Normal Sorting but Defective Endocytosis of the Low Density Lipoprotein Receptor in Mice with Autosomal Recessive Hypercholesterolemia

Autosomal recessive hypercholesterolemia (ARH) is a genetic form of hypercholesterolemia that clinically resembles familial hypercholesterolemia (FH). As in FH, the rate of clearance of circulating low density lipoprotein (LDL) by the LDL receptor (LDLR) in the liver is markedly reduced in ARH. Unlike FH, LDL uptake in cultured fibroblasts from ARH patients is normal or only slightly impaired. The gene defective in ARH encodes a putative adaptor protein that has been implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in the liver, ARH-deficient mice were developed. Plasma levels of LDL-cholesterol were elevated in the chow-fed Arh–/– mice (83 ± 8 mg/dl versus 68 ± 8 mg/dl) but were lower than those of mice expressing no LDLR (Ldlr–/–) (197 ± 8 mg/dl). Cholesterol feeding elevated plasma cholesterol levels in both strains. The fractional clearance rate of radiolabeled LDL was reduced to similar levels in the Arh–/– and Ldlr–/– mice, whereas the rate of removal of α2-macroglobulin by the LDLR-related protein, which also interacts with ARH, was unchanged. Immunolocalization studies revealed that a much greater proportion of immunodetectable LDLR, but not LDLR-related protein, was present on the sinusoidal surface of hepatocytes in the Arh–/– mice. Taken together, these results are consistent with ARH playing a critical and specific role in LDLR endocytosis in the liver. Autosomal recessive hypercholesterolemia (ARH) is a genetic form of hypercholesterolemia that clinically resembles familial hypercholesterolemia (FH). As in FH, the rate of clearance of circulating low density lipoprotein (LDL) by the LDL receptor (LDLR) in the liver is markedly reduced in ARH. Unlike FH, LDL uptake in cultured fibroblasts from ARH patients is normal or only slightly impaired. The gene defective in ARH encodes a putative adaptor protein that has been implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in the liver, ARH-deficient mice were developed. Plasma levels of LDL-cholesterol were elevated in the chow-fed Arh–/– mice (83 ± 8 mg/dl versus 68 ± 8 mg/dl) but were lower than those of mice expressing no LDLR (Ldlr–/–) (197 ± 8 mg/dl). Cholesterol feeding elevated plasma cholesterol levels in both strains. The fractional clearance rate of radiolabeled LDL was reduced to similar levels in the Arh–/– and Ldlr–/– mice, whereas the rate of removal of α2-macroglobulin by the LDLR-related protein, which also interacts with ARH, was unchanged. Immunolocalization studies revealed that a much greater proportion of immunodetectable LDLR, but not LDLR-related protein, was present on the sinusoidal surface of hepatocytes in the Arh–/– mice. Taken together, these results are consistent with ARH playing a critical and specific role in LDLR endocytosis in the liver. Low density lipoproteins (LDL) 1The abbreviations used are: LDL, low density lipoprotein; LDL-C, LDL cholesterol; LDLR, LDL receptor; LRP, LDLR-related protein; VLDL, very low density lipoprotein; α2M, α2-macroglobulin; ARH, autosomal recessive hypercholesterolemia; FH, familial hypercholesterolemia; PTB, phosphotyrosine binding. particles are the major cholesterol transport vehicle in the circulation. Approximately 70% of cholesterol in human plasma is contained in the LDL fraction. The major fate of circulating LDL is uptake into the liver by LDLR-mediated endocytosis (1Osono Y. Woollett L.A. Herz J. Dietschy J.M. J. Clin. Invest. 1995; 95: 1124-1132Crossref PubMed Scopus (164) Google Scholar, 2Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Vol. II. McGraw-Hill Inc., New York2001: 2683-2913Google Scholar). The rate of removal of LDL from the plasma is a key determinant of plasma cholesterol levels. Reductions in hepatic LDLR number or activity result in elevated plasma levels of LDL-C. The most common monogenic cause of severe hypercholesterolemia is the autosomal dominant disorder, familial hypercholesterolemia (FH), caused by mutations in LDLR (2Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Vol. II. McGraw-Hill Inc., New York2001: 2683-2913Google Scholar, 3Goldstein J.L. Brown M.S. Science. 2001; 292: 1310-1312Crossref PubMed Scopus (202) Google Scholar). FH homozygotes, who have mutations in both LDLR alleles, have a markedly decreased rate of removal of circulating LDL and a dramatic increase in plasma cholesterol levels (2Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Vol. II. McGraw-Hill Inc., New York2001: 2683-2913Google Scholar). The hypercholesterolemia results in deposition of cholesterol in body tissues, including skin and tendons (xanthomas), and coronary arteries, causing premature coronary atherosclerosis (2Goldstein J. Hobbs H. Brown M. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. Vol. II. McGraw-Hill Inc., New York2001: 2683-2913Google Scholar). Recently, the molecular defect for a recessive form of hypercholesterolemia that clinically resembles FH but is not due to mutations in LDLR was identified (4Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar). This disorder, called autosomal recessive hypercholesterolemia, is caused by mutations in the putative adaptor protein ARH (5Wilund K.R. Yi M. Campagna F. Arca M. Zuliani G. Fellin R. Ho Y.K. Garcia J.V. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (92) Google Scholar). Patients lacking ARH clear LDL from the circulation at rates as low as those observed in patients with no functioning LDLRs (6Zuliani G. Arca M. Signore A. Bader G. Fazio S. Chianelli M. Bellosta S. Campagna F. Montali A. Maioli M. Pacifico A. Ricci G. Fellin R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar). Immortalized lymphoblasts from ARH patients exhibit impaired LDL internalization, despite expressing 2-fold more LDLRs on the cell surface than lymphoblasts from unaffected individuals (5Wilund K.R. Yi M. Campagna F. Arca M. Zuliani G. Fellin R. Ho Y.K. Garcia J.V. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (92) Google Scholar, 7Norman D. Sun X.-M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Invest. 1999; 104: 619-628Crossref PubMed Scopus (73) Google Scholar). In contrast to ARH-deficient lymphoblasts and hepatocytes, cultured fibroblasts from ARH patients have preserved LDLR function, with levels of LDLR activity ranging from 30 to 100% of normal (8Arca M. Zuliani G. Wilund K. Campagna F. Fellin R. Bertolini S. Calandra S. Ricci G. Glorioso N. Maioli M. Pintus P. Carru C. Cossu F. Cohen J. Hobbs H.H. Lancet. 2002; 359: 841-847Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Although ARH and FH homozygotes have similar rates of LDL clearance (6Zuliani G. Arca M. Signore A. Bader G. Fazio S. Chianelli M. Bellosta S. Campagna F. Montali A. Maioli M. Pacifico A. Ricci G. Fellin R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar), ARH patients generally have lower plasma cholesterol levels and later onset of cardiovascular disease than FH homozygotes (8Arca M. Zuliani G. Wilund K. Campagna F. Fellin R. Bertolini S. Calandra S. Ricci G. Glorioso N. Maioli M. Pintus P. Carru C. Cossu F. Cohen J. Hobbs H.H. Lancet. 2002; 359: 841-847Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 9Zuliani G. Vigna G.B. Corsini A. Maioli M. Romagnoni F. Fellin R. Eur. J. Clin. Invest. 1995; 25: 322-331Crossref PubMed Scopus (49) Google Scholar). ARH contains a single phosphotyrosine-binding (PTB) domain, which is capable of binding the LDLR cytoplasmic tail in vitro (10He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Mishra S.K. Watkins S.C. Traub L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16099-16104Crossref PubMed Scopus (148) Google Scholar). Adaptor proteins containing PTB domains bind the conserved NPXY sequence motif located in the cytoplasmic domains of various cell surface receptors and mediate diverse cellular functions, including receptor trafficking and endocytosis (12Forman-Kay J.D. Pawson T. Curr. Opin. Struct. Biol. 1999; 9: 690-695Crossref PubMed Scopus (107) Google Scholar, 13Yan K.S. Kuti M. Zhou M.-M. FEBS Lett. 2002; 513: 67-70Crossref PubMed Scopus (60) Google Scholar). The LDLR cytoplasmic tail contains a single NPXY motif that is required for clustering and endocytosis of the receptors in fibroblasts (14Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar, 15Davis C.G. Lehrman M.A. Russell D.W. Anderson R.G. Brown M.S. Goldstein J.L. Cell. 1986; 45: 15-24Abstract Full Text PDF PubMed Scopus (241) Google Scholar). Point mutations in this highly conserved sequence cause FH and eliminate binding of ARH to LDLR in vitro (10He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Mishra S.K. Watkins S.C. Traub L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16099-16104Crossref PubMed Scopus (148) Google Scholar, 15Davis C.G. Lehrman M.A. Russell D.W. Anderson R.G. Brown M.S. Goldstein J.L. Cell. 1986; 45: 15-24Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 16Garcia-Garcia A.B. Real J.T. Puig O. Cebolla E. Marin-Garcia P. Martinez Ferrandis J.I. Garcia-Sogo M. Civera M. Ascaso J.F. Carmena R. Armengod M.E. Chaves F.J. Hum. Mutat. 2001; 18: 458-459Crossref PubMed Scopus (31) Google Scholar). In addition to binding the LDLR tail, ARH also binds the β2-adaptin subunit of AP-2 and the terminal domain of clathrin in a sequence-specific manner in vitro, so it has been proposed to link the LDLR to the endocytic machinery (4Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar, 10He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In support of this scenario, immortalized lymphocytes from ARH subjects are defective in LDL internalization (7Norman D. Sun X.-M. Bourbon M. Knight B.L. Naoumova R.P. Soutar A.K. J. Clin. Invest. 1999; 104: 619-628Crossref PubMed Scopus (73) Google Scholar). Although human studies suggest that ARH is required specifically for normal LDLR function, it may also be involved in the internalization of other proteins containing an NPXY motif in the cytoplasmic tail, such as other members of the LDLR gene family. For instance, the LDLR-related protein (LRP) contains two NPXY motifs. One of them binds the PTB domain protein Dab1, an adaptor protein that is involved in the control of neuronal migration and signaling through LDLR family members and that also interacts with the LDLR (17Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 18Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar). LRP is abundantly expressed in the liver, where it functions in concert with the LDLR in the clearance of chylomicron remnants (19Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (255) Google Scholar, 20Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (400) Google Scholar), but it is unknown whether LRP also functionally interacts with ARH. In the initial three ARH patients examined, an accumulation of chylomicron remnants was not reported (6Zuliani G. Arca M. Signore A. Bader G. Fazio S. Chianelli M. Bellosta S. Campagna F. Montali A. Maioli M. Pacifico A. Ricci G. Fellin R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (85) Google Scholar), suggesting that ARH may not be required for normal LRP endocytosis. The currently available limited data from human subjects suggest that ARH function may be tissue-specific, inasmuch as LDL internalization in ARH patients was found to be defective in the liver and in lymphoblasts, but is apparently largely normal in fibroblasts from the same patients. To facilitate the investigation of ARH function in the liver, the central organ that regulates LDL metabolism, we have now generated ARH-deficient mice by gene targeting. These animals replicate central features of the human disease phenotype, including abnormal sequestration of the LDLR at the cell surface and reduced internalization of LDLR but not LRP. ARH-deficient mice are currently the only physiological model system on which the role of this specialized adaptor protein in receptor-mediated endocytosis can be experimentally investigated. General Methods—Unless otherwise indicated, DNA manipulations were performed by standard techniques (21Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). Cholesterol and triglycerides were determined enzymatically with assay kits obtained from Roche Applied Science and Sigma, respectively. Mouse LDL (d 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation (22Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar) from pooled plasma obtained from Ldlr–/– mice (23Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1273) Google Scholar) that had been fasted >6 h. Lipoproteins were radiolabeled with 125I by the iodine monochloride method (22Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Methylamine-activated α2-macroglobulin was radiolabeled with 125I using the iodogen method as described previously (24Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar). Antibodies—Rabbit polyclonal antibodies against Rab5 (KAP-GP006), EEA1 (324610), and GRP78 (BiP) (SPA-826) were purchased from StressGen Biotechnologies (Victoria, BC, Canada) and Calbiochem. Rabbit polyclonal antibodies against Lamp1 (H-228) and Rab11 (H-87) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies were raised in rabbits against the PTB domain of mouse ARH and the C-terminal 13 residues of mouse LDLR. Rabbit polyclonal antibodies against LRP and purified LDLR from bovine adrenal have been described elsewhere (25Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (738) Google Scholar, 26Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (218) Google Scholar). Cloning of Mouse ARH cDNA—Murine ARH cDNA was amplified from a commercially available mouse liver cDNA library (Clontech) using the following primers: 5′-ATTCTAGACATGGACGCGCTCAAGTCGGCG-3′ and 5′-TTAAGCTTTCAGAAGGTGAAGACGTCATCATC-3′. Amplification products were TA-cloned into pCR2.1-TOPO (Invitrogen) and sequenced. Generation of ARH Knockout Mice—Two fragments were amplified from genomic DNA from mouse liver (129S6/SvEv strain) using long range PCR (Takara Biochemical, Inc., Berkeley, CA). An 8.5-kb fragment, corresponding to a region extending from intron 4 into the 3′-untranslated region, was derived using primers 5LA1 (5′-ACGGCGGCCGCCTGTGTGGCCTGAGTCCCTCCCTGG-3′) and 3LA1 (5′-CCTGCGGCCGCCGCCAGCATGAGCAAGC-3′), and a 1-kb fragment containing part of intron 3 was amplified using primers 5SA (5′-ACGCTCGAGGTGTGCCAGTGGGGAACCAGGAAGG-3′) and 3SA (5′-TGCCTCGAGGGGAGATGGAAATAAGAAATGAAGGAAGC-3′). Bold characters indicate inserted restriction sites. The two fragments were cloned into the targeting vector (27Gotthardt M. Hammer R.E. Hubner N. Monti J. Witt C.C. McNabb M. Richardson J.A. Granzier H. Labeit S. Herz J. J. Biol. Chem. 2003; 278: 6059-6065Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) on either side of a PGKneobpA expression cassette (28Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1799) Google Scholar) using NotI and XhoI sites, respectively. The vector also contained two copies of the herpes simplex virus thymidine kinase gene (29Mansour S.L. Thomas K.R. Capecchi M.R. Nature. 1988; 336: 348-352Crossref PubMed Scopus (1325) Google Scholar) in tandem at the 5′ end of the short arm. The linearized vector was electroporated into murine embryonic SM1 stem cells, and recombinant clones were selected using G418 and ganciclovir, as described (30Willnow T.E. Herz J. Methods Cell Biol. 1994; 43: 305-334Crossref PubMed Scopus (29) Google Scholar). Homologous recombination was identified by PCR (28Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1799) Google Scholar) using primers ARH-T (5′-ACGCTCGAGCAGCCCAAATCCATGCTATCCATGGAC-3′) and Neo-36 (5′-CAGGACAGCAAGGGGGAGGATTGGGAAGAC-3′) and confirmed by Southern blot analysis after EcoRI and SacI digestion. Twelve independent stem cell clones containing a disrupted arh allele were injected into C57Bl/6 blastocysts, yielding a total of 18 chimeric males. Of these, 17 were fertile, and 6 gave offspring that carried the disrupted allele. Two separate lines were established for experimentation. No differences were detected between the two lines. Animals were genotyped by allele-specific PCR, using three primers. Primer ARH-T was used as the upstream oligonucleotide, whereas primers Neo-36 and ARH-GW (5′-CCTGTACTCCCAGACTACTTCATGATCCCCAC-3′) were used as the downstream primers for the disrupted and wild-type alleles, respectively. Animal Housing and Feeding—Mice were housed on a 12-h dark/12-h light cycle and given standard chow (number 7002; Harlan Teklad, Madison, WI) and water ad libitum. In cholesterol feeding experiments, animals were either fed the Paigen high cholesterol diet (1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% cholic acid) or fed a Western-type diet containing 0.2% cholesterol (31Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). All experiments were performed with F2 or F3 generation descendents, which were hybrids between C57Bl/6 and 129Sv strains. No differences in the results were observed when littermates were used in the experiments or when animals from vertically inbred lines were compared. Clearance of LDL and α2-Macroglobulin from Plasma—Clearance of lipoprotein and α2-macroglobulin (α2M) was determined as described previously (23Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1273) Google Scholar, 32Choi S.Y. Cooper A.D. J. Biol. Chem. 1993; 268: 15804-15811Abstract Full Text PDF PubMed Google Scholar). Briefly, mice were anesthetized with sodium pentobarbital (80 mg/kg) and injected via the external jugular vein with a 0.2-ml intravenous bolus of either 15 μg of 125I-LDL or 5 μg of 125I-α2M in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.2% (w/v) bovine serum albumin. At various time points, blood samples were drawn from the retro-orbital plexus into EDTA-coated tubes (Microvette 500 KE, Sarstedt, Newton, NC). The plasma content of 125I-labeled protein was measured by trichloroacetic acid precipitation followed by γ counting. The amount of LDL or α2M remaining in the blood was expressed as a percent of the initial blood concentration, measured as the average 125I-radioactivity in the plasma 2 min after injection. Interaction of ARH PTB Domain with Lipoprotein Receptor Cytoplasmic Tails—The LDLR family cytoplasmic tails/LexA fusion protein constructs and the Dab1 prey construct have been described previously (33Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). The ARH-PTB domain was amplified from the cloned cDNA using the primers 5APY (5′-ATGGAATTCAGCCTCAAGTACCTTGGTATGACG-3′) and 3APY (5′-TTGTCGACTCAGGACACCTGCCAAAACTCAAAGGC-3′). Bold characters indicate inserted restriction sites. The resulting PCR product was digested with EcoRI and SalI, inserted into the EcoRI-XhoI-digested prey vector pB42AD (MATCHMAKER system, Clontech), and sequenced. Yeast transformations and matings were performed following the manufacturer's instructions in the MATCHMAKER manual, and interactions were assessed as described previously (33Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Immunohistochemistry—Anesthetized mice were perfused by cardiac puncture with warm Hank's balanced salt solution followed by 4% (w/v) paraformaldehyde in phosphate-buffered saline. The livers were removed and divided into 0.5-cm2 sections. The tissue was fixed for an additional hour at 25 °C in 4% (w/v) paraformaldehyde in phosphate-buffered saline followed by an overnight incubation in 30% (w/v) sucrose solution. The tissue was frozen in OCT compound 4583 (Miles Laboratories, Elkhart, IN) over dry ice and stored at –70 °C until cutting. Sections of 7 μm were cut on a Leitz Cryostat (E. Leitz, Inc., Rockleigh, NJ) at –20 °C and mounted onto poly-l-lysine-coated slides. Samples were blocked by incubation for 1 h with 10 mm Tris-HCl, pH 9.0, 150 mm NaCl (Tris-buffered saline) containing 20% (v/v) normal goat serum and 1% (w/v) bovine serum albumin. Sections were then incubated overnight with rabbit antiserum raised against the PTB domain of ARH (1:800 dilution), rabbit antiserum against LRP (25Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (738) Google Scholar) (1:200), or polyclonal rabbit IgG directed against the LDLR (26Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (218) Google Scholar) (1:400). Slides were washed three times in Tris-buffered saline/0.1% bovine serum albumin, and bound primary antibody was detected by incubation for 2 h with 20 μg/ml Alexa-Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Slides were washed three times in Tris-buffered saline/0.1% bovine serum albumin, rinsed once with water, and mounted under a coverslip with Immu-mount (Shandon, Pittsburgh, PA). Images were taken using the 63 × 0.70 objective on a Leica confocal microscope. Sucrose Gradients—Male wild-type, Arh–/–, and Ldlr–/– mice aged 15 weeks were fasted for 6 h and sacrificed. The livers were removed, rinsed briefly in cold phosphate-buffered saline, and minced in a weigh boat over ice. The minced liver was homogenized at 20% (w/v) in cold homogenization buffer (50 mm Tris-HCl, pH 7.4, 250 mm sucrose, 25 mm KCl, 5 mm MgCl2, 3 mm imidazole, Roche protease inhibitor mixture) with 20 strokes in a Dounce homogenizer. Nuclei and other debris were removed by low speed centrifugation at 1000 × g for 10 min at 4 °C. 300 μl of the supernatant was loaded on a 4-ml continuous 10–40% sucrose gradient and centrifuged using a Beckman SW60 Ti rotor for 16 h at 40,000 rpm, as described previously (34Stockinger W. Sailler B. Strasser V. Recheis B. Fasching D. Kahr L. Schneider W.J. Nimpf J. EMBO J. 2002; 21: 4259-4267Crossref PubMed Scopus (103) Google Scholar). A 20-gauge needle was used to puncture the bottom of each tube, and ∼200-μl fractions were collected. Protein was precipitated with trichloroacetic acid, neutralized with NaOH, and dissolved in 100 μl of sample buffer. A 10-μl aliquot was used for Western blotting. Generation of ARH-deficient Mice—To disrupt the Arh gene in the mouse, targeted homologous recombination was used to replace exon 4 of Arh with a neomycin resistance cassette (Fig. 1A). Gene disruption was confirmed by Southern blotting (Fig. 1B). Deletion of exon 4 disrupts the PTB domain and is predicted to render the protein unable to bind the NPXY motifs of LDLR family members. If splicing occurs between exons 3 and 5, a frameshift and nonsense mutation would be introduced upstream of the putative binding sites for clathrin and AP-2 (10He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). No immunoreactive ARH protein was detected in liver lysates of mice homozygous for the disruption using an antibody directed against the PTB domain (Fig. 1C). Levels of both LDLR and LRP protein were unchanged in the Arh–/– mice. Arh–/– mice were fertile and had normal litter sizes. Arh–/– Mice Are Hypercholesterolemic and Sensitive to Dietary Cholesterol—Plasma cholesterol levels were only mildly elevated in the chow-fed Arh–/– mice and not significantly different in heterozygous mice when compared with wild-type littermate controls (Table I and Fig. 2A). Female Arh+/–, Arh–/–, Ldlr–/–, and wild-type mice were fed chow alone, chow supplemented with 0.2% cholesterol-containing Western-type diet, or chow supplemented with 1.25% cholesterol (Paigen diet) for 4 weeks. On the chow diet, the mean plasma level of cholesterol in the ARH–/– mice was intermediate (83 mg/dl) to the wild-type (68 mg/dl) and Ldlr–/– mice (196 mg/dl). On the Western diet, plasma cholesterol levels were increased to a similar level in the Arh–/– and Ldlr–/– mice (Table I and Fig. 2A). Feeding of the Paigen diet resulted in dramatic elevations of plasma cholesterol levels to an average of 1270 mg/dl for the Arh–/– mice and 1442 mg/dl for the Ldlr–/– mice. No significant differences were seen in plasma lipid levels between the Arh+/– mice and wild-type mice on any of the diets, which is consistent with the autosomal recessive inheritance pattern of ARH in humans. Liver cholesterol and triglyceride levels were not elevated in the Arh–/– mice over their wild-type littermates (Table I).Table ICholesterol and triglyceride levels in response to cholesterol feeding0.02% cholesterol0.2% cholesterol1.25% cholesterol/0.5% cholic acidPlasma cholesterol (mg/dl)Wild type67.7 ± 8.1125.9 ± 38.5184.9 ± 35.8ARH+/-94.6 ± 18.5157.1 ± 22.3155.7 ± 25.2ARH-/-83.1 ± 7.9307.9 ± 62.91270.0 ± 7.9LDLR-/-196.4 ± 7.6239.1 ± 80.51442.4 ± 42.0Plasma triglycerides (mg/dl)Wild type58.8 ± 4.556.6 ± 9.647.2 ± 5.0ARH+/-56.2 ± 9.361.4 ± 6.546.2 ± 1.8ARH-/-58.7 ± 7.8100.0 ± 12.549.0 ± 10.4LDLR-/-97.4 ± 12.194.8 ± 22.7129.2 ± 1.4Liver tissue cholesterol (mg/g)Wild type2.64 ± 0.056.80 ± 1.7319.58 ± 0.75ARH+/-2.69 ± 0.1010.44 ± 2.6415.97 ± 2.00ARH-/-2.67 ± 0.156.76 ± 0.7523.23 ± 0.21LDLR-/-3.27 ± 0.334.46 ± 0.4020.96 ± 0.76Liver tissue triglycerides (mg/g)Wild type26.0 ± 3.127.3 ± 6.729.0 ± 1.2ARH+/-22.3 ± 7.533.5 ± 2.419.9 ± 7.0ARH-/-22.9 ± 8.338.6 ± 10.821.5 ± 7.7LDLR-/-26.4 ± 6.136.3 ± 11.111.3 ± 0.7 Open table in a new tab To determine the distribution of cholesterol in the lipoprotein fractions, pooled plasma from mice of the same genotype on the different diets was subjected to fast protein liquid chromatography. On the regular chow diet, there was very little accumulation of LDL in the Arh–/– animals (Fig. 2B). On the cholesterol-enriched diets, most of the cholesterol was in the LDL fraction in Arh–/– and in Ldlr–/– mice (Fig. 2, C and D). The relative increase in LDL-C in the mice of different genotypes was estimated by taking the sum of the cholesterol content of each column fraction in the LDL peak. On the chow diet, LDL-C was increased 1.4-fold in the Arh–/– mice relative to wild-type littermates, and the difference between the plasma cholesterol levels of these two strains increased further after cholesterol feeding. On a Western diet, the mean LDL-C level was 4.7-fold higher in the Arh–/– mice than in the littermate controls and 7.3-fold higher than the Arh–/– mice on a chow diet. In contrast to these results, plasma LDL-C levels increased only 2-fold in Ldlr–/– mice fed with the Western-type diet. Plasma LDL-C levels were increased 11-fold in the Arh–/– mice on the Paigen diet when compared with wild-type littermates and 42-fold when compared with chow-fed Arh–/– mice. Ldlr–/– mice on the Paigen diet had LDL-C levels that were similar to those of Arh–/– mice on the same diet. LDL-C levels of Ldlr–/– animals were 12-fold increased over wild-type mice fed the same diet and 11-fold increased over Ldlr–/– mice that had been fed a 0.2% cholesterol-containing Western-diet. Thus, cholesterol feeding was associated with a more dramatic elevation of cholesterol in the Arh–/– mice. Clearance of 125I-labeled LDL Is Reduced in Arh–/– Mice—To determine the effect of ARH deficiency on hepatic LDLR function, we compared the rates of removal of 125I-labeled LDL from the circulation of Arh–/– mice to wild-type and Ldlr–/– mice. The half-time f