Impaired cell proliferation in regenerating liver of 3 ß-hydroxysterol Δ14-reductase (TM7SF2) knock-out mice.

The liver is the most important organ in cholesterol metabolism, which is instrumental in regulating cell proliferation and differentiation. The gene Tm7sf2 codifies for 3 ß-hydroxysterol-Δ14-reductase (C14-SR), an endoplasmic reticulum resident protein catalyzing the reduction of C14-unsaturated sterols during cholesterol biosynthesis from lanosterol. In this study we analyzed the role of C14-SR in vivo during cell proliferation by evaluating liver regeneration in Tm7sf2 knockout (KO) and wild-type (WT) mice. Tm7sf2 KO mice showed no alteration in cholesterol content. However, accumulation and delayed catabolism of hepatic triglycerides was observed, resulting in persistent steatosis at all times post hepatectomy. Moreover, delayed cell cycle progression to the G1/S phase was observed in Tm7sf2 KO mice, resulting in reduced cell division at the time points examined. This was associated to abnormal ER stress response, leading to alteration in p53 content and, consequently, induction of p21 expression in Tm7sf2 KO mice. In conclusion, our results indicate that Tm7sf2 deficiency during liver regeneration alters lipid metabolism and generates a stress condition, which, in turn, transiently unbalances hepatocytes cell cycle progression.

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box.

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Sterol dependent regulation of human TM7SF2 gene expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis.

3Beta-hydroxysterol Delta(14)-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3beta-hydroxysterol Delta(14)-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3beta-hydroxysterol Delta(14)-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3beta-hydroxysterol Delta(14)-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Delta(14)-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3beta-hydroxysterol Delta(14)-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3beta-hydroxysterol Delta(14)-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.

Cloning and expression of sterol Delta 14-reductase from bovine liver.

Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes. Sterol Delta 14-reductase (Delta 14-SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals. Amino-acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene, and with the C-terminal domain of human lamin B receptor. A cDNA encoding the 38-kDa protein, similar to human TM7SF2, was identified by analysis of a bovine expressed sequence tag (EST) database. The cDNA was synthesized by RT-PCR, cloned, and sequenced. The cDNA encodes a 418 amino-acid polypeptide with nine predicted transmembrane domains. The deduced amino-acid sequence exhibits high similarity with Delta 14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta 14-SR. Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain. The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells. Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited Delta 14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta 14-SR and provide evidence that the human TM7SF2 gene encodes Delta 14-SR.