Phosphorylation of an RNA-Binding Protein Rck/Me31b by Hippo Is Essential for Adipose Tissue Aging

Abstract

The metazoan lifespan is determined in part by a complex signaling network that regulates energy metabolism and stress responses. Key signaling hubs in this network include insulin/IGF-1, AMPK, mTOR, and sirtuins. The Hippo/Mammalian Ste20-like Kinase1 (MST1) pathway has been reported to maintain lifespan in Caenorhabditis elegans, but its role has not been studied in higher metazoans. In this study, we report that overexpression of Hpo, the MST1 homolog in Drosophila melanogaster, decreased lifespan with concomitant changes in lipid metabolism and aging-associated gene expression, while RNAi Hpo depletion increased lifespan. These effects were mediated primarily by Hpo-induced transcriptional activation of the RNA-binding protein maternal expression at 31B (Me31b)/RCK, resulting in stabilization of mRNA-encoding a lipolytic hormone, Akh. In mouse adipocytes, Hpo/Mst1 mediated adipocyte differentiation, phosphorylation of RNA-binding proteins such as Rck, decapping MRNA 2 (Dcp2), enhancer Of MRNA decapping 3 (Edc3), nucleolin (NCL), and glucagon mRNA stability by interacting with Rck. Decreased lifespan in Hpo-overexpressing Drosophila lines required expression of Me31b, but not DCP2, which was potentially mediated by recovering expression of lipid metabolic genes and formation of lipid droplets. Taken together, our findings suggest that Hpo/Mst1 plays a conserved role in longevity by regulating adipogenesis and fatty acid metabolism.

Keywords: adipocyte; differentiation; fat body; hippo; lifespan; lipid metabolism; mRNA decay.

FIGURE 1

Hippo overexpression decrease of lifespan and promotion of lipolysis in Drosophila. (A) Survivorship analysis of wild‐type (Dcg/+), Hippo‐overexpressing (Dcg > Hpo), and Hippo RNAi (Dcg > Hpo‐RNAi) D. melanogaster lines from newly eclosed male and female flies. N = 80. (B) Levels of TAG, DAG, and FFA species measured by TAG assay and HPLC‐MS/MS analyses of third instar larval homogenates isolated from Dcg/+ and Dcg > Hpo lines. Error bars represent SD of three independent experiments. N = 3; **p < 0.01; NS, not significant from Student's t‐test. (C, D) Level of DAG (C) and FFA (D) species measured by HPLC‐MS/MS of larval homogenates isolated from Dcg/+ and Dcg > Hpo lines. Error bars represent the SD of three independent experiments. N = 3; ***p < 0.001; **p < 0.01; *p < 0.05; NS, not significant from Student's t‐test. (E) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo lines using nile red (red) and DAPI labeling of nuclei (blue). Quantification of lipid droplet (LD) diameter in Dcg/+ and Dcg > Hpo animals. Each point represents a single LD. Error bars represent SD. N = 3, ***p < 0.001 from Student's t‐test. Scale bar, 50 μm. (F) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo animals using anti‐Pxn antibody as a marker of infiltrating immune cells. Scale bar, 50 μm. (G) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo animals using anti‐gamma‐H2AX antibody as a marker of senescence. Scale bar, 50 μm. (H) mRNA levels of dap and p53 from RT‐qPCR data of Dcg > Hpo. Error bars represent the SD of three independent experiments. N = 3, **p < 0.01, *p < 0.05 from Student's t‐test.

FIGURE 2

Hippo overexpression upregulation of lipid metabolism and RNA‐binding protein genes in Drosophila. (A) Heatmap of RNA‐seq using total RNA isolated from fat bodies of third instar larvae of Dcg/+ and Dcg > Hpo animals. Relative abundance of transcripts encoding lipid metabolism genes (top) and hpo were plotted in Dcg > Hpo animals. (B) Immunostaining of fat bodies of third instar larvae isolated from Dcg/+ and Dcg > Hpo animals with Akh (red) and DAPI (blue). Scale bar, 50 μm. (C–E) Protein and mRNA levels were measured by immunoblot (Akh, Myc, and Act5C) and RT‐qPCR (Akh mRNA) in fat bodies of third instar larvae of Dcg/+ and Dcg > Hpo animals. Akh mRNA stability was measured after transcription inhibition using actinomycin D in Dcg/+ and Dcg > Hpo animals. N = 3, ***p < 0.001 from Student's t‐test.

FIGURE 3

Hippo overexpression upregulates Akh expression level. (A) Relative abundances of transcripts encoding RNA‐binding proteins (top) are plotted according to their degrees of fold changes. (B) Akh mRNA pull‐down (left) and Western blot of Me31b‐GFP (right) using S2 cell lysates. Akh mRNA RT‐qPCR of me31b‐GFP immunopellets from lysates of S2 cells transfected with empty vector or a Hpo‐Myc‐expressing plasmid. N = 3, ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant from Student's t‐test. (C) Akh mRNA level in S2 cells transfected with plasmids expressing me31b‐GFP or Hpo‐Myc relative to empty vector control. N = 3, **p < 0.01, *p < 0.05 from Student's t‐test. (D) Akh mRNA level in fat bodies of third instar larvae of Dcg/+, Dcg > Hpo, Dcg > Hpo/Me31b RNAi, and Dcg > Hpo/Dcp2 RNAi animals. N = 3, **p < 0.01 from Student's t‐test.

FIGURE 4

Hippo promotion of mouse adipocyte differentiation via RNA‐binding protein phosphorylation. (A) Phase contrast images (left) and total DAG levels (right) of primary mouse adipocytes on days 0, 2, 6, and 8 after differentiation and co‐treatment with the Mst1 inhibitor Xmu‐mp‐1 (5 μM) or vehicle (DMSO) beginning on day 0. N = 3, ***p < 0.001, from Student's t‐test. (B) mRNA (RT‐qPCR, right) and protein (Western blot, left) levels of Lep, Fabp4, Adipoq, and Gcg mRNAs and proteins from pre‐adipocytes (Day 0) and mature adipocytes (Day 8) after treatment with the Mst1 inhibitor or vehicle. Values are expressed as mean ± SD of three independent experiments (p < 0.001, Student's t‐test). N = 3, ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant from Student's t‐test. (C) mRNA (RT‐qPCR, right) and protein (Western blot, left) levels of p16, p21, and Actb mRNAs and proteins from pre‐adipocytes (Day 0) and mature adipocytes (Day 8) after treatment with the Mst1 inhibitor or vehicle. Values are expressed as mean ± SD of three independent experiments (p < 0.001, Student's t‐test). N = 3, *p < 0.05, NS, not significant from Student's t‐test. (D) Gcg and Fabp4 mRNA stabilities were measured after inhibiting transcription with Actinomycin D in mouse 3 T3‐L1 cells before and after differentiation and with or without treatment with the Mst1 inhibitor. Data were normalized to 18S rRNA.

FIGURE 5

Prediction of human RCK structure. (A) Western blot analysis of p‐MST1, MST1, HuR, Rck, DCP2, and Actb in cell lysates derived from pre‐adipocytes (Day 0) and mature adipocytes (Day 8) from Student's t‐test. (B) RT‐qPCR levels of Fabp4 and Gcg mRNAs normalized to Gapdh mRNA purified from Rck or IgG immunopellets from cell lysates of precursor and mature adipocytes. Data are expressed as mean ± SD of three independent experiments. N = 3, **p < 0.01, NS, not significant from Student's t‐test. (C) Human RCK structure predicted using the AlphaFold database provided by Deepmind and EMBL. The colors indicate the accuracy of the model based on a per‐residue confidence score (pLDDT) between 0 and 100. Blue represents pLDDT values over 90 (Very high). Sky blue indicates pLDDT values between 70 and 90 (Confident). Yellow color represents pLDDT values between 50 and 60 (Low). Deep orange color indicates the pLDDT values below 50 (Very low). The phosphorylation sites, Thr 36 and Thr 75, are indicated by the red circles based on the direction of the side chain. (D) mRNA (RT‐qPCR, right) and protein (Western blot, left) levels of Fabp4, Gcg, and Actb mRNAs and proteins from pre‐adipocytes (Day 0) and mature adipocytes (Day 8) after transfection of the Rck shRNA or control. Values are expressed as mean ± SD of three independent experiments (p < 0.001, Student's t‐test). N = 3, ***p < 0.001, NS, not significant from Student's t‐test.

FIGURE 6

Requirement of Me31b/RCK for Hippo‐mediated decrease of lifespan. (A) Survivorship analysis of wild‐type (Dcg > +) and Hippo‐overexpressing (Dcg > Hpo) animals crossed with Me31b RNAi (Dcg > Hpo/Me31b‐RNAi), and Dcp2 RNAi (Dcg > Hpo/Dcp2‐RNAi) D. melanogaster . N = 80 (Dcg > +), 54 (Dcg > Hpo), 50 (Dcg > Hpo, Me31b RNAi) and 70 (Dcg > Hpo, Dcp2 RNAi; Dcg > Me31b RNAi; Dcg > Dcp2 RNAi and Dcg > Sqd RNAi). (B) Lipid droplet staining of fat bodies of third instar larvae from Dcg/+, Dcg > Hpo, Dcg > Hpo/Me31b RNAi and Dcg > Hpo/Dcp2 RNAi animals using nile red and DAPI labeling of nuclei (blue). Scale bar, 50 μm. Quantification of LD diameter, with each point representing a single LD. Data are expressed as mean ± SD. N = 3; ***p < 0.001; NS, not significant from Student's t‐test. (C) Tri‐acyl glycerol (TAG) levels in homogenates purified from third instar larvae of Dcg/+, Dcg > Hpo, Dcg > Hpo/Me31b RNAi and Dcg > Hpo/Dcp2 RNAi animals. Data are expressed as mean ± SD of three independent experiments. N = 3, **p < 0.01, *p < 0.05 from Student's t‐test.

FIGURE 7

Proposed model for Hpo regulation of lifespan. Proposed model for Hpo regulation of lifespan.