Genetic and Developmental Regulation of the

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 264. No. 3,Ianue of January 25. pp. 1473-1482.1969 Printed in U.S.A.

Genetic and Developmental Regulation of the Lipoprotein LipaseGene: Loci Both Distaland Proximal to the Lipoprotein LipaseStructural Gene Control Enzyme Expression* (Received for publication, June 23,1988)

Todd G . KirchgessnerS, ReneeC. LeBoeufg, Carol A. Langnernll, SusanZollmanS, Chris H. Changs, Benjamin A. Taylor$$, Michael C. Schotzg, Jeffrey I. Gordonn**Q§,and Aldons J. Lusis$$jl[T From the $Departments of Medicine and Microbiology, University of California, Los Angeles, California 90024, §Research, Veterans Administration WadsworthMedical Center, Los Angeles, California 90073, the Departments of (Biological Chemistry and **Medicine. Washington University School of Medicine, St. Louis, Missouri 63110, and $’$.TheJackson Laboratory, Bar Harbor, Maine 04609

of LPL in We report here a study of the developmental and netic mutations resulting in deficiencies genetic controlof tissue-specific expressionof lipopro- mice, the W mutation on chromosome 6 and the cld tein lipase, the enzyme responsible for hydrolysis of mutation onmouse chromosome 17,do not involvethe triglycerides in chylomicrons and very low density LPL structural gene locus. Finally, we show that the lipoproteins. Lipoprotein lipase (LPL) mRNA is pre- gene €or hepatic lipase, a member of a gene familywith sent in a wide variety of adult rat and mouse tissues LPL, is unlinked to the gene for LPL. This indicates examined, albeitat very different levels. A remarkable that combined deficiencies of LPL and hepatic lipase, strains increase in the levels of LPL mRNA occurs in heart observed in humansas well as in certain mutant of a cluster over a period of several weeksfollowing birth, closely of mice, do not result from focal disruptions paralleling developmental changes in lipase activity of lipase genes. and myocardial &oxidation capacity. Large increases in LPL mRNA alsooccur duringdifferentiation of 3T3L1 cells to adipocytes. As previously reported, at Lipoprotein lipase (LPL)’ plays a major role in the metableast two separate genetic loci control the tissue-specific expression of LPL activity in mice. One of the olism and transport of lipids (1).It is responsible for the loci, controlling LPL activity in heart, is associated hydrolysis of core triglycerides in chylomicrons and very low with an alteration in LPL mRNA size, whilethe other, density lipoproteins, thereby providing fatty acids to periphcontrolling LPL activity in adipose tissue, appears to eral tissues for storage (adipose tissue) or oxidation (muscle affect the translationo r post-translational expression and other tissues). This hydrolysis is also important for the of LPL. To examine whether these genetic variations maturation of lipoprotein particles, andthusthe enzyme are due to mutations of the LPL structural locus, we affects cholesterol as well as triglyceride metabolism. Individmapped the LPL gene ato region of mouse chromosome uals with rare deficiencies of LPL, or of its required cofactor 8 using restrictionfragment-length polymorphisms apolipoprotein CII, exhibit massive accumulation of blood and analysis of hamster-mouse somatic cell hybrids, chylomicrons and corresponding increases in blood triglycerThis regionis homologous to the region of human chro- ides, a condition termed type1 hyperlipoproteinemia (2). The mosome 8 which contains the human LPL gene as physiologically active form of LPL is localized at thesurface judged by the conservationof linked genetic markers, of the vascular endothelium, where it is anchored by a memGenetic variations affecting LPL expression in heart brane-bound glycosaminoglycan chain. The synthesis of the cosegregated with the LPLgene, while variations affecting LPL expression in adipose tissue did not.Fur- enzyme, however, occurs in parenchymal cells, from which it is secreted andthen transported by a poorly understood thermore, Southern blotting analysis indicates that LPL is encoded by a single gene and, thus, the genetic process to the lumenal surface of endothelial cells. The endifferences are not a consequence of independent reg- zyme has been identified in a wide variety of extrahepatic ulation of two separategenes in the twotissues. These tissuesand cells, including adipose tissue, heart, skeletal results suggest the existenceof cis-acting elements for muscle, lung, lactating mammary gland, brain, and macroLPL gene expression that operate in heart but not phages (1).Recently, we isolated complementary DNAs for adipose tissue. Our results also indicate that two ge- both mouse (3) and human (4) LPL. Sequence comparisons indicate that LPL is a member of a gene family including hepatic lipase and pancreatic lipase (3, 5). *This work was supported by Grants HL28481, GM18684, One intriguing aspect of LPL expression relates toits AM30292, and AM37960 from the National Institutes of Health, the Veterans Administration, andthe American Heart Association, tissue-specific regulation. During postnataldevelopment, the Greater Los Angeles Affiliate (Grants 492-IG15 and 816F1 to R. C. enzyme activity in certain tissues, including heart, undergoes L.). The costs of publication of this article were defrayed in part by large changes (6). Similarly, LPL activity exhibits striking the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 increases (several orders of magnitude) during the differentiation of certain preadipocyte cell lines in uitro (7, 8). Thus, solely to indicate this fact. 11 Recipient of a Josiah Macy Foundation pre-doctoral fellowship. LPL is highly regulated in a tissue-specific manner during

$5 Established Investigator of the American Heart Association.

MI To whom correspondence should be addressed Dept. of Medi-

cine, UCLA, Los Angeles, CA 90024.

* The abbreviations used are: LPL, lipoprotein lipase; RI, recombinant inbred; kb, kilobase pairs.

1473

1474

Genetic and Developmental Control of the Lipoprotein Lipase Gene

development, although it is not known at what level(s) of gene expression this regulation occurs. In fact, although LPL is present in avariety of tissues, the sites of enzyme synthesis are unclear. Enzyme activity in adults is regulated in a complex manner in response to dietary and hormonal changes, and this also occurs in a tissue-specific manner. For example, adipose LPL activity is high in fed animals and low during fasting (9),whereas the reciprocal relationship exists in heart and skeletal muscle (1, 10). In addition, genetic studies of polymorphisms among various inbred strainsof mice indicate that the enzyme activity in different tissues is under independent genetic control. For instance, strain C57BL/6 has higher activity than strainBALB/c in heart but lower activity in adipose tissue. Studies of genetic crosses between the two strains indicate that heart and adipose activity are controlled by separate major genetic loci (11). Independent genetic control of LPL activity in different tissues hasalso been observed in humans. While most LPL-deficient patients lack activity in all tissues, a minority of patients exhibit the deficiency in only some tissues (12). Finally, although LPL is expressed principally in extrahepatic tissues and hepatic lipase is expressed only in liver, mutations have been identified in both mice (13-15) andhumans (12) which result in combined deficiencies of both enzymes. It has been speculated that such mutations could result from focaldisruptions of the two genes if present as a gene cluster (12). Alternatively, the mutations may disrupt regulatory or enzyme processing pathways shared by LPL andhepatic lipase. Here, we report studies of LPL tissue-specific expression addressing the following questions: What is the relative abundance of LPL mRNA in various tissues? Do changes in LPL activity during development of tissues and differentiation of cell lines reflect changes in the expression of LPL mRNA? What is the molecular basis for the independent genetic regulation of LPL indifferent tissues? What is the natureof mutations resulting in deficiencies of LPL? EXPERIMENTALPROCEDURES

Animals-Mice were obtained from Jackson Laboratory (Bar Harbor, ME). The strains used were AKR/J (AKR), BALB/cJ (BALB), C3H/HeJ (C3H), C57BL/6J (C57BL/6), DBA/23 (DBA/2), NZB/ B l N J (NZB), SWR/J (SWR), 129/J (129), PL/J (PL), and various recombinant inbred strains. Female mice 4 months of age were used. All mice werefasted overnight (16-18 h) prior to collection of tissues. Sprague-Dawley rats were obtained from Sasco (St. Louis, MO). Animals were fedad libitum a standard chow diet (Rodent LabChow 500, Ralston Purina). For developmental studies, timed pregnant female, neonatal, and adult male rats were purchased from Sasco. Littermates were decapitated between 1000 a.m. and 2:OO p.m., and the tissues wereremoved and quickly frozen in liquid nitrogen. Tissues were pooled from 10-40animals at each developmental time surveyed, and total cellular RNA was extracted from pooled, pulverized specimens as described below. Cultured Cell Lines-Total cellular RNA was isolated from murine BC3H1 cells prior to andafter differentiation. Undifferentiated cells were grown in DME supplemented with heat-inactivated fetal calf serum (final concentration 20'%),L-glutamine (2 mM), streptomycin (100 pglml), and penicillin (100 units/ml). RNA was isolated from these cells afterthey had grown to about 50% confluency. The protocol used for inducing differentiation to a myocyte-like cell by serum deprivation was identical to that described by Lathrop et al. (16). Differentiation was verified by measuring creatine phosphokinase levels (16). RNA was extracted from murine 3T3L1 cells prior to andafter differentiation to adipocytes as described by Bernlohr et al. (17). cDNA Probes-Mouse LPL cDNA inserts, mL5, mLl1, and mL31 (3), and human LPL cDNA insert LPL46 (4) were isolated by gel electrophoresis and labeled with [32P]dCTPto a specific activity of about 1 X lo9 cpmlpg using an oligonucleotide random priming

procedure (18). Studies of hepatic lipase utilized a full-length rat hepatic lipase cDNA (19). Southern Blotting-Mouse and human genomic DNA wasisolated from spleen cell and lymphocyte nuclei (20), respectively, by incubation in 10 mM EDTA, pH 8.0, 0.2% sodium dodecyl sulfate with proteinase K (600 pg/ml) at 37"C for 24 h followedby phenol extraction and ethanol precipitation. Southern blots were prepared using nylon filters and hybridized to 32P-labeledLPL cDNA or 32Plabeled hepatic lipase cDNA as previously described (21). RNA Blot Hybridization Analysis-Total tissue RNA with the exception of adipose, was extracted from pulverized specimens with guanidinium thiocyanate followed bycentrifugation through a cesium chloride gradient (22). Adipose RNA was isolated as described by Rosen et al. (23). The integrity of each RNA preparation was verified by denaturing gel electrophoresis (24). Northern blottingand hybridization were performed as previously described (3, 25). RNA dot blots were constructed as previously described (26). Briefly, four aliquots (0.5, 1, 2, and 3 pg) of each RNA preparation were applied to nitrocellulose filters using a template manifold (Bethesda Research Labs). Yeast tRNA was added to tissue RNA samples prior to their denaturation so that the amount of RNA contained in each dot would be identical (3 pg total). Stringencies employed for filter hybridization and washing were as in Demmer et al. (26). The relative abundance of LPL mRNAs in tissue RNA preparations was calculated by scanning filter autoradiographs with an LKBUltroscan XL laser densitometer. To determine if signals were in the linear range of film sensitivity, total cellular adult heart and liver RNA was applied to filters at concentrations ranging from 0.03 to 3 pg/dot. These standards were also used to normalize signals between filters probed with the same preparation of 32P-labeledcDNA and todetermine the limits of sensitivity of LPL mRNA detection in samples of total RNA. Somatic Cell Hybrid Studies-A panel of mouse-hamster somatic cell hybrids was generously provided by Dr. T. Mohandas, Division of Medical Genetics, Harbor-UCLA Medical Center. These were prepared by fusion of thymidine kinase-deficient hamster fibroblasts (RJK) and mouse spleen cells using a previously described protocol (27). A panel of 10 clones was grown up for DNA extraction and chromosome studies. Chromosome analysis was performed on a minimum of 30 metaphases per hybrid clone. DNA samples were digested with either Hind111 or EcoRI and 15 pg from each digest was fractionated by electrophoresis through 1.2% agarose followed byblotting onto nylon filters. Hybridization with mouse LPL cDNAor rat hepatic lipase cDNA was performed as described above. RESULTS

TissueDistribution of LPL mRNA in AdultRodentsTwelve rat and 9 mouse tissues were surveyed from animals fed standard chow diets adlibitum. When dot blots of rat and mouse tissue RNA samples were probed with a murine LPL cDNA the results shown in Fig. 1and Table I were obtained. Among the adult (7O-day-old, 250-g) malerat tissues studied, the highest concentration of reactive mRNA was found in heart. RNA prepared from soleus muscle, a slow twitch, skeletal muscle which depends primarily on fatty acid oxidation for energy (28, 29), produced a signal which was essentially equivalent (98%) to that produced by heart RNA. A representative fast twitch muscle (psoas) which depends more on glucose for its energy requirements (28,30),contained only 21% as much reactive mRNA as that found in heart. RNA from adipose, a tissue that stores fatty acids derived in part from circulating lipoproteins through the action of lipoprotein lipase, produced a signal that was 88% that found in heart, while adult adrenalRNA produced a signal that was 41% that found in heart.RNA isolated from testis, an organ known to oxidize and esterify fatty acids (31), produced a signal that was only 2% of the reference heart RNA. Adult brain, which does not effectively use fatty acids as an energy source (32), had LPL mRNA levels only 1% of those in heart. Although fatty acids are a major energy source in the renal cortex (33, 34), only trace amountsof hybridizable mRNA werediscerned

Genetic and Developmental Controlof the Lipoprotein LipaseGene

1475

RNA. We did not detect LPL mRNA in adult rat or mouse liver (a tissue which oxidizesfatty acids) in this dot blot assay, however, we have previouslydetected low levels inmouse liver by Northern analysis (3). The limits of sensitivity of the dot blot assay were determined by noting that signals could be detected in dots containing as little as 30 ng of total heart RNA (data not shown). The specificity of the signals produced by rat tissue RNAs was assessed by Northern blot hybridizations. TheLPL cDNA probe reacted with a single 3.6-kb mRNA species in samples of total cellular adrenal, heart, psoas, soleus, and testicular RNAs (e.g. see Lane 1 in Fig. 1C). This finding is consistent with our earlier observation (3) that a single LPL mRNA species is derived from the rat LPLgene. The pattern of tissue-specific expression of the LPL gene in adult BALB/c mice was qualitatively similar to that observed in Sprague Dawley rats (Table I). However,some quantitative differences in relative tissue distribution were FIG.1. Distribution of LPL mRNA in adult rat tissues. Total noted. For example, LPL mRNA was relatively more abuncellular RNAwas isolated from tissues of 10 adult male Sprague dant in mouse kidney compared to rat kidneyRNA (11% Dawley rats fed a standard chow diet ad libitum. Panel A , an autoradiograph of a dot blot containing various tissue RNA samples after uersus 4 % of reference heart values) whileit was less abunit was probed with a 32P-labeledmurine LPL cDNA. Stringencies dant in mouse skeletal muscle comparedto rat. An additional used for hybridization and washing were identical to those described difference in LPL gene expression betweenthese two rodent by Demmer et al. (26).Panel B, relative concentrations of LPL mRNA species is that two LPL mRNA species exist in these mouse in adult tissue RNA samples calculated after quantitative scanning laser densitometry of the dot blot shown in Panel A (see “Experimen- tissues, one identical to the rat mRNA (3.6 kb) and other tal Procedures”). Lanes I and 2 in Panel C illustrate the results other 200 nucleotides smaller (3). Among mousetissue RNAs obtained when a Northern blot was prepared after formaldehyde- the relative amounts of the two species appeared constant. agarose electrophoresis of t o t a l cellular RNA extracted from rat testis Based on studies of human LPL it islikely that the two (Lane 1 ) and murine 3T3L1 cells after differentiation to adipocytes mRNA species in mice are due to differences in the length of (15 pg of RNA was applied to each slot). The nitrocellulose blot was probed with the same LPL cDNAused for the dot blot studies their 3”nontranslated regions and are derived by alternate employing identical hybridization and washing stringencies. Note use of two polyadenylationsignals (4). that Lane 1 represents a 46-h exposure of the autoradiograph, while LPL mRNA Expression in Preadipocyte and Myocyte Cell Lane 2 represents results obtained after a 6-hexposure. Lines: Induction during Preadipocyte Differentiation-Murine 3T3L1 cells can be inducedto differentiate into adipocytes at TABLE I high frequency(35,36). Once inducedthese cells exhibit many Relative LPL mRNA concentrations in samples of RNA prepared of the morphological and biochemical characteristics of adifrom a variety of adult rodent tissues and cell lines pocytes. Differentiation isaccompanied by induction of a Values were determined by quantitative scanning laser densitom- number of genes (17,37-39) including those encoding glyceretry of RNA dot blots (see “Experimental Procedures” and Fig. 1). Data have been expressed as a percentage of the signal produced from ophosphate dehydrogenase, an enzyme involved in triglyceride synthesis, as well as aP2, a protein which exhibits rethe reference adult heart RNA samdes. markable sequence similarity to a fatty acid binding protein Tissues and cell lines Sprague Dawley rat BALB/c mouse found inheart andskeletal muscle (40). Dot blot hybridization ?6 7% studies disclosed that differentiation of 3T3L1 cells to adiHeart 100 100 pocytes was accompanied by a 7-foldrise in LPL mRNA Skeletal muscle 13 levels (Fig. 2 and Table I). While clearly subconfluent, the Soleus 98 Psoas 21 3T3-Ll pre-adipocytes from which RNA was prepared were Adrenal 41 at a density such that thecells were likely committed and had Testes 2 already begun to differentiate (50-70% confluent). With LPL Lung 3 4 being a very early marker in 3T3-Ll differentiation (37), this Spleen 1 Brain 1 Not detectable would explain the relatively low-foldinduction in LPL mRNA Small intestine Colon Liver Kidney Adipose 3T3 L1cells Undifferentiated Adipocyte-like BC3H1 cells Undifferentiated Myocyte-like

2 c1 Not detectable