TSH induce changes in genes related to thyroid specific function, as well as genes involved in the adaptation of the cell in order to work as a thyroid cell. For example, TSH acts on lipid biosynthetic pathways needed for thyrocites proliferation. TSH as well as the cAMP agonist, forskolin, transcriptionally induce the enzime 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR, Aloj et al., 1990), which represent the limiting step in cholesterol synthesis, and the malic enzyme, involved in the synthesis of fatty acids. Similar to the effect mediated by CREB3L1 in response to TSH, it was reported that TSH induces the activation of the sterol regulatory element-binding proteins (SREBPs), which are the main transcriptional modulators the lipid synthesis. In addition, the SREPB-1c and SREBP-2 factors also activate the transcription of genes involved in the synthesis of thyroid hormones, such as NIS, TPO and TG (Rauer et al., 2014; Ringseis et al., 2013).
Based on our results, which indicate that CREB3L1 induces an increase in Golgi volume, and results obtained by other laboratories, we hypothesize that CREB3L1 would participate in the cross-regulatory talk between the lipid and protein biosynthesis pathways necessary to increase the secretory capacity of the cell. To analyze cross-regulatory mechanisms between the lipid and protein biosynthetic pathways, we evaluated the lipid composition of the rat thyroid follicular cell line PCCL3, stimulated with TSH at different times by lipidomics assays, a technique based on high performance liquid chromatography coupled to mass spectrometry. This was done in collaboration with the laboratory of Giovani D'Angelo (Laboratory of Lipid Biology, EPFL, Lausanne, Switzerland). Assays were performed on PCCL3 cells that were incubated under growth conditions for 24 h, subsequently deprived of TSH (starvation conditions) for 72 h, and then stimulated with TSH for 24 h and 48 h. Before performing the lipidomics assays, TSH response was confirmed by Western blot using anit-CREB3L1, GM130 and NIS antibodies (Figure 1).
Figure 1. Western blot analysis of PCCL3 cells stimulated with TSH at different times: Western blot of whole protein extracts from PCCL3 cells. Cells under growth conditions (Ctrl), deprived from TSH for 72 h (0 h) and stimulated with TSH for 24 h and 48 h. Labels on the right side of the Western blot indicate the relative electrophoretic mobility of the corresponding NIS polypeptide according to its glycosylation state: immature glycosylation (~60kDa, Band A), and fully glycosylated (~100kDa, Band B).
Lipidomics assays revealed a great difference in the composition of the lipids studied (Figure 2) in the different conditions. In the absence of TSH, some lipids decrease, such as Diacylglycerols (DAG), Triacylglycerols (TAG), Sphingomyelin (SM), Glucosylceramide (GlcCer) while others increase, such as Phosphatidylcholine (PC), Phosphatidylserine (PS) and Cardiolipins (CL) compared to cells grown under growth conditions (control).
On the other hand, upon TSH stimulation (24 h and 48 h, Figure 2), especially after 48 h, an increase in DAG, TAG and lysobisphosphatidic acid (LBPA), a group of lipids associated with late endosomes, is detected. Furthermore, the group of sphingolipids (SLs) that include ceramides (CER), glucosylceramides (GlcCer) and sphingomyelins (SM) also increases. SLs contribute to Golgi function, participating in signaling, protein transport, and membrane deformation (Halter et al., 2007). SLs such as SM and GlcCer decrease their levels considerably after TSH deprivation, and increase when stimulated with TSH (Figure 2). It stands out that the lipids that decrease are those that increased after TSH deprivation, such as PC, PS and CL.
Figure 2. Lipidomics assays in PCCL3 cells reveal heterogeneous behavior in lipid changes: PCCL3 cells under growth conditions (Ctrl), TSH-deprived for 72 h (0 h), and TSH-stimulated for 24 h and 48 h. Lipids were extracted using methyl-tert-butyl ether (MTBE) as solvent and then introduced into liquid chromatography coupled to mass spectrometry (LC-MS). Heatmap of the dataset obtained containing all the lipids analyzed in an experiment with 3 biological replicates.
Quantitative comparison between TSH (0h) vs TSH (48h) conditions after considering a cut-off 4-fold increase or decrease (Figure 3 A) confirmed what was qualitatively shown in Figure 2.
Enrichment analysis using the software LION: "Lipid Ontology Enrichment Analysis” [http://www.lipidontology.com, (Molenaar et al., 2019)] revealed the chemical and functional identity of several lipid metabolic pathways involved in the response to TSH (Figure 3 B and C). Within the classifications associated to chemical structure are neutral head lipids, glycerolipids, triacylglycerols and diacylglycerols represented by the large amount of TAG and DAG increased in this condition. This result agrees with previous reports where it was shown that the increase in TAG is mediated by TSH in adipocytes (Ma et al., 2015). Among the classifications associated with biological functions are "lipid storage" and "lipid droplets", mainly caused by the increase in TAG, which is the main component of these structures that function as energy reservoirs within the cell. In addition, the metabolic pathways related to hexosylceramide, the precursor of several sphingolipids, are also enriched against stimulation with TSH (Figure 3 B), although to a lesser extent.
On the other hand, among the lipid metabolic pathways that decrease in response to TSH, those related to phospholipids such as PC and PS are represented (Figure 3 C). The chemical classifications of the lipids involved are: headgroup with positive charge, and glycerophosphocholines and diacylglycerophosphocholines, precursors of PC and PS, among others. The main classification that involves biological characteristics is ER (endoplasmic reticulum) due to the large number of phospholipids involved in the structure of this organelle (van Meer et al., 2008). Finally, several pathways related to physicochemical properties that refer to the role of phospholipids in membrane composition and function (such as neutral intrinsic curvature, low transition temperature, high lateral diffusion) (Figure 3 C) were also identified.
Figure 3. TSH-regulated metabolic pathways: (A) “Volcano plot” type representation representing the “Heatmap” data from Figure 2. A fold change (fc) cut-off of 4, or log2 fc of + /- 2 was set to identify those lipids whose changes were significant. (B and C) Lipid Ontology Analysis of lipids whose changes were significant using the LION software (http://www.lipidontology.com).
To corroborate the effect of TSH on sphingolipid synthesis within the cell, we performed immunofluorescence analyzes in TSH-stimulated PCCL3 cells to identify two sphingolipids representative of different metabolic pathways: GM1, a ganglio-series sphingolipid whose function is related to cell differentiation (Russo et al., 2018), and SM, mainly involved in intracellular trafficking processes (Hannun & Obeid, 2018). Cholera toxin subunit B (isolated from Vibrio cholerae) and echinotoxin-II (isolated from the sea anemone Actinia equina) were used for the specific labeling of GM1 and SM, respectively (Russo et al. al., 2018). It was observed that, compared to cells cultured in growth medium, TSH deprivation for 72 h, TSH (0 h), increases the fluorescence signal of GM1 (Cholera Tx, Figure 4), while, in the case of SM, labeled with echinotoxin-II (Equina Tx), the opposite happens. In contrast, after stimulation with TSH, a decrease in the fluorescence intensity of Cholera Tx, and an increase in Equina Tx signal are observed (Figure 4). The increase in SM levels could be a consequence of the increase in Golgi volume induced by TSH stimulation. These results were corroborated using flow cytometry in cells deprived from TSH for 72 h and then stimulated for 24 h. As can be seen in Figure 5, TSH deprivation for 72 h causes an increase in GM1 levels (-TSH, Cholera Tx, Figure 5), as well as a decrease in SM (-TSH, Equina Tx, Figure 5) . In agreement with the results of the confocal images, the stimulation with TSH for 24 h produces a slight decrease in the levels of GM1 (+TSH, Cholera Tx, Figure 5), while no changes were observed in the levels of SM (+TSH , Equine Tx, Figure 5).
Figure 4. Immunofluorescence analysis of GM1 and SM expressed in TSH-stimulated PCCL3 cells: Confocal analysis of PCCL3 cells under growth conditions (Ctrl), deprived from TSH for 72 h (0 h) and stimulated with TSH for 24 h and 48 h, stained with cholera toxin (Cholera Tx, green) and echinotoxin II (Equina Tx, cyan). The nuclei were labeled with Hoechst 33258.
Figure 5. Flow cytometry (FACS) analysis ic PCCL3 cells stimulated with TSH: Analysis by flow cytometry of PCCL3 cells of cells stained with Equina Tx (SM) and Cholera Tx (GM1) under growth conditions (Control ), deprived of TSH for 72 h (-TSH 72 h) and stimulated with TSH for 24 h (+ TSH 24 h).
To evaluate whether the production of SM, and sphingolipids as a whole, is necessary for the adaptation of the thyroid cell to TSH stimulation, the effect of the inhibition of sphingolipid synthesis in this process was analyzed. For this, two specific inhibitors that act at different stages of the metabolic pathway of sphingolipid synthesis were used (Figure 6): Myriocin, a serine palmitoyltransferase (SPT) inhibitor, virtually blocks the synthesis of all sphingolipids by inhibiting the production of CER (Hojjati et al., 2005); and PDMP, an inhibitor of ceramide glucosyltransferase (GCS), blocks the synthesis of GlcCer, and therefore of all the glycosylated sphingolipids (glycosphingolipids) derived from it. PDMP also causes accumulation of SM and Galactosylceramide.
Figure 6. Representative scheme of the sphingolipid synthesis metabolic pathway. Adapted from van Etchen et al. 2018
PCCL3 cells were incubated for eight days with the inhibitor, PDMP or Myriocin, and from the fifth day of incubation the scheme of deprivation and 24 h stimulation with TSH was started, as previously described (Figure 1), maintaining the presence of the inhibitors throughout the process. As shown in Figure 7, in the absence of inhibitors (lines 1-4), CREB3L1 and NIS levels decrease after TSH deprivation, and increase upon the stimulus at 24 h and 48 h post TSH (Figure 7, lines 1 – 4). In the presence of PDMP, TSH induced a very mild activation of CREB3L1 without modifying NIS levels (Figure 7, lines 5-6). In addition, incubation with Myriocin prevented both CREB3L1 activation and TSH-mediated increase in NIS (Figure 7, lanes 7-8). These results agree with previous publications where it was reported that, in tumor cells, the activation of CREB3L1 induced by Doxorubicin is mediated by ceramide and this effect is blocked by Myriocin (Denard et al., 2012).
Figure 7. Effect of PDMDP and Myriocin inhibitors on TSH-stimulated PCCL3 cells: PCCL3 cells under growth conditions (Ctrl) were incubated in the absence, or presence of PDMP (20 μM) or Myriocin (2.5 μM) by a period of 5 days, then they were deprived of TSH for 72 h (0 h) and stimulated with TSH for 24 h in the presence of the inhibitors.
To assess whether CREB3L1 modulates lipid changes caused by TSH stimulation, we performed inhibition and overexpression assays of CREB3L1, using a specific siRNA for this transcription factor or transfection with a constitutively active version (CREB3L1 CA). PCCL3 cells under growth conditions were transfected with siCREB3L1 and siScramble as control (Figure 8 A), and CREB3L1 CA or pcDNA 3.1 as control (Figure 8 B). Here, the cells were not subjected to a TSH deprivation/stimulation process. As seen in Figure 8 A, the inhibition of CREB3L1 using a specific siRNA produced a slight decrease in this transcription factor, with minimal changes in GM130 and NIS, proteins positively regulated by CREB3L1. CREB3L1 CA transfected cells exhibit higher levels of CREB3L1 than pcDNA transfected cells, and higher expression levels of GM130 and NIS are also detected.
Figure 8. Western blot of PCCL3 cells transfected with siCREB3L1 and CREB3L1 CA: Whole protein extracts of PCCL3 cells incubated in growth conditions, transfected for 72 h with siCREB3L1 or siScramble (A) or CREB3L1 CA or pcDNA (B). Images represent three biological replicates.
Furthermore, as previously shown, CREB3L1 CA overesxpression increase the Golgi volume (arrowheads and inset, Figure 9). These results suggest that lipid metabolism, associated with changes in the Golgi complex, could be modified by the increase in active CREB3L1.
Figure 9. CREB3L1 modulates Golgi volume: Immunofluorescence of PCCL3 cells transfected with CREB3L1 CA or pcDNA for 72 h, stained with anti-CREB3L1 (green) and anti-GM130 (red). Arrowheads in GM130 panels indicate transfected cells with enlarged Golgi phenotype. The nuclei were labeled with Hoechst 33258.
Lipidomics assays performed on cells transfected with siCREB3L1 or CREB3L1 CA (Figure 10), indicate that changes in CREB3L1 expression induce changes in lipids. Cells transfected with siCREB3L1 have higher expression levels of certain PC and LysoPC species (dotted box, Figure 10 A), this is comparable to what occurs in the absence of TSH (Figure 2), where CREB3L1 levels are low. On the other hand, cells transfected with CREB3L1CA display higher levels of TAG, DAG, LBPA, CER, GlcCer and SM compared to cells transfected with pcDNA (dotted line box, Figure 10 B), similar to what occurs upon stimulation with TSH for 48 h.
Figure 10. Effect of changes in CREB3L1 expression on the lipid profile of PCCL3 cells: PCCL3 cells under growth conditions were transfected with siCREB3L1 or siScramble (A), and CREBL31 CA or pcDNA 3.1 (B). Lipids were extracted using methyl-tert-butyl ether (MTBE) as solvent and then introduced into liquid chromatography coupled to mass spectrometry (LC-MS) equipment. “Heatmap” of the data set obtained from an experiment carried out with three biological replicates.
Similarly to what was done previously, we made a comparative analysis of the inhibition and overexpression of CREB3L1 with respect to its corresponding controls (Figure 11 A and 12 A). Different groups of lipids are decreased in cells transfected with siCREB3L1, as this is the case for LBPA and TAG mainly. In addition, some PC species are increased (Figure 11 A). Enrichment analysis of lipids upregulated under this condition revealed a predominance of PC, endoplasmic reticulum lipids and others such as glycerophospholipids (Figure 11 B). In contrast, in lipid species decreased by the inhibition of CREB3L1, the metabolic pathways involved revealed by the analysis show a certain similarity with those found upon stimulation with TSH, such as the lipids of the endo-lysosomal pathway and sphingolipids (Figure 11 C).
Figure 11. Metabolic pathways affected in response to CREB3L1 inhibition: (A) Volcano plot of lipid species in siCREB3L1/si Scramble transfected cells. A log2 fold change cut off of +/- 1 (or fc=2) was selected to identify those lipids whose changes were significant (A). (B and D) Lipid ontology analysis (performed with the LION software) of increased (B) and decreased (C) lipids whose changes were significant.
In CREB3L1 CA transfected cells (Figure 12 A), a predominance in increased lipids is observed with a notable absence of decreased lipids when compared to control cells. Furthermore, increased lipids correlate with decreased lipids in siCREB3L1-transfected cells (Figure 11 A) and with increased lipids upon TSH stimulation by 48 h (Figure 3 A). Among the metabolic pathways revealed by enrichment analysis of increased lipids in cells transfected with CREB3L1 CA, are those related to endosomes/lysosomes, and glycerophospholipids (Figure 12 B), due to the marked increase in LBPA and TAG. These results highlight the importance of CREB3L1 in the reprogramming of lipid metabolism in thyroid cells.
Figure 12. Metabolic pathways affected in response to CREB3L1 CA transfection: (A) Volcano plot of lipid species in CREB3L1 CA transfected cells, pcDNA was used as a control. A fold change (fc) cutoff of 4, or log2 fc of +/- 2, was selected to identify significant changes between lipids. (B) Lipid ontology analysis (performed with LION software) of upregulated lipids whose changes were significant.
3 CREB3L1 modulates the levels of transcription factors responsible for lipid remodeling upon stimulation with TSH
To further study the role of CREB3L1 in TSH-induced lipid metabolism reprogramming, we performed qPCR assays to analyze the levels of the SREBPs transcription factors (SREBP-1C and SREBP-2). The SREBPs have been postulated as master regulators of enzymes required for the synthesis of cholesterol, fatty acids, triacylglycerols, and phospholipids (Eberlé et al., 2004). Among the enzymes that regulate the SREBPs, it has been reported that fatty acid synthase (FAS) and the glycerol-3-phosphate acyltransferase (GPAT) are modulated by SREBP-1c, while SREBP-2 is responsible for coordinating the synthesis of the LDL receptor (LDLR) and HMGCo-A reductase (HMGCR) (Ringseis et al., 2013). For this, FRTL-5 cells were transfected with siCREB3L1, or siScramble as a control, as previously described. After 24 h post-transfection, cells were deprived from TSH for 72 h and then stimulated with TSH for a period of 16 h. As shown in Figure 13 A, upon TSH stimulation, cells transfected with siCREB3L1 have lower expression levels of SREBP-1C, but no differences are observed in SREBP-2, when compared to control cells (si Scramble). Moreover, when analyzed the effect of CREB3L1 inhibition on the genes regulated by SREBP-1C and SREBP-2, CREB3L1 seems to affect only the LDLR, which is regulated by SREBP-2 (Figure 13 B). This puzzling effect may be the result of analysing these genes at the wrong time, where mRNA return to its basal levels. Nevertheless, these results highlight the importance of CREB3L1 as a transcription factor that regulates thyroid cell lipid metabolism at the transcriptional level. Subsequent studies will be necessary to elucidate whether CREB3L1 regulates these genes directly or indirectly.
Figure 13. CREB3L1 inhibition reduces the levels of genes involved in lipid synthesis: FRTL-5 cells were transfected with siScramble or siCREB3L1 for 24 h, then TSH deprived for 72 h and finally stimulated with TSH for 16 h. Quantification of mRNA levels of indicated transcripts by qPCR, using total RNA from FRTL-5 cells transfected with siScramble or siCREB3L1. Results are normalized to β-Actin levels, expressed by the 2-∆∆Ct method relative to levels of TSH-deprived siScramble cells (set as one).
These results show the change in lipid metabolism carried out by the thyroid cell upon TSH stimulation and suggest that CREB3L1 may be playing a key role in this process. From these results it was concluded that:
- In the process of TSH stimulation, the thyroid cell undergoes lipid reprogramming, from a composition rich in certain lipids such as PC, PS and CL in the absence of TSH, to a composition rich in TAG, DAG, LBPA and sphingolipids in the presence of TSH. Consequently, lipids whose levels are high in the absence of TSH decrease with stimulation, and vice versa.
- In particular, in the group of sphingolipids, this reprogramming occurs in such a way that when the cells are deprived of TSH, the levels of the ganglio-sphingolipid GM1 increase and those of SM, in the plasma membrane, decrease. When stimulated with TSH, the cell begins to decrease GM1 levels and increase SM levels. Reprogramming at the sphingolipid levels has been previously described in neuronal differentiation models (Russo et al., 2018).
- The inhibition in the production of ceramide, and therefore of its sphingolipid derivatives, decreases the levels of CREB3L1 and its activation through stimulation with TSH. In addition, it was observed that NIS levels are altered and do not increase in response to TSH stimulation in the presence of inhibitors. Hence, it is concluded that the inhibition of sphingolipid production blocks the normal adaptation of the thyroid cell upon TSH stimulation.
- CREB3L1 regulates lipid production in thyroid cells. The lipids that decrease in CREB3L1 silenced cells, and increase with the expression of CREB3L1 CA, correlate with the increased lipids upon TSH stimulation, as well as the metabolic processes associated with these lipids. In contrast, the lipids that increase in cells where CREB3L1 is silenced bear some degree of similarity to those that increase in TSH-deprived cells.
- CREB3L1 modulates the levels of the SREBP-1C transcription factors, key modulator of the lipid synthesis.