B-type lamins (lamins B1 and B2) have been considered to be essential for many crucial functions in the cell nucleus (e.g., DNA replication and mitotic spindle formation). However, this view has been challenged by the observation that an absence of both B-type lamins in keratinocytes had no effect on cell proliferation or the development of skin and hair. The latter findings raised the possibility that the functions of B-type lamins are subserved by lamins A and C. To explore that idea, we created mice lacking all nuclear lamins in keratinocytes. Those mice developed ichthyosis and a skin barrier defect, which led to death from dehydration within a few days after birth. Microscopy of nuclear-lamin-deficient skin revealed hyperkeratosis and a disordered stratum corneum with an accumulation of neutral lipid droplets; however, BrdU incorporation into keratinocytes was normal. Skin grafting experiments confirmed the stratum corneum abnormalities and normal BrdU uptake. Interestingly, the absence of nuclear lamins in keratinocytes resulted in an interspersion of nuclear/endoplasmic reticulum membranes with the chromatin. Thus, a key function of the nuclear lamina is to serve as a “fence” and prevent the incursion of cytoplasmic organelles into the nuclear chromatin.
Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (LmnaPLAO-5NT) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3′ UTR, and a second (LmnaPLAO-UTR) in which prelamin A's 3′ UTR was replaced with lamin C's 3′ UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the LmnaPLAO-UTR allele than with the LmnaPLAO-5NT allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3′ UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson–Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.
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.
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.
Protein phosphatase 2A (PP2A), the major serine/threonine phosphatase in eukaryotic cells, is a heterotrimeric protein composed of structural, catalytic, and targeting subunits. PP2A assembly is governed by a variety of mechanisms, one of which is carboxyl-terminal methylation of the catalytic subunit by the leucine carboxyl methyltransferase LCMT1. PP2A is nearly stoichiometrically methylated in the cytosol, and although some PP2A targeting subunits bind independently of methylation, this modification is required for the binding of others. To examine the role of this methylation reaction in mammalian tissues, we generated a mouse harboring a gene-trap cassette within intron 1 of Lcmt1. Due to splicing around the insertion, Lcmt1 transcript and LCMT1 protein levels were reduced but not eliminated. LCMT1 activity and methylation of PP2A were reduced in a coordinate fashion, suggesting that LCMT1 is the only PP2A methyltransferase. These mice exhibited an insulin-resistance phenotype, indicating a role for this methyltransferase in signaling in insulin-sensitive tissues. Tissues from these animals will be vital for the in vivo identification of methylation-sensitive substrates of PP2A and how they respond to differing physiological conditions.
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
A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.
Nuclear lamins are usually classified as A-type (lamins A and C) or B-type (lamins B1 and B2). A-type lamins have been implicated in multiple genetic diseases but are not required for cell growth or development. In contrast, B-type lamins have been considered essential in eukaryotic cells, with crucial roles in DNA replication and in the formation of the mitotic spindle. Knocking down the genes for B-type lamins (LMNB1, LMNB2) in HeLa cells has been reported to cause apoptosis. In the current study, we created conditional knockout alleles for mouse Lmnb1 and Lmnb2, with the goal of testing the hypothesis that B-type lamins are crucial for the growth and viability of mammalian cells in vivo. Using the keratin 14-Cre transgene, we bred mice lacking the expression of both Lmnb1 and Lmnb2 in skin keratinocytes (Lmnb1Δ/ΔLmnb2Δ/Δ). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1Δ/ΔLmnb2Δ/Δ mice, and lamin B1 and lamin B2 proteins were undetectable. But despite an absence of B-type lamins in keratinocytes, the skin and hair of Lmnb1Δ/ΔLmnb2Δ/Δ mice developed normally and were free of histological abnormalities, even in 2-year-old mice. After an intraperitoneal injection of bromodeoxyuridine (BrdU), similar numbers of BrdU-positive keratinocytes were observed in the skin of wild-type and Lmnb1Δ/ΔLmnb2Δ/Δ mice. Lmnb1Δ/ΔLmnb2Δ/Δ keratinocytes did not exhibit aneuploidy, and their growth rate was normal in culture. These studies challenge the concept that B-type lamins are essential for proliferation and vitality of eukaryotic cells.
Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.
Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1–deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.
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 lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries represents one of the most persistent mysteries of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.
Lamin A is formed from prelamin A by four post-translational processing steps—farnesylation, release of the last three amino acids of the protein, methylation of the farnesylcysteine and the endoproteolytic release of the C-terminal 15 amino acids (including the farnesylcysteine methyl ester). When the final processing step does not occur, a farnesylated and methylated prelamin A accumulates in cells, causing a severe progeroid disease, restrictive dermopathy (RD). Whether RD is caused by the retention of farnesyl lipid on prelamin A, or by the retention of the last 15 amino acids of the protein, is unknown. To address this issue, we created knock-in mice harboring a mutant Lmna allele (LmnanPLAO) that yields exclusively non-farnesylated prelamin A (and no lamin C). These mice had no evidence of progeria but succumbed to cardiomyopathy. We suspected that the non-farnesylated prelamin A in the tissues of these mice would be strikingly mislocalized to the nucleoplasm, but this was not the case; most was at the nuclear rim (indistinguishable from the lamin A in wild-type mice). The cardiomyopathy could not be ascribed to an absence of lamin C because mice expressing an otherwise identical knock-in allele yielding only wild-type prelamin A appeared normal. We conclude that lamin C synthesis is dispensable in mice and that the failure to convert prelamin A to mature lamin A causes cardiomyopathy (at least in the absence of lamin C). The latter finding is potentially relevant to the long-term use of protein farnesyltransferase inhibitors, which lead to an accumulation of non-farnesylated prelamin A.
Previously, we identified the E3 ubiquitin ligase Idol (inducible degrader of the low-density lipoprotein [LDL] receptor [LDLR]) as a posttranscriptional regulator of the LDLR pathway. Idol stimulates LDLR degradation through ubiquitination of its C-terminal domain, thereby limiting cholesterol uptake. Here we report the generation and characterization of mouse embryonic stem cells homozygous for a null mutation in the Idol gene. Cells lacking Idol exhibit markedly elevated levels of the LDLR protein and increased rates of LDL uptake. Furthermore, despite an intact sterol responsive element-binding protein (SREBP) pathway, Idol-null cells exhibit an altered response to multiple regulators of sterol metabolism, including serum, oxysterols, and synthetic liver X receptor (LXR) agonists. The ability of oxysterols and lipoprotein-containing serum to suppress LDLR protein levels is reduced, and the time course of suppression is delayed, in cells lacking Idol. LXR ligands have no effect on LDLR levels in Idol-null cells, indicating that Idol is required for LXR-dependent inhibition of the LDLR pathway. In line with these results, the half-life of the LDLR protein is prolonged in the absence of Idol. Finally, the ability of statins and PCSK9 to alter LDLR levels is independent of, and additive with, the LXR-Idol pathway. These results demonstrate that the LXR-Idol pathway is an important contributor to feedback inhibition of the LDLR by sterols and a biological determinant of cellular LDL uptake.
The modification of proteins with farnesyl or geranylgeranyl lipids, a process called protein prenylation, facilitates interactions of proteins with membrane surfaces. Protein prenylation is carried out by a pair of cytosolic enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). FTase and GGTase-I have attracted interest as therapeutic targets for both cancer and progeria, but very little information exists on the importance of these enzymes for homeostasis of normal tissues. One study actually suggested that FTase is entirely dispensable. To explore the importance of the protein prenyltransferases for normal tissues, we used conditional knockout alleles for Fntb and Pggt1b (which encode the β-subunits of FTase and GGTase-I, respectively) and a keratin 14–Cre transgene to create mice lacking FTase or GGTase-I in skin keratinocytes. Keratinocyte-specific Fntb knockout mice were viable but developed severe alopecia. Although hair follicles appeared normal during development, they were morphologically abnormal after birth, and ultrastructural and immunohistochemical studies revealed many apoptotic cells. The interfollicular epidermis of Fntb-deficient mice appeared normal; however, keratinocytes from these mice could not proliferate in culture. As expected, non-farnesylated prelamin A and non-farnesylated DNAJA1 accumulated in Fntb-deficient keratinocytes. Keratinocyte-specific Pggt1b knockout mice survived development but died shortly after birth. Like Fntb-deficient keratinocytes, Pggt1b-deficient keratinocytes did not proliferate in culture. Thus, both FTase and GGTase-I are required for the homeostasis of skin keratinocytes.
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