Cholesterol modulates type I/II TGF-β receptor complexes and alters the balance between Smad and Akt signaling in hepatocytes

Abstract

Cholesterol mediates membrane compartmentalization, affecting signaling via differential distribution of receptors and signaling mediators. While excessive cholesterol and aberrant transforming growth factor-β (TGF-β) signaling characterize multiple liver diseases, their linkage to canonical vs. non-canonical TGF-β signaling remained unclear. Here, we subjected murine hepatocytes to cholesterol depletion (CD) or enrichment (CE), followed by biophysical studies on TGF-β receptor heterocomplex formation, and output to Smad2/3 vs. Akt pathways. Prior to ligand addition, raft-dependent preformed heteromeric receptor complexes were observed. Smad2/3 phosphorylation persisted following CD or CE. CD enhanced phospho-Akt (pAkt) formation by TGF-β or epidermal growth factor (EGF) at 5 min, while reducing it at later time points. Conversely, pAkt formation by TGF-β or EGF was inhibited by CE, suggesting a direct effect on the Akt pathway. The modulation of the balance between TGF-β signaling to Smad2/3 vs. pAkt (by TGF-β or EGF) has potential implications for hepatic diseases and malignancies.

Fig. 1. TβRI and TβRII form ligand-independent complexes which are enhanced by TGF-β1.

AML12 cells were transfected with expression vectors encoding myc-TβRI, HA-TβRII, or both. When a single construct was transfected, the second one was replaced by an empty vector. After 24 h, they were serum-starved (30 min) and subjected to fluorescent labeling by monovalent Fab’ fragments (myc-TβRI) and/or IgG patching/cross-linking (CL) (HA-TβRII) as described under “Methods”. The patching/CL protocol results in HA-TβRII patched and labeled by Alexa 488-GαR IgG (designated “IgG αHA”), and myc-TβRI labeled by monovalent Fab’ (murine αmyc Fab’ followed by Alexa 546-GαM Fab’). In control experiments without crosslinking, the HA-TβRII was labeled by rabbit αHA Fab’ and Alexa 488-GαR Fab’. Where indicated, TGF-β1 (100 pM) was added during the last fluorescent labeling step, and maintained throughout the measurement. FRAP studies were conducted as described under “Methods”. Solid lines represent the best-fit of non-linear regression analysis to the lateral diffusion equation (“Methods”). Representative FRAP curves of singly-expressed myc-TβRI (a), myc-TβRI coexpressed with Fab’-labeled HA-TβRII (b), HA-TβRII immobilized by IgG crosslinking (c), and myc-TβRI coexpressed with IgG-crosslinked HA-TβRII (d). Average R_f (e) and D (f) values of multiple FRAP measurements. Bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Some of these numbers are lower for the D values, since accurate D values cannot be derived from FRAP curves with recovery below 20%. Asterisks indicate significant differences between the R_f values of the pairs indicated by brackets (*p < 0.05; **p < 10⁻³; ****p < 10⁻⁵; one^-way ANOVA and Bonferroni post hoc test). No significant differences were observed between the D values.

Fig. 2. Effect of cholesterol depletion or enhancement on the cholesterol content of AML12 cells.

The cholesterol (chol.) level in AML12 cells was reduced (cholesterol depletion; CD) by treatment with lovastatin and mevalonate (50 μM each) in medium containing 10% LPDS (for control samples, 10% FCS), or elevated (cholesterol enrichment; CE) by incubation with cholesterol-MβCD complex (5 mM MβCD, 300 μg/ml cholesterol) in complete growth medium for 16 h, followed by 30 min serum starvation under the same conditions (see “Methods”). The level of free (non-esterified) and total cholesterol was determined using the Abcam cholesterol assay kit (“Methods”). In each experiment, the values obtained in untreated (control) cells were taken as 100%. a Cholesterol depletion. Data are mean ± SEM of four independent experiments. b Cholesterol enrichment. Bars are mean ± SEM of six independent experiments. The results show similar levels of reduction (a) or elevation (b) in total and free cholesterol. c Effect of statin-mediated CD on free cholesterol level at the plasma membrane. After the CD treatment, the membrane cholesterol was extracted by short incubation with a high level of MβCD (30 mM, 30 min, 37 °C), and the free cholesterol level was assayed. Data are mean ± SEM of four independent experiments. d Effect of CE on the free plasma membrane cholesterol. The CE treatment was followed by extraction of the membrane cholesterol by a short exposure to MβCD as described in (c). Data are mean ± SEM of three independent experiments. Asterisks indicate significant differences between the pairs indicated by the brackets. ns not significant. In (a, b), significance was calculated using Student’s two-tailed t test. In (c, d), significance was evaluated using one-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3. Cholesterol depletion disrupts TβRI/TβRII preformed but not ligand mediated complexes.

At 6 h post-transfection with myc-TβRI alone or together with HA-TβRII as in Fig. 1, AML12 cells were subjected to CD (16 h; see “Methods”), serum-starved for 30 min, and taken for patch/FRAP studies (the patching/labeling protocol is described under “Methods” and in Fig. 1). Where shown (CL: IgG αHA), HA-TβRII was immobilized by IgG-CL. Where indicated (dark orange bars), 100 pM TGF-β1 were added during the last fluorescent antibody labeling step of the patch/FRAP protocol, and maintained throughout. The lateral mobility of Fab’-labeled myc-TβRI was measured by FRAP. a Average R_f values; b Average D values. Bars are mean ± SEM; the number of measurements (on different cells) is depicted within each bar. Of note, the reduction in R_f of myc-TβRI upon coexpression with HA-TβRII with or without immobilization of HA-TβRII, which was seen in untreated cells in the absence of ligand (Fig. 1e, white bars), was completely abolished by CD (a, light orange bars). On the other hand, addition of TGF-β1 fully restored the reduction in myc-TβRI R_f values by HA-TβRII coexpression and/or CL (a, dark orange bars), yielding results similar to those obtained for untreated cells in the presence of ligand (Fig. 1e, black bars). No significant differences were observed between the D values of myc-TβRI under all conditions (b). These results indicate that the TβRI/TβRII PFCs are disrupted by the CD treatment, but TGF-β1-mediated complex formation is unperturbed. Asterisks indicate significant differences between the R_f values of the pairs indicated by brackets (**p < 10⁻³; ****p < 10⁻⁵; one-way ANOVA followed by Bonferroni post hoc test).

Fig. 4. Cholesterol enrichment enhances TβRI/TβRII preformed complexes.

AML12 cells were transfected with myc-TβRI alone or together with HA-TβRII. They were then labeled with fluorescent Fab’ and/or IgG and subjected to patch/FRAP studies exactly as in Fig. 3, except that the cholesterol-altering treatment employed was CE (5 mM MβCD complexed with 300 μg/ml cholesterol, 16 h; see “Methods”). CL: IgG αHA designates experiments where HA-TβRII was immobilized by IgG. Patch/FRAP experiments were conducted either without ligand (light blue bars) or with 100 pM TGF-β1 (dark blue bars), added during the last fluorescent antibody labeling step and maintained throughout. The lateral mobility of Fab’-labeled myc-TβRI was measured by FRAP. a Average R_f values; b Average D values. Bars are mean ± SEM; the number of measurements (each conducted on a different cell) is given within the bars. The dotted line in (a) depicts R_f obtained for myc-TβRI coexpressed with IgG crosslinked HA-TβRII in untreated cells (taken from Fig. 1e, third bar from the left, as indicated in the margin to the right). The further reduction in R_f of myc-TβRI upon CE (third light blue bar in a) suggests that CE enhances the formation of TβRI/TβRII PFCs. These stronger interactions could mask additional incremental changes in R_f in the presence of ligand, as suggested by the similar differences between the R_f values obtained upon incubation with TGF-β1 (a, dark blue bars). No significant differences were found between D values of myc-TβRI under all conditions (b). Asterisks indicate significant differences between the R_f values of the pairs indicated by brackets (**p < 10⁻³; ****p < 10⁻⁵; one-way ANOVA followed by Bonferroni post hoc test).

Fig. 5. Cholesterol depletion reduces basal pSmad2/3, while cholesterol enrichment enhances TGF-β1-mediated pSmad2/3 formation at long stimulation times.

AML12 cells were subjected (or not; control) to cholesterol depletion (CD) or enrichment (CE) treatments for 12 h as detailed under cholesterol treatments for signaling studies (“Methods”). After treatment, they were serum-starved (16 h) in the same medium used for the specific treatment (with 0.5% FCS for control or CE, or 0.5% LPDS for CD). The cells were then incubated without or with TGF-β1 (100 pM) for the indicated times, lysed, and analyzed by immunoblotting for phospho Smad2/3 (pSmad2/3), total Smad2/3 (tSmad2/3) and β-actin. The bands were quantified by ECL and densitometry. a A representative blot of pSmad2/3 formation in AML12 cells without (zero time) and with stimulation by 100 pM TGF-β1 for the indicated times. b–e Quantification of TGF-β1 signaling to pSmad2/3 relative to tSmad2/3. b–e depict stimulation for 0, 5, 20 and 45 min. Data are mean ± SEM of 12 independent experiments. The value obtained for TGF-β1 stimulation of untreated cells at 45 min was taken as 100. Asterisks indicate significant differences between the indicated pairs, using one-way ANOVA followed by Bonferroni post hoc test. Comparison of the different treatments at the same time points demonstrated a significant reduction in the basal (time 0) pSmad2/3 levels following CD, but not CE (b). This reduction was still noticeable at 5 and 20 min (c, d; *p < 0.05). On the other hand, CE treatment did not lead to significant differences from untreated cells at 0 or 5 min, but enhanced pSmad2/3 at 20 and 45 min (d, e; *p < 0.05, **p < 0.01). Of note, comparison of each specific treatment at different time points (using ANOVA on all bars together to compare all the white bars, orange bars, and blue bars at different time points) demonstrated significant stimulation at 20 and 45 min under all conditions (for simplicity, only the statistics for the 45 min point are shown; ****p < 10⁻⁵). ns not significant.

Fig. 6. Cholesterol depletion enhances TGF-β1-mediated pAkt formation at 5 min and accelerates its loss at longer times, whereas cholesterol enrichment inhibits pAkt formation.

AML12 cells were subjected (or not; control) to CD or CE followed by serum starvation exactly as in Fig. 5. The cells were then incubated ± TGF-β1 (100 pM) for the indicated times, lysed, and analyzed by immunoblotting for pAkt (pSer473 or pThr308), total Akt (tAkt) and β-actin. The bands were quantified by ECL and densitometry. a, b Representative blots of pAkt (pS473) formation (a) and of pAkt (pT308) formation (b). ns non specific band. c–f Quantification of TGF-β1 signaling to pAkt (pS473) relative to tAkt. c–f depict stimulation for 0, 5, 20 and 45 min. Data are mean ± SEM of 12 independent experiments. The values obtained for TGF-β1 stimulation of untreated cells at 5 min were set to 100. g–j Quantification of TGF-β1 signaling to pAkt (pT308) relative to tAkt. Data are mean ± SEM of six independent experiments. pT308 levels of TGF-β1 stimulated untreated cells at 5 min were set to 100. Asterisks indicate significant differences between the indicated pairs, using one-way ANOVA followed by Bonferroni post hoc test. Comparison between the different treatments at the same time points demonstrated a significant increase in pAkt following CD at 5 min (d, h; *p < 0.05; ***p < 0.001), followed by accelerated loss at 20 and/or 45 min (compare orange bars in d with e and f, and in h with i and j; **p < 0.01, ***p < 0.001; ****p < 10⁻⁵). Conversely, CE treatment led to inhibition of pAkt formation at all time points (compare blue bars in c with d–f, and in g with h–j; *p < 0.05; **p < 0.01, ***p < 0.001). Comparison of each specific treatment at different time points (using ANOVA on all bars together, as in Fig. 5) demonstrated a high TGF-β-mediated phosphorylation of Akt on pS473 at 5 min, with lower but still significant stimulation at 20 min for untreated or CD-treated (but not CE-treated) cells (for simplicity, only the statistics for the 5 min point are shown; ****p < 10⁻⁵). Similar comparison for Akt phosphorylation on pT308 showed also for untreated cells a high level of pT308 at 5 min, which was lower but still significant at 20 min. In CD-treated cells, only the time point at 5 min was significant due to faster loss of pT308, which was evident at 20 min. CE treatment abrogated the TGF-β-mediated pT308 at all time points, as in the case of pS473. For simplicity, only the statistics for the 5 min points are shown; ****p < 10⁻⁵).

Fig. 7. EGF-mediated pAkt formation is affected by cholesterol depletion or enrichment similar to TGF-β-induced pAkt formation.

Experiments were conducted on AML12 cells as in Fig. 6, except that stimulation was with 10 nM EGF. Cells were then lysed, immunoblotted for pAkt (pSer473 or pThr308), tAkt and β-actin. Representative experiments of pAkt (pS473) formation in response to EGF (a) and of pAkt (pT308) formation (b). ns non specific band. c–f Quantification of EGF-stimulated pAkt (pS473) formation. c–f depict stimulation for 0, 5, 20 and 45 min. Data are mean ± SEM of five independent experiments. The values obtained for EGF stimulation of untreated cells at 5 min were taken as 100. g–j Quantification of EGF signaling to pAkt (pT308). Data are mean ± SEM of eight independent experiments. pT308 levels of EGF stimulated untreated cells at 5 min were set to 100. Asterisks indicate significant differences between the indicated pairs, using one-way ANOVA followed by Bonferroni post hoc test. Comparison between cholesterol treatments at the same time points showed a significant increase in pAkt (both pS473 and pT308) mediated by CD at 5 min (d, h; **p < 0.01; ****p < 10⁻⁵), followed by accelerated loss at 20 and/or 45 min (compare orange bars in d with e and f, and in h with i and j; *p < 0.05; **p < 0.01; ***p < 0.001). Conversely, CE treatment partially inhibited EGF-induced pAkt formation at all time points (compare blue bars in c with d–f, and in g with h–j; *p < 0.05; **p < 0.01). Comparison of each treatment at different time points (using ANOVA on all bars together) showed a high EGF-mediated pS473 formation in untreated cells at all time points. For CD-treated cells, the pS473 level was high at 5 min, becoming lower but still significant at 20 min. Analogous comparison for EGF-mediated pT308 yielded a similar pattern for both untreated cells and the effects of CD and CE at all time points. For simplicity, only the statistics for the 5 min points are shown (***p < 10⁻³; ****p < 10⁻⁵).

Fig. 8. Stimulation of pErk1/2 by EGF persists following cholesterol enrichment.

Studies were conducted on AML12 cells as in Fig. 7. After stimulation with EGF (10 nM), the cells were lysed and immunoblotted for pErk1/2, tErk1/2 and β-actin. a A representative immunoblot. b Quantification of EGF-stimulated pErk1/2 formation relative to tErk1/2. Panels i, ii, iii, iv depict stimulation for 0, 5, 20 and 45 min. Data are mean ± SEM of five independent experiments. The values obtained for EGF stimulation of untreated cells at 5 min were taken as 100. Asterisks indicate significant differences between the indicated pairs, using one-way ANOVA followed by Bonferroni post hoc test. Comparison of untreated, CD- and CE-treated cells at the same time points demonstrated a significant increase in the basal (time 0; unstimulated) pErk1/2 levels following either CD or CE (bi; *p < 0.05, **p < 0.01), and at 5 min stimulation following CD (bii; **p < 0.01). Of note, CE treatment had no significant effect (ns) on EGF-mediated pErk1/2 formation at all time points.

Fig. 9. Schematic representation of the effects of cholesterol on the balance between TGF-β signaling to pSmad2/3 vs. pAkt.

The effects of CD (orange) or CE (blue) on TGF-β- or EGF-mediated levels of the indicated pathways at early (5 min) or late (45 min) stimulation time points are depicted as ↑ (increase), ↓ (decrease), or − (no change). The outcomes on EGF-mediated signaling to pAkt are shown as control. For TGF-β, the balance of (pSmad2/3)/pAkt signaling is altered depending on the treatment (CD vs. CE), as well as on stimulation time. Thus, at early times, CD reduces pSmad2/3 and increases pAkt, while at late times, this effect is reversed (pSmad2/3 remains unchanged, while pAkt is reduced). CE has no effect on pSmad2/3 stimulation at early times but reduces pAkt, an effect opposite to that of CD, while at later times it retains the same effect on pAkt and raises pSmad2/3 even higher. For EGF, the effects of CD and CE on pAkt are similar to those of TGF-β.