Fibroblast growth factor-21 (FGF21) regulates low-density lipoprotein receptor (LDLR) levels in cells via the E3-ubiquitin ligase Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting saposin-like protein (Msap)

Abstract

The LDLR is a critical factor in the regulation of blood cholesterol levels that are altered in different human diseases. The level of LDLR in the cell is regulated by both transcriptional and post-transcriptional events. The E3 ubiquitin ligase, myosin regulatory light chain-interacting protein (Mylip)/inducible degrader of the LDL-R (Idol) was shown to induce degradation of LDLR via protein ubiquitination. We have here studied novel factors and mechanisms that may regulate Mylip/Idol in human hepatocyte cells and in mouse macrophages. We observed that FGF21 that is present in serum in different conditions reduced Mylip/Idol at the RNA and protein level, and increased LDLR levels and stability in the cells. FGF21 also enhanced expression of Canopy2 (Cnpy2)/MIR-interacting Saposin-like protein (Msap) that is known to interact with Mylip/Idol. Overexpression of Cnpy2/Msap increased LDLRs, and knockdown experiments showed that Cnpy2/Msap is crucial for the FGF21 effect on LDLRs. Experiments using DiI-labeled LDL particles showed that FGF21 increased lipoprotein uptake and the effect of FGF21 was additive to that of statins. Our results are consistent with an important role of FGF21 and Cnpy2/Msap in the regulation of LDLRs in cultured cells, which warrants further studies using human samples.

FIGURE 1.

Cnpy2/Msap increases LDLR levels and decreases Mylip/Idol levels in cells. Human hepatocytes (Huh7) and mouse macrophages (RAW) cell lines were transfected with 4 μg of EGFP or Cnpy2/Msap or shRNA-expressing plasmids and incubated for 24 h. Immunoblots were done as described under “Experimental Procedures” using antibodies against LDLR, Cnpy2/Msap, and Mylip/Idol. β-Actin was used as control. Values are means ± S.D., n = 3. **, p < 0.01 for Msap versus C. A, LDLR levels were increased by Cnpy2/Msap (MSAP) in both cell types as shown by immunoblots (left) and by using quantification (right). B and C, Mylip/Idol levels. Overexpression of Cnpy2/Msap decreased (B), whereas down-regulation of Cnpy2/Msap using shRNA (C) increased Mylip/Idol in Raw cells. Values are means ± S.D., n = 3. *, p < 0.05 for Msap versus GFP transfection. **, p < 0.01 for shRNA-Msap versus C. D, control experiment. Raw cells were transfected with 4 μg of scrambled shRNA (sc), and immunoblotting was done as above. No change in expression levels was observed.

FIGURE 2.

Cnpy2/Msap affects the ubiquitination of Mylip/Idol. N2A cells were transfected with 4 μg V5-Mylip/Idol for 24 h alone or together with 4 μg of Cnpy2/Msap. All cells also expressed GFP-ubiquitin. 5 μm MG132 was added for 4 h to inhibit proteasomes. Immunoprecipitation (IP) was done using anti-V5 antibodies as described under “Experimental Procedures” followed by immunoblotting (IB) using specific antibodies. Note a decrease of Mylip/Idol in Cnpy2/Msap-expressing cells. Ubiquitination of Mylip/Idol was increased by MG132 in control and in Cnpy2/Msap-expressing cells. A typical experiment is shown and was repeated three times.

FIGURE 3.

FGF21 increases LDLR and Cnpy2/Msap in cells. Cells were treated as below. Left, immunoblots; Right, quantification. Values are means ± S.D., n = 3. *, p < 0.05 and **, p < 0.01 for FGF21 versus C. A and B, raw cells were treated with 50 ng/ml FGF21 for different times and LDLR (A) and Cnpy/Msap (B) levels were determined by immunoblots. β-Actin was used as control. LDLR levels. Left, immunoblots; Right, quantification. C and D, Huh7 cells were treated for 24 h using different FGF21 concentrations followed by immunblotting using LDLR (C) and Cnpy2/Msap (D) antibodies.

FIGURE 4.

FGF21 decreases Mylip/Idol and increases Cnpy2/Msap at the RNA level. A, Huh7 cells were treated for 24 h with different concentrations of FGF21 and Mylip/Idol was determined. Left, immunoblots; Right, quantification. Values are means ± S.D., n = 3. **, p < 0.01 for treated versus C. B, quantitative PCR. Mylip/Idol-mRNA levels decreased in Raw cells after treatment with 50 ng/ml FGF21. Values are means ± S.D., n = 4. **, p < 0.01 for treated versus C. C, overexepression of Cnpy2/Msap for 24 h decreased Mylip/Idol-mRNA levels. Values are means ±S.D., n = 3. *, p < 0.05 for treated versus C. D, raw cells were transfected with the Mylip/Idol-promoter luciferase construct and further treated for 24 h with 50 ng/ml FGF21 alone or together with 1 μm GW3695 (GW). Luciferease activity was measured as described and corrected for that of Renilla. Mylip/Idol promoter activity was decreased by FGF21 and was increased by GW. Values are means ± S.D., n = 3. **, p < 0.01 and ***, p < 0.01 for treated versus C. E and F, quantitative PCR. Raw cells were stimulated with 50 ng/ml FGF21and the mRNA levels of Cnpy2/Msap and LDLR determined. Values are means ± S.D., n = 3. *, p < 0.05 and **, p < 0.01 for treated versus C.

FIGURE 5.

FGF21 increases the stability of LDLRs in cells. Raw cells were left untreated (controls) or treated with 50 ng/ml FGF21 for 20h. 30 ng/ml actinomycin-D was then added to block gene transcription, and cells were incubated further for various periods of time. LDLR levels were determined. Note that the control immunoblot shown was exposed longer time to reveal the 24 h-LDLR value. Values are means ± S.D., n = 3. *, p < 005 and **, p < 0.01 for treated versus C. Left, control cells. Calculated half-life of LDLR was about 6 h. Right, in FGF21-treated cells, LDLR was stable for more than 6 h, and the half-life was about 15 h.

FIGURE 6.

Down-regulation of Cnpy2/Msap inhibits the effect of FGF21 on LDLR and lipid uptake. Raw cells were transfected with 4 μg shRNA against Cnpy2/Msap for 24 h and half of the cells were further stimulated for 20 h with 50 ng/ml FGF21 followed by immunoblotting. Values are means ± S.D., n = 3. *, p < 0.05 and **, p < 0.01 for treated versus control. A, Cnpy2/Msap. Left, immunoblots. Right, quantification. Note down-regulation of Cnpy2/Msap by shRNA. The increase in Cnpy2/Msap by FGF21 was blocked by the shRNA. B, LDLR. Left, immunoblots; Right, quantification. Down-regulation of Cnpy2/Msap by shRNA decreased LDLR levels and reduced the increase in LDLR by FGF21. C, control experiment. Cells were transfected with 4 μg of scrambled shRNA (sc), and immunoblotting was done as above. FGF21 increased LDLR levels in sc-treated cells. D, Mylip/Idol down-regulation. Cells were transfected with 4 μg of shRNA against Mylip/Idol. Note a decrease in Mylip/Idol and an increase in LDLR by the shRNA. Treatment with 50 ng/ml FGF21 for 24 h further reduced Mylip/Idol levels. E, lipoprotein uptake. Cnpy2/Msap was down-regulated using shRNA for 24 h, and cells were incubated with 50 ng/ml FGF21 for an additional 20 h. 5 μg/ml DiI-labeled LDL was then added for 6 h, and the uptake was determined as described under “Experimental Procedures.” Values are means ± S.D., n = 3. **, p < 0.01 and ***, p < 0.001 and for treated versus C.

FIGURE 7.

FGF21 and statin increase LDLR levels and lipid uptake in an additive manner. A, raw cells (upper panel) and Huh7 cells (lower panel) were treated for 20 h with 50 ng/ml FGF21 alone or together with 3 μm simvastatin. LDLR levels were determined. B, quantification shown for Huh7 cells. Values are means ± S.D., n = 3. **, p < 0.01 and ***, p < 0.001 for treated versus C. C, Huh7 cells were incubated for 20 h with 50 ng/ml FGF21 alone or together with 3 μm simvastatin 5 μg/ml DiI-labeled LDL was added for 6 h, and the uptake was determined. Values are means ± S.D., n = 3. **, p < 0.01 and ***, p < 0.001 for treated versus C. NC, is a negative control in the presence of Dynasore to inhibit endocytosis.

FIGURE 8.

Summary of the effects of FGF21 and Cnpy2/Msap on LDLR. Schematic drawing of the roles of FGF21 and Cnpy2/Msap in the regulation of LDLRs. FGF21 increases Cnpy2/Msap and LDLR levels, and decreases Mylip/Idol. Cnpy2/Msap interacts with Mylip/Idol and decreases its levels. Reduced Mylip/Idol levels stabilize LDLR and increases LDL uptake into cells in an additive manner to that of statins.