Premature senescence in cells from patients with autosomal recessive hypercholesterolemia (ARH): evidence for a role for ARH in mitosis.

OBJECTIVE: The defective gene causing autosomal recessive hypercholesterolemia (ARH) encodes ARH, a clathrin-associated 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, γ-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. CONCLUSIONS: 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.

Peroxisome proliferator-activated receptor alpha deficiency abolishes the response of lipogenic gene expression to re-feeding: restoration of the normal response by activation of liver X receptor alpha.

The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor alpha (LXRalpha). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARalpha-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARalpha-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARalpha were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRalpha was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARalpha deficiency was abolished during re-feeding. However, although re-feeding the LXRalpha ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARalpha-deficient mice. We conclude that intact PPARalpha is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRalpha.