Much of the work on nuclear lamins during the past 15 years has focused on mutations in LMNA (the gene for prelamin A and lamin C) that cause particular muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These disorders, often called “laminopathies,” mainly affect mesenchymal tissues (e.g., striated muscle, bone, and fibrous tissue). Recently, however, a series of papers have identified important roles for nuclear lamins in the central nervous system. Studies of knockout mice uncovered a key role for B-type lamins (lamins B1 and B2) in neuronal migration in the developing brain. Also, duplications of LMNB1 (the gene for lamin B1) have been shown to cause autosome-dominant leukodystrophy. Finally, recent studies have uncovered a peculiar pattern of nuclear lamin expression in the brain. Lamin C transcripts are present at high levels in the brain, but prelamin A expression levels are very low—due to regulation of prelamin A transcripts by microRNA 9. This form of prelamin A regulation likely explains why “prelamin A diseases” such as Hutchinson-Gilford progeria syndrome spare the central nervous system. In this review, we summarize recent progress in elucidating links between nuclear lamins and neurobiology.
Mutations in SLURP1 cause mal de Meleda, a rare palmoplantar keratoderma (PPK). SLURP1 is a secreted protein that is expressed highly in keratinocytes but has also been identified elsewhere (e.g., spinal cord neurons). Here, we examined Slurp1-deficient mice (Slurp1−/−) created by replacing exon 2 with β-gal and neo cassettes. Slurp1−/− mice developed severe PPK characterized by increased keratinocyte proliferation, an accumulation of lipid droplets in the stratum corneum, and a water barrier defect. In addition, Slurp1−/− mice exhibited reduced adiposity, protection from obesity on a high-fat diet, low plasma lipid levels, and a neuromuscular abnormality (hind limb clasping). Initially, it was unclear whether the metabolic and neuromuscular phenotypes were due to Slurp1 deficiency because we found that the targeted Slurp1 mutation reduced the expression of several neighboring genes (e.g., Slurp2, Lypd2). We therefore created a new line of knockout mice (Slurp1X−/− mice) with a simple nonsense mutation in exon 2. The Slurp1X mutation did not reduce the expression of adjacent genes, but Slurp1X−/− mice exhibited all of the phenotypes observed in the original line of knockout mice. Thus, Slurp1 deficiency in mice elicits metabolic and neuromuscular abnormalities in addition to PPK.
To assess the redundancy of lamins B1 and B2, knock-in lines were created that produce lamin B2 from the Lmnb1 locus and lamin B1 from the Lmnb2 locus. Both lines developed severe neurodevelopmental abnormalities, indicating that the abnormalities elicited by the loss of one B-type lamin cannot be prevented by increased synthesis of the other.
Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated “reciprocal knock-in mice”—mice that make lamin B2 from the Lmnb1 locus (Lmnb1B2/B2) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2B1/B1). Lmnb1B2/B2 mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1B2/B2 mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2B1/B1 mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.
Lipoprotein lipase (LPL) is produced by parenchymal cells, mainly adipocytes and myocytes, but its role in hydrolyzing triglycerides in plasma lipoproteins occurs at the capillary lumen. For decades, the mechanism by which LPL reached its site of action in capillaries was unclear, but this mystery was recently solved. GPIHBP1, a GPI-anchored protein of capillary endothelial cells, picks up LPL from the interstitial spaces and shuttles it across endothelial cells to the capillary lumen. When GPIHBP1 is absent, LPL is mislocalized to the interstitial spaces, leading to severe hypertriglyceridemia. Some cases of hypertriglyceridemia in humans are caused by GPIHBP1 mutations that interfere with GPIHBP1's ability to bind LPL, and some are caused by LPL mutations that impair LPL's ability to bind to GPIHBP1. This review will cover recent progress in understanding GPIHBP1's role in health and disease and will discuss some remaining mysteries surrounding the processing of triglyceride-rich lipoproteins.
hypertriglyceridemia; chylomicronemia; GPIHBP1; lipoprotein lipase; endothelial cells; lymphocyte antigen 6 proteins
The nuclear lamina is an intermediate filament meshwork composed largely of four nuclear lamins—lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Located immediately adjacent to the inner nuclear membrane, the nuclear lamina provides a structural scaffolding for the cell nucleus. It also interacts with both nuclear membrane proteins and the chromatin and is thought to participate in many important functions within the cell nucleus. Defects in A-type lamins cause cardiomyopathy, muscular dystrophy, peripheral neuropathy, lipodystrophy, and progeroid disorders. In contrast, the only bona fide link between the B-type lamins and human disease is a rare demyelinating disease of the central nervous system—adult-onset autosomal-dominant leukoencephalopathy, caused by a duplication of the gene for lamin B1. However, this leukoencephalopathy is not the only association between the brain and B-type nuclear lamins. Studies of conventional and tissue-specific knockout mice have demonstrated that B-type lamins play essential roles in neuronal migration in the developing brain and in neuronal survival. The importance of A-type lamin expression in the brain is unclear, but it is intriguing that the adult brain preferentially expresses lamin C rather than lamin A, very likely due to microRNA-mediated removal of prelamin A transcripts. Here, we review recent studies on nuclear lamins, focusing on the function and regulation of the nuclear lamins in the central nervous system.
Nuclear lamina; brain development; A-type lamins; B-type lamins; differential gene expression
Inducible Degrader Of the Low-density lipoprotein receptor (IDOL) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the low-density lipoprotein receptor (LDLR). IDOL expression is controlled at the transcriptional level by the cholesterol-sensing nuclear receptor LXR. In response to rising cellular sterol levels, activated LXR induces IDOL production, thereby limiting further uptake of exogenous cholesterol through the LDLR pathway. The LXR–IDOL–LDLR mechanism for feedback inhibition of cholesterol uptake is independent of and complementary to the SREBP pathway. Since the initial description of the LXR–IDOL pathway, biochemical studies have helped to define the structural basis for both IDOL target recognition and LDLR ubiquitin transfer. Recent work has also suggested links between IDOL and human lipid metabolism.
The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes.
The endoplasmic reticulum (ER) is a structure that performs a variety of functions within eukaryotic cells. It can be divided into two regions: the surface of the rough ER is coated with ribosomes that manufacture various proteins, while the smooth ER is involved in activities such as lipid synthesis and carbohydrate metabolism. Proteins synthesized by the ribosomes attached to the rough ER are generally transferred to another structure within the cell, the Golgi apparatus, where they undergo further processing and packaging before being secreted or transported to another location within the cell.
Proteins are shuttled from the ER to the Golgi apparatus by vesicles covered with coat protein complex II (COPII). This complex is composed of an inner and outer coat, each of which is assembled primarily with two different SEC proteins: the SEC23/SEC24 protein heterodimer forms the inner coat of the COPII vesicle, and plays a key role in recruiting the appropriate protein cargos to the transport vesicle, while the SEC13/SEC31 protein heterotetramer forms the outer coat and is generally responsible for regulating vesicle size and rigidity.
Previous work found that mammals, including humans and mice, harbor multiple copies of several SEC protein genes, including two copies of SEC23 and four copies of SEC24. Both copies of SEC23 are derived from the same ancestral gene, and all four copies of SEC24 are derived from a different ancestral gene, and the availability of these copies potentially expands the range of properties that the vesicles can have. Insight into the roles of each SEC protein has come from work with SEC mutants. For example, a mutation in SEC23A was found to cause skeletal abnormalities in humans.
Here, Chen et al. report the results of experiments which showed that mice with an inactive Sec24a gene could develop normally. However, these mice experienced a 45% reduction in their plasma cholesterol levels because they were not able to recruit and transport a secretory protein called PCSK9, which is a critical regulator of blood cholesterol levels.
The work of Chen et al. reveals a previously unappreciated complexity in the recruitment of secretory proteins to the COPII vesicle and suggests that the various combinations of SEC proteins influence the proteins selected for transport to the Golgi apparatus. The work also identifies Sec24a as a potential therapeutic target for the reduction of plasma cholesterol, a finding that could be of interest to researchers working on heart disease and other conditions exacerbated by high cholesterol.
Secretory pathway; COP II; Cholesterol metabolism; Mouse
Gpihbp1-deficient mice (Gpihbp1−/−) lack the ability to transport lipoprotein lipase to the capillary lumen, resulting in mislocalization of LPL within tissues, defective lipolysis of triglyceride-rich lipoproteins, and chylomicronemia. We asked whether GPIHBP1 deficiency and mislocalization of catalytically active LPL would alter the composition of triglycerides in adipose tissue or perturb the expression of lipid biosynthetic genes. We also asked whether perturbations in adipose tissue composition and gene expression, if they occur, would be accompanied by reciprocal metabolic changes in the liver.
Methods and Results
The chylomicronemia in Gpihbp1−/− mice was associated with reduced levels of essential fatty acids in adipose tissue triglycerides and increased expression of lipid biosynthetic genes. The liver exhibited the opposite changes—increased levels of essential fatty acids in triglycerides and reduced expression of lipid biosynthetic genes.
Defective lipolysis in Gpihbp1−/− mice causes reciprocal metabolic perturbations in adipose tissue and liver. In adipose tissue, the essential fatty acid content of triglycerides is reduced and lipid biosynthetic gene expression is increased, while the opposite changes occur in the liver.
lipoprotein lipase; hypertriglyceridemia; lipolysis; essential fatty acids; lipid biosynthetic genes
The B-type lamins are widely assumed to be essential for mammalian cells. In part, this assumption is based on a highly cited study that found that RNAi-mediated knockdown of lamin B1 or lamin B2 in HeLa cells arrested cell growth and led to apoptosis. Studies indicating that B-type lamins play roles in DNA replication, the formation of the mitotic spindle, chromatin organization and regulation of gene expression have fueled the notion that B-type lamins must be essential. But surprisingly, this idea had never been tested with genetic approaches. Earlier this year, a research group from UCLA reported the development of genetically modified mice that lack expression of both Lmnb1 and Lmnb2 in skin keratinocytes (a cell type that proliferates rapidly and participates in complex developmental programs). They reasoned that if lamins B1 and B2 were truly essential, then keratinocyte-specific lamin B1/lamin B2 knockout mice would exhibit severe pathology. Contrary to expectations, the skin and hair of lamin B1/lamin B2-deficient mice were quite normal, indicating that the B-type lamins are dispensable in some cell types. The same UCLA research group has gone on to show that lamin B1 and lamin B2 are critical for neuronal migration in the developing brain and for neuronal survival. The absence of either lamin B1 or lamin B2, or the absence of both B-type lamins, results in severe neurodevelopmental abnormalities.
lamin B1; lamin B2; nuclear envelope; nuclear lamina
Members of the lipin protein family are phosphatidate phosphatase (PAP) enzymes, which catalyze the dephosphorylation of phosphatidic acid to diacylglycerol, the penultimate step in TAG synthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in that they also promote fatty acid oxidation through their activity as co-regulators of gene expression by DNA-bound transcription factors. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins—lipin-1, lipin-2, and lipin-3. In mice and humans, the levels of lipin activity are a determinant of TAG storage in diverse cell types, and humans with deficiency in lipin-1 or lipin-2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of “lipinopathies” in mouse models and in humans has revealed an important role for lipin activity in the regulation of lipid intermediates (phosphatidate and diacylglycerol), which influence fundamental cellular processes including adipocyte and nerve cell differentiation, adipocyte lipolysis, and hepatic insulin signaling. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.
GPIHBP1, a glycosylphosphatidylinositol-anchored Ly6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. We sought to define the ability of GPIHBP1 to bind other lipase family members as well as other apolipoproteins and lipoproteins.
Methods and Results
As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1 but other members of the lipase family do not. We also examined the binding of apoAV–phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1’s amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind HDL. Chylomicrons binds avidly to GPIHBP1-transfected CHO cells, but this binding is dependent on GPIHBP1’s ability to bind LPL within the cell culture medium.
GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but did not bind apoAI or HDL. The ability of GPIHBP1-transfected CHO cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium.
lipoprotein lipase; chylomicronemia; hypertriglyceridemia; GPIHBP1
Adult GPIHBP1-deficient mice (Gpihbp1−/−) have severe hypertriglyceridemia; however, the plasma triglyceride levels are only mildly elevated during the suckling phase when lipoprotein lipase (Lpl) is expressed at high levels in the liver. Lpl expression in the liver can be induced in adult mice with dietary cholesterol. We therefore hypothesized that plasma triglyceride levels in adult Gpihbp1−/− mice would be sensitive to cholesterol intake.
Methods and Results
After 4–8 weeks on a western diet containing 0.15% cholesterol, plasma triglyceride levels in Gpihbp1−/− mice were 10,000–12,000 mg/dl. When 0.005% ezetimibe was added to the diet to block cholesterol absorption, Lpl expression in the liver was reduced significantly, and the plasma triglyceride levels were significantly higher (>15,000 mg/dl). We also assessed plasma triglyceride levels in Gpihbp1−/− mice fed western diets containing either high (1.3%) or low (0.05%) amounts of cholesterol. The high-cholesterol diet significantly increased Lpl expression in the liver and lowered plasma triglyceride levels.
Treatment of Gpihbp1−/− mice with ezetimibe lowers Lpl expression in the liver and increases plasma triglyceride levels. A high-cholesterol diet had the opposite effects. Thus, cholesterol intake modulates plasma triglyceride levels in Gpihbp1−/− mice.
lipoprotein lipase; chylomicronemia; hypertriglyceridemia; GPIHBP1
The risk of atherosclerosis in the setting of chylomicronemia has been a topic of debate. In this study, we examined susceptibility to atherosclerosis in Gpihbp1-deficient mice (Gpihbp1−/−), which manifest severe chylomicronemia as a result of defective lipolysis.
Methods and Results
Gpihbp1−/− mice on a chow diet have plasma triglyceride and cholesterol levels of 2812 ± 209 and 319 ± 27 mg/dl, respectively. Even though nearly all of the lipids were contained in large lipoproteins (50–135 nm), the mice developed progressive aortic atherosclerosis. In other experiments, we found that both Gpihbp1-deficient “apo-B48–only” mice and Gpihbp1-deficient “apo-B100–only” mice manifest severe chylomicronemia. Thus, GPIHBP1 is required for the processing of both apo-B48– and apo-B100–containing lipoproteins.
Chylomicronemia causes atherosclerosis in mice. Also, we found that GPIHBP1 is required for the lipolytic processing of both apo-B48– and apo-B100–containing lipoproteins.
lipoprotein lipase; chylomicronemia; lipolysis; GPIHBP1
Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I–deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor–deficient mice.
Methods and Results
Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b∆/∆) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b∆/∆ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I–deficient macrophages revealed upregulation of the cyclooxygenase 2–peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I– and CD36-mediated basal and high-density lipoprotein–stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo.
Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.
atherosclerosis; cholesterol; hydroxymethylglutaryl-CoA reductase inhibitors; macrophages; prenylation; statins
The main function of the nuclear lamina, an intermediate filament meshwork lying primarily beneath the inner nuclear membrane, is to provide structural scaffolding for the cell nucleus. However, the lamina also serves other functions, such as having a role in chromatin organization, connecting the nucleus to the cytoplasm, gene transcription, and mitosis. In somatic cells, the main protein constituents of the nuclear lamina are lamins A, C, B1, and B2. Interest in the nuclear lamins increased dramatically in recent years with the realization that mutations in LMNA, the gene encoding lamins A and C, cause a panoply of human diseases (“laminopathies”), including muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. Here, we review the laminopathies and the long strange trip from basic cell biology to therapeutic approaches for these diseases.
A common strategy for conditional knockout alleles is to “flox” (flank with loxP sites) a 5′ exon within the target gene. Typically, the floxed exon does not contain a unit number of codons so that the Cre-mediated recombination event yields a frameshift and a null allele. Documenting recombination within the genomic DNA is often regarded as sufficient proof of a frameshift, and the analysis of transcripts is neglected. We evaluated a previously reported conditional knockout allele for the β-subunit of protein farnesyltransferase. The recombination event in that allele—the excision of exon 3—was predicted to yield a frameshift. However, following the excision of exon 3, exon 4 was skipped by the mRNA splicing machinery, and the predominant transcript from the mutant allele lacked exon 3 and exon 4 sequences. The “Δexon 3–4 transcript” does not contain a frameshift but rather is predicted to encode a protein with a short in-frame deletion. This represents a significant concern when studying an enzyme, since an enzyme with partial function could lead to erroneous conclusions. With thousands of new conditional knockout alleles under construction within mouse mutagenesis consortiums, the protein farnesyltransferase allele holds an important lesson—to characterize knockout alleles at both the DNA and RNA levels.
protein prenylation; farnesylation; prelamin A; HDJ-2; knockout mice
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by multiple aging-like disease phenotypes. We recently reported that a protein farnesyltransferase inhibitor (FTI) improved several disease phenotypes in mice with a HGPS mutation (LmnaHG/+). Here, we investigated the impact of an FTI on the survival of LmnaHG/+ mice. The FTI significantly improved the survival of both male and female LmnaHG/+ mice. Treatment with the FTI also improved body weight curves and reduced the number of spontaneous rib fractures. This study provides further evidence for a beneficial effect of an FTI in HGPS.
progeria; aging; protein farnesyltransferase inhibitor; knock-in mice
GPIHBP1 is an endothelial cell protein that serves as a platform for lipoprotein lipase–mediated processing of triglyceride-rich lipoproteins within the capillaries of heart, adipose tissue, and skeletal muscle. The absence of GPIHBP1 causes severe chylomicronemia. A hallmark of GPIHBP1 is the ability to bind lipoprotein lipase, chylomicrons, and apolipoprotein (apo-) AV. A homozygous G56R mutation in GPIHBP1 was recently identified in two brothers with chylomicronemia, and the authors of that study suggested that the G56R substitution was responsible for the hyperlipidemia. In this study, we created a human GPIHBP1 expression vector, introduced the G56R mutation, and tested the ability of the mutant GPIHBP1 to reach the cell surface and bind lipoprotein lipase, chylomicrons, and apo-AV. Our studies revealed that the G56R substitution did not affect the ability of GPIHBP1 to reach the cell surface, nor did the amino acid substitution have any discernible effect on the binding of lipoprotein lipase, chylomicrons, or apo-AV.
chylomicronemia; GPIHBP1; hypertriglyceridemia; apolipoprotein AV; lipoprotein lipase
Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.
Transcriptional effectors of white adipocyte-selective gene expression have not been described. Here we show that TLE3 is a white-selective cofactor that acts reciprocally with the brown-selective cofactor Prdm16 to specify lipid storage and thermogenic gene programs. Occupancy of TLE3 and Prdm16 on certain promoters is mutually exclusive, due to the ability of TLE3 to disrupt the physical interaction between Prdm16 and PPARγ. When expressed at elevated levels in brown fat, TLE3 counters Prdm16, suppressing brown-selective genes and inducing white-selective genes, resulting in impaired fatty acid oxidation and thermogenesis. Conversely, mice lacking TLE3 in adipose tissue show enhanced thermogenesis in inguinal white adipose depots and are protected from age-dependent deterioration of brown adipose tissue function. Our results suggest that the establishment of distinct adipocyte phenotypes with different capacities for thermogenesis and lipid storage is accomplished in part through the cell type–selective recruitment of TLE3 or Prdm16 to key adipocyte target genes.
A clinical trial of a protein farnesyltransferase inhibitor (lonafarnib) for the treatment of Hutchinson-Gilford progeria syndrome (HGPS) was recently completed. Here, we discuss the mutation that causes HGPS, the rationale for inhibiting protein farnesyltransferase, the potential limitations of this therapeutic approach, and new potential strategies for treating the disease.
Hutchinson-Gilford progeria syndrome (HGPS) is caused by the production of a truncated prelamin A, called progerin, which is farnesylated at its carboxyl terminus. Progerin is targeted to the nuclear envelope and causes misshapen nuclei. Protein farnesyltransferase inhibitors (FTI) mislocalize progerin away from the nuclear envelope and reduce the frequency of misshapen nuclei. To determine whether an FTI would ameliorate disease phenotypes in vivo, we created gene-targeted mice with an HGPS mutation (LmnaHG/+) and then examined the effect of an FTI on disease phenotypes. LmnaHG/+ mice exhibited phenotypes similar to those in human HGPS patients, including retarded growth, reduced amounts of adipose tissue, micrognathia, osteoporosis, and osteolytic lesions in bone. Osteolytic lesions in the ribs led to spontaneous bone fractures. Treatment with an FTI increased adipose tissue mass, improved body weight curves, reduced the number of rib fractures, and improved bone mineralization and bone cortical thickness. These studies suggest that FTIs could be useful for treating humans with HGPS.
Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C–only mice (Lmna+/+), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna+/+ mice were entirely healthy, and Lmna+/+ cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl–prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A–related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single LmnaLCO allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24–/– mice. Moreover, treating Zmpste24–/– cells with a prelamin A–specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.
Lipoprotein lipase (LPL) is a 448-amino-acid head-to-tail dimeric enzyme that hydrolyzes triglycerides within capillaries. LPL is secreted by parenchymal cells into the interstitial spaces; it then binds to GPIHBP1 (glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1) on the basolateral face of endothelial cells and is transported to the capillary lumen. A pair of amino acid substitutions, C418Y and E421K, abolish LPL binding to GPIHBP1, suggesting that the C-terminal portion of LPL is important for GPIHBP1 binding. However, a role for LPL's N terminus has not been excluded, and published evidence has suggested that only full-length homodimers are capable of binding GPIHBP1. Here, we show that LPL's C-terminal domain is sufficient for GPIHBP1 binding. We found, serendipitously, that two LPL missense mutations, G409R and E410V, render LPL susceptible to cleavage at residue 297 (a known furin cleavage site). The C terminus of these mutants (residues 298–448), bound to GPIHBP1 avidly, independent of the N-terminal fragment. We also generated an LPL construct with an in-frame deletion of the N-terminal catalytic domain (residues 50–289); this mutant was secreted but also was cleaved at residue 297. Once again, the C-terminal domain (residues 298–448) bound GPIHBP1 avidly. The binding of the C-terminal fragment to GPIHBP1 was eliminated by C418Y or E421K mutations. After exposure to denaturing conditions, the C-terminal fragment of LPL refolds and binds GPIHBP1 avidly. Thus, the binding of LPL to GPIHBP1 requires only the C-terminal portion of LPL and does not depend on full-length LPL homodimers.