Premature Senescence in Cells From Patients With Autosomal Recessive Hypercholesterolemia (ARH)

Abstract

Objective-The defective gene causing autosomal recessive hypercholesterolemia (ARH) encodes ARH, a clathrinassociated adaptor protein required for low-density-lipoprotein receptor endocytosis in most cells but not in skin fibroblasts. The aim here was to elucidate why ARH fibroblasts grow slowly and undergo premature senescence. Methods and Results-Knockdown of ARH by RNA interference in IMR90 cells produces the same phenotype, indicated by increased p16 expression, ␥-H2AX-positive foci, and enlarged flattened morphology. We showed that ARH contributes to several aspects of mitosis: it localizes to mitotic microtubules, with lamin B1 on the nuclear envelope and spindle matrix, and with clathrin heavy chain on mitotic spindles. Second, ARH is phosphorylated in G 2 /M phase by a roscovitine-sensitive kinase, probably cdc2. Third, cells lacking ARH show disfigured nuclei and defective mitotic spindles. Defects are most marked in ARH W22X cells, where translation starts at Met46, so the protein lacks a phosphorylation site at Ser14, identified by mass spectrometry of wild-type ARH. Conclusion-The ARH protein is involved in cell cycle progression, possibly by affecting nuclear membrane formation through interaction with lamin B1 or other mitotic proteins, and its absence affects cell proliferation and induces premature senescence, which may play a role in the development of atherosclerosis in ARH. Key Words: gene mutations Ⅲ lipoproteins Ⅲ receptors Ⅲ cell division Ⅲ mitosis T he autosomal recessive hypercholesterolemia protein (ARH) is a 32-kDa endocytic adaptor protein involved in low-density-lipoprotein receptor (LDLR) endocytosis. The N terminus of ARH contains a highly conserved sequence of Ϸ40 amino acids with unidentified function, followed by a phosphotyrosine-binding domain of Ϸ130 residues. The C terminus is less conserved, but it contains a clathrin box sequence (LLDLE) and a binding motif for the ␤2 subunit of adaptor protein 2 and a PDZ binding motif with the potential to bind to many signaling proteins (reviewed by Soutar et al 1 ).The uptake of plasma low-density lipoprotein (LDL) by eukaryotic cells occurs via clathrin-dependent LDLRmediated endocytosis. Structure-function studies have shown that ARH regulates endocytosis of LDLR through binding of its phosphotyrosine-binding domain to the NPXY motif in the cytoplasmic tail of the LDLR and other members of its family and binding of its C-terminal motifs to clathrin heavy chain (CHC) and adaptor protein 2. This role is well established from studies in homozygous ARH patients, whose lymphoblasts in culture fail to internalize LDLR and who exhibit severe hypercholesterolemia and premature atherosclerosis because LDL uptake is defective in the liver. 2,3 ARH knockout mice also show impaired endocytosis of LDLR and hypercholesterolemia. 4 Surprisingly, cultured skin fibroblasts from ARH patients exhibit normal internalization of LDLR, 1 possibly because Dab2 can compensate for the absence of ARH in these cells. 5,6 Although it is clear that ARH functions a...

Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics

“…Thus, the putative CDK1 target site pS14 is up-regulated in mitosis and its deletion or mutation leads to defective mitotic spindle formation suggesting an important role for Ldlrap1 in the mitotic cell cycle [36]. We find pS14 robustly down-regulated again supporting the idea of cAMP-induced cell cycle arrest.…”

Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC

“…Figures 8A and 8B show that infection of human VSMC with Ad133a results in significantly less proliferation of those cells, compared with scrambled control virus (98.6 ×10 3 +/−9.5 × 10 3 vs 50.6 × 10 3 +/−3.2 × 10 3 , P<0.01). Limited literature suggests LDLRAP1 may have cell-specific functions unrelated to LDLR internalization [40]. To determine if LDLRAP1 played a role in VSMC proliferation, human VSMC were transfected with LDLRAP1-specific siRNA, and VSMC proliferation quantitated.…”

“…This hypercholesterolemia is assumed to be a result of impaired LDL internalization by hepatocytes, and disruption of LDLRAP1 results in 80% reduction in LDL internalization in cultured hepatocytes [31,32,35]. Importantly, LDLRAP1 function has been posited to be tissue specific [40], as hepatocytes and lymphoblasts from ARH patients display impaired LDL internalization, but internalization is normal in fibroblasts from the same patients. The transformation of VSMC into foam cells is a key step in atherogenesis [2].…”

“…Indeed, limited literature suggests LDLRAP1 has functions outside of LDLR internalization. For example, fibroblasts from some ARH patients grow more slowly, and one report suggests LDLRAP1 may participate in mitosis [40]. VSMC in which LDLRAP1 is knocked down are comparable to ARH patients which have a non-functional LDLRAP1 protein, suggesting a function for LDLRAP1 in VSMC proliferation.…”

Autosomal Recessive Hypercholesterolemia: A Mild Phenotype of Familial Hypercholesterolemia: Insight from the Kinetic Study using Stable Isotope and Animal Studies

“…The function of LDLRAP1 in the kidneys has been described as playing a key role in potassium homeostasis, at least in mice 21) . Another article indicated that LDLRAP1 proteins are involved in cell cycle progression, possibly via effects on nuclear membrane formation through their interactions with mitotic proteins 22) . However, there is no evidence of primary potassium retention, hyperkalemia or premature aging in human ARH subjects.…”