thyroid tissue, too.
The amount of active cysteine peptidases is enhanced in Lat2-deficient thyroid glands
It is known that autophagy induction and subsequent autophagosome-lysosome fusion results in increased acidification and proteolytic activity of lysosomal proteases, thereby supporting enhanced autophagic flux (Yim & Mizushima 2020). Therefore, in order to study whether the amounts of active proteases are altered in autophagy-triggered Lat2-deficient thyroid glands, thyroid tissue lysates were prepared from Lat2−/− and WT mice in lysis buffer containing the biotin-conjugated activity-based probe DCG-04, which exclusively binds to proteolytically active cysteine peptidases in an equimolar manner (Greenbaum et al. 2000). Samples separated on horizontal gradient SDS-gels and transferred onto nitrocellulose membrane were subsequently incubated with HRP-conjugated streptavidin to detect proteases that were accessible to DCG-04. In accordance with previously published results (Venugopalan et al. 2021a), high molecular mass bands representing endogenous biotinylated proteins were observed above those denoting DCG-04-labeled, that is proteolytically active cysteine cathepsins (Fig. 8A). Blots were analyzed by densitometry and by normalizing DCG-04-recognised bands to total protein per lane determined by Ponceau staining (Fig. 8B).
Results revealed that Lat2 deficiency led to a significant two-fold increase in the amounts of DCG-04 reactive cysteine peptidases compared to WT controls, further confirming autophagy induction and pointing toward increased lysosomal activity in Lat2−/− mice.
Discussion
The results of this study suggest that it is the insufficient Lat2 protein function that induces autophagy in Ctsk−/−/Mct8−/y/Mct10−/− mice. Accordingly, autophagy induction subsequently causes enhanced biosynthesis of endo-lysosomal proteins and increased endo-lysosomal protease activities, thereby exacerbating the already drastic dysregulation of proteostasis within thyrocytes in this mouse model of non-canonical thyroid auto-regulation (Weber et al. 2017, Venugopalan et al. 2021a). Therefore, we propose that enhanced cathepsin-mediated Tg degradation and, possibly, altered Lat2 mRNA translation efficiency, along with the absence of Mct8 and Mct10, causes the observed severe self-thyrotoxicity in Ctsk−/−/Mct8−/y/Mct10−/− mice. Lat2 mRNA in the murine thyroid amounts to only 0.5% of that of Mct8 transcripts (Di Cosmo et al. 2010). However, the data of this study suggest that Lat2 functionality is important to maintain thyroid homeostasis in mice by non-canonical means of thyroid auto-regulation.
Significance of balanced Tg degradation and TH export from thyroid follicles
Tg degradation for TH liberation is enabled by cathepsins before TH are exported from thyroid follicles into the bloodstream by different TH transporters such as Mct8. The functional link between cathepsins and TH transporters has been evidenced in our previous studies, whereby murine models lacking cathepsins show increased TH transporter protein amounts, while mice lacking TH transporters show increased protein amounts and activities of cathepsins in a non-classical pathway of thyroid gland regulation (Weber et al. 2017, Venugopalan et al. 2021a, b). However, the molecular mechanism behind this functional link was not fully understood.
Recently, we have demonstrated that mice lacking TH transporters Mct8 and Mct10 in a cathepsin K-deficient background persistently degrade Tg, leading to increased TH liberation despite impaired TH export and, consequently, toxic intrathyroidal accumulation of TH in these animals results in thyrocyte death and altered thyroid gland morphology (Venugopalan et al. 2021a,b). This detrimental thyroid phenotype in Ctsk−/−/Mct8−/y/Mct10−/− mice is caused by autophagy-induced biogenesis of lysosomal proteins, which comprise cathepsins (Venugopalan et al. 2021a). These results opened the question of whether TH transporters potentially behave as ‘transceptors’, that is transporters that act dually as receptors and also relay nutrient concentrations, to involve in signaling pathways (Hundal & Taylor 2009, Van Zeebroeck et al. 2009, Diallinas 2017). Hence, we hypothesized that in the thyroid gland, TH transporters possibly contribute to the regulation of cathepsin-mediated Tg proteolysis albeit in a counter-intuitive fashion. An absence of TH transporters mediating TH export from the follicles would fail to shut down Tg-processing and could lead to persistent TH liberation.
Lat2 has been suggested as a potential nutrient-driven transceptor in specific tissues, for example, in rat parietal epithelial cells of the renal glomerulus, whereby amino acid transport by Lat2 regulates the mTORC1 complex (Kurayama et al. 2011). The downstream effects of the mTORC1 signaling pathway include induction of anabolic processes like general transcription capacity, ribosome biogenesis, and global protein synthesis for cell growth while limiting catabolic processes such as autophagy (Deleyto-Seldas & Efeyan 2021). Mechanistically, mTORC1 activation is assumed to occur through Lat2-mediated leucine import into the cell and simultaneous efflux of Lat2’s intracellular substrates (Nicklin et al. 2009, Gallagher et al. 2016).
Because TH as iodothyronines represent amino acid derivatives, we propose that Lat2-mediated amino acid and TH transport across endo-lysosomal membranes might be involved in regulating autophagy in the thyroid, depending on intrathyroidal cytosolic TH concentrations or nutrient states. Thus, in thyroid glands of WT mice, euthyroid TH status and normal amino acid transport by Lat2 would lead to mTORC1 activation, that is autophagy pathways are not activated. In contrast, diminished Lat2-mediated transport would render mTORC1 inactive and induce autophagy. Therefore, we propose Lat2 acts as a putative endo-lysosomal amino acid- and TH-driven transceptor, that is Lat2 mediates transport of amino acids and TH that arise from Tg degradation by cathepsins within endo-lysosomes, and Lat2 relays its transport activity through mTORC1. As long as amino acids and TH are generated by Tg proteolysis in a regular manner, Lat2 does not activate autophagy. However, in the absence or when Lat2 function is compromised, the extent of Tg degradation is no longer sensed and autophagy is induced. This effect would be independent of fully operational T4 export by, for example, Mct8, and hence, would add to the severe intrathyroidal thyrotoxicity caused by reduced or absent TH transport across the basolateral plasma membranes of thyrocytes (see Fig. 9).
Such a scenario of amino acid transport at the lysosomal membrane influencing mTOR activity has been suggested earlier for amino acid transporter Lat1 (Milkereit et al. 2015). As the related Lat1 and Lat2 are expressed in murine thyroid tissue (Di Cosmo et al. 2010), it is possible that thyroidal transceptors exhibit functional redundancy. Hence, in the absence of Lat2 or upon its down-regulation as observed in this study for the triple-deficient mice, Lat1 may compensate for reduced Lat2 function. The role of Lat1 in the thyroid gland needs to be addressed in future experiments, for example, in mice lacking Lat1 in the thyroid gland, only.
While it would indeed be interesting to study thyroid Lat1 in more detail, several lines of evidence argue against redundancy among the amino acid and TH transporters in mice. Deficiency in the main thyroidal TH transporter Mct8 does not alter transcript levels of either Mct10, Lat1 or Lat2 (Di Cosmo et al. 2010). Furthermore, Mct8 deficiency shows a drastic thyroid phenotype (Trajkovic-Arsic et al. 2010, Wirth et al. 2011), generally arguing against functional compensation, that is redundancy among TH-transporting molecules. Similarly, no signs of TH transporter redundancy were observed in the triple-deficient murine model used in this study (Venugopalan et al. 2021a). Thus, although Lat1 is approximately twice as abundant as Lat2 in murine thyroid tissue (Di Cosmo et al. 2010), Lat2 bears a specific function acting as a transceptor in self-regulation of murine thyroid function (this study, see Fig. 9).
Significance of Lat2 for thyroid epithelial cell physiology
Our previous studies already indicated that Lat2−/− mice do not show any systemic alterations in thyroid function because the serum TH and TSH concentrations remain normal, suggesting that thyroidal Lat2 is possibly dispensable for canonical thyroid regulation (Braun et al. 2011b). However, exploring Lat2’s contributions in regulating autophagy in the thyroid gland, as shown in this study, unravels an important role of Lat2 in non-canonical thyroid auto-regulation. At the transcriptional level, lysosomal biogenesis is induced by nuclear localization of transcription factor EB (TFEB) and its subsequent binding to a 10-base-pair motif (GTCACGTGAC) known as the coordinated lysosomal expression and regulation (CLEAR) element, which is present in the promoter regions of genes encoding lysosomal proteins, including cathepsins (Sardiello et al. 2009). Therefore, not unexpectedly, thyroid tissue from Lat2-deficient mice showed upregulation of cathepsins B, D, and L. Furthermore, cathepsin-positive vesicles were enlarged in Lat2−/− thyroid cryo-sections when compared to WT controls, confirming autophagy initiation and induction of lysosomal biogenesis. Interestingly, similar results regarding autophagy induction, increased sizes of endo-lysosomal compartments, and cathepsin activities were previously observed also for Ctsk−/−/Mct8−/y/Mct10−/− mice (Venugopalan et al. 2021a). Thus, taken together, we propose endo-lysosomal enlargement in Lat2−/− thyrocytes is promoted by the loss of Lat2 transport function at their endo-lysosomal membranes.
Conclusions and perspectives
Cumulatively, the results of this and previous studies (Braun et al. 2011b, Weber et al. 2017, Venugopalan et al. 2021a, b) suggest that diminished substrate transport across endo-lysosomal membranes due to reduced Lat2 protein amounts fails to activate mTORC1, consequently activating autophagy. As a direct consequence of autophagy induction, the biogenesis of lysosomal proteins (i.e. cathepsins) results in excessive Tg degradation and TH liberation (Venugopalan et al. 2021a). The lack of TH export from the triple-deficient thyroid follicles due to lack of Mct8 and Mct10 worsens the phenotype, thereby leading to increased intrathyroidal TH accumulation, thus furthering thyrotoxic stress-induced autophagy in Ctsk−/−/Mct8−/y/Mct10−/− animals (see Fig. 9).
It is of note that the thyroid status of Lat2−/− mice is characterized by normal TH concentrations in blood serum and the canonical HPT-axis regulation is not affected in these animals (Braun et al. 2011b). Hence, we propose that the presence of functional Mct8 and/or Mct10 maintains TH export from thyroid follicles in Lat2−/−, but clearly not in Ctsk−/−/Mct8−/y/Mct10−/− mice where insufficient Lat2 protein amounts lead to autophagy induction.
Studies addressing the signaling potential of Lat2 in thyrocytes, in particular regarding the mTORC1 pathway and lysosomal activity, would be the next logical step in experimentation to reach a more comprehensive understanding of the regulatory capabilities promoted by amino acid and TH transporters in the thyroid gland. We suggest to use thyroid epithelial cell culture models to perform transient Lat2 inhibition in future studies to unravel the mechanism of autophagy induction in a step-wise manner in induced pluripotent stem cell-derived thyroid organoids or 3D cultures of thyroid epithelial cells (Kopp et al. 2015, Antonica et al. 2017).
This study shows that it is essentially important to decipher molecular mechanisms underlying canonical and non-canonical thyroid regulation by the use of murine models (Brix et al. 2020) notwithstanding that it is highly unlikely to observe a thyroid infliction in humans which would reflect deficiencies in TH-liberating and TH-transporting molecules like cathepsin K, Mct8, and Mct10, further combined with reduced Lat2 transceptor function, as studied herein.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-22-0060.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the Deutsche Forschungsgemeinschaft (DFG), Germany, in the framework of the priority program SPP 1629/1 and 2 ‘Thyroid Trans Act,’ in particular, BR1308/11-1 and 11-2 to K B, HO 2140/6-2 to G H, SCHW914/6-1 to U S, and HE 3418/7-1, and 7-2 to H H. The laboratory of F V was supported by Swiss National Science Foundation grant #31_166430/1. The laboratory of J K was funded by DFG: KI-1988/5-1 and KI-1988/7-1. The funding to the laboratory of E K W was given by Charité-Universitätsmedizin Berlin to E K W (Rahel-Hirsch stipend).
Ethics statement
Animal studies were performed in accordance with the regulations in S1 laboratories of Jacobs University Bremen (Senatorin für Gesundheit, Frauen und Verbraucherschutz der Hansestadt Bremen, Germany, Az. 513-30-00/2-15-32 including K B as project leader, and Az. 0515_2040_15 to K B authorizing animal breeding, housing and sacrifice of genetically engineered mice).
Author contribution statement
Conceptualization, V V and K B; Methodology, V V, M R, J G, V R, G H and K B; Formal Analysis, V V, M R, J W, L R, J G, V R; Investigation, V V, M R, J W, L R, J G, V R; Resources, U V, F V, J K, H H, U S, E K W and K B; Data curation, V V and M R; Writing – original draft preparation, V V and K B; Writing – review and editing, V V, M R, J W, L R, A A H, J G, V R, G H, U V, F V, J K, H H, U S, D B, E K W and K B; Visualization, V V and K B; Supervision, M R, H H, E K W and K B; Funding Acquisition, K B, G H, U S, H H, E K W. All authors have read and agreed to the submitted version of the manuscript.
Acknowledgements
The authors thank Prof Dr. Matthew S. Bogyo (Department of Pathology, Stanford University School of Medicine, USA) for his generous sharing of activity-based probe DCG-04 used in immunoblotting experiments. The authors are indebted to Thomas Ströhlein (Jacobs University Bremen, Germany) for his excellent support in animal keeping.
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