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Targeted Disruption of the PDZK1 Gene in Mice Causes Tissue-specific Depletion of the High Density Lipoprotein Receptor Scavenger Receptor Class B Type I and Altered Lipoprotein Metabolism

2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês

10.1074/jbc.m310482200

1083-351X

Olivier Kocher, Ayce Yesilaltay, Christine Cirovic, Rinku Pal, Attilio Rigotti, Monty Krieger,

Protein Kinase Regulation and GTPase Signaling

PDZK1, a multi-PDZ domain containing adaptor protein, interacts with various membrane proteins, including the high density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI). Here we show that PDZK1 controls in a tissue-specific and post-transcriptional fashion the expression of SR-BI in vivo. SR-BI protein expression in PDZK1 knock-out (KO) mice was reduced by 95% in the liver, 50% in the proximal intestine, and not affected in steroidogenic organs (adrenal, ovary, and testis). Thus, PDZK1 joins a growing list of adaptors that control tissue-specific activity of cell surface receptors. Hepatic expression of SR-BII, a minor splice variant with an alternative C-terminal cytoplasmic domain, was not affected in PDZK1 KO mice, suggesting that binding of PDZK1 to SR-BI is required for controlling hepatic SR-BI expression. The loss of hepatic SR-BI was the likely cause of the elevation in plasma total and HDL cholesterol and the increase in HDL particle size in PDZK1 KO mice, phenotypes similar to those observed in SR-BI KO mice. PDZK1 KO mice differed from SR-BI KO mice in that the ratio of unesterified to total plasma cholesterol was normal, females were fertile, and cholesteryl ester stores in steroidogenic organs were essentially unaffected. These differences may be due to nearly normal extrahepatic expression of SR-BI in PDZK1 KO mice. The PDZK1-dependent regulation of hepatic SR-BI and, thus, lipoprotein metabolism supports the proposal that this adaptor may represent a new target for therapeutic intervention in cardiovascular disease. PDZK1, a multi-PDZ domain containing adaptor protein, interacts with various membrane proteins, including the high density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI). Here we show that PDZK1 controls in a tissue-specific and post-transcriptional fashion the expression of SR-BI in vivo. SR-BI protein expression in PDZK1 knock-out (KO) mice was reduced by 95% in the liver, 50% in the proximal intestine, and not affected in steroidogenic organs (adrenal, ovary, and testis). Thus, PDZK1 joins a growing list of adaptors that control tissue-specific activity of cell surface receptors. Hepatic expression of SR-BII, a minor splice variant with an alternative C-terminal cytoplasmic domain, was not affected in PDZK1 KO mice, suggesting that binding of PDZK1 to SR-BI is required for controlling hepatic SR-BI expression. The loss of hepatic SR-BI was the likely cause of the elevation in plasma total and HDL cholesterol and the increase in HDL particle size in PDZK1 KO mice, phenotypes similar to those observed in SR-BI KO mice. PDZK1 KO mice differed from SR-BI KO mice in that the ratio of unesterified to total plasma cholesterol was normal, females were fertile, and cholesteryl ester stores in steroidogenic organs were essentially unaffected. These differences may be due to nearly normal extrahepatic expression of SR-BI in PDZK1 KO mice. The PDZK1-dependent regulation of hepatic SR-BI and, thus, lipoprotein metabolism supports the proposal that this adaptor may represent a new target for therapeutic intervention in cardiovascular disease. Cytoplasmic adaptor proteins that bind directly to cell surface receptors or to receptor-associated proteins play crucial roles in regulating various biological processes including signal transduction, adhesion, membrane trafficking, and cellular transport (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). They usually consist of evolutionarily shuffled combinations of modular protein interaction domains such as src-homology (SH2, SH3), phosphotyrosine binding, and PDZ domains that recognize short peptide or phosphopeptide epitopes (e.g. PDZ domains usually bind to the C-terminal 3–4 residues of interacting proteins) (2Hung A. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). These adaptors help link one or more integral membranes and otherwise non-membranous proteins into functional complexes (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). For example, PDZK1, a four PDZ domain-containing protein whose expression is increased in a significant number of human kidney, colon, lung, and breast carcinomas, interacts with a number of membrane-associated transporter proteins, including cMOAT/MRP2 (3Kocher O. Comella N. Gilchrist A. Pal R. Tognazzi K. Brown L.F. Knoll J.H.M. Lab. Invest. 1999; 79: 1161-1170PubMed Google Scholar), the multidrug resistance-associated protein, the type IIa Na+/Pi cotransporter (4Gisler S.M. Stagljar I. Traebert M. Bacic D. Biber J. Murer H. J. Biol. Chem. 2001; 276: 9206-9213Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), the chloride channel ClC-3B (5Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), the cystic fibrosis transmembrane conductance regulator (5Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Wang S. Yue H. Derin R. Guggino W. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and the high density lipoprotein (HDL) 1The abbreviations used are: HDLhigh density lipoproteinIDLintermediate density lipoproteinLDLlow density lipoproteinSR-BI and SR-BIIscavenger receptor class B types I and II, respectivelyARHautosomal recessive hypercholesterolemiaKOknock-out. receptor called scavenger receptor class B type I (SR-BI) (7Ikemoto M. Arai H. Feng D. Tanaka K. Aoki J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar, 8Silver D.L. J. Biol. Chem. 2002; 277: 34042-34047Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The multiple PDZ domains in PDZK1 may mediate its simultaneous interaction with several target proteins and, thus, permit it to orchestrate complex biological functions by acting as a scaffolding protein (9Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar). high density lipoprotein intermediate density lipoprotein low density lipoprotein scavenger receptor class B types I and II, respectively autosomal recessive hypercholesterolemia knock-out. Recently the phosphotyrosine binding domain-containing adaptor ARH (product of the defective gene in autosomal recessive hypercholesterolemia (10Zuliani 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)) was shown to bind to the LDL receptor and components of the clathrin endocytic machinery and to control LDL receptor endocytic activity in selective cell types (hepatocytes and lymphocytes but not fibroblasts) (11He 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, 12Garcia 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 (468) Google Scholar). In mice and humans, ARH deficiency causes loss of hepatic LDL receptor endocytic activity and, consequently, hypercholesterolemia (13Wilund K. Yi M. Campagna F. Arca M. Zuliani G. Fellin R. Ho Y. Garcia J. Hobbs H.H. Cohen J.C. Hum. Mol. Genet. 2002; 11: 3019-3030Crossref PubMed Scopus (93) Google Scholar). The cell type-specific requirement for ARH suggests that the activities of other adaptors can substitute for that of ARH in some cells (e.g. fibroblasts), that these alternative adaptors are not expressed in or do not function effectively in ARH-sensitive cells (e.g. hepatocytes and/or lymphocytes), or that ARH plays some as yet undefined critical and dominant roles in controlling the endocytosis of LDL receptor in ARH-sensitive but not ARH-insensitive cells. These observations also raise the possibility that other adaptor proteins might control the function of other cell surface receptors in a similarly tissue-specific fashion. Manipulation of the activities of such adaptors might provide new insights into receptor physiology (e.g. new approach for tissue-specific knock-out/knockdown of receptor activity) and possibly attractive targets for pharmaceutical intervention in related disease processes (e.g. atherosclerosis) (12Garcia 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 (468) Google Scholar). Here we have used recently described homozygous null PDZK1 knock-out (KO) mice (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar) to show that PDZK1 controls, in a tissue-specific, post-transcriptional fashion, the expression of the HDL receptor SR-BI and HDL metabolism. In PDZK1 KO mice SR-BI expression was dramatically reduced in the liver, mildly decreased in the proximal intestine, and not affected in the steroidogenic organs (adrenal, ovary, and testis). The loss of hepatic SR-BI expression in these mice is the likely cause of the elevation in total plasma cholesterol levels (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar) and the presence of abnormally large HDL particles, as in the case for SR-BI knock-out mice (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar), wild-type mice in which hepatic SR-BI protein expression is abolished by treatment with the peroxisome proliferator-activated receptor-α agonist ciprofibrate (16Mardones P. Pilon A. Bouly M. Duran D. Nishimoto T. Arai H. Kozarsky K.F. Altoyo M. Miquel J. Luc G. Clavey V. Staels B. Rigotti A. J. Biol. Chem. 2003; 278: 7884-7890Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and mice in which hepatic SR-BI and PDZK1 protein expression was dramatically suppressed by transgenic hepatic overexpression of the small PDZK1-interacting protein MAP17 (3Kocher O. Comella N. Gilchrist A. Pal R. Tognazzi K. Brown L.F. Knoll J.H.M. Lab. Invest. 1999; 79: 1161-1170PubMed Google Scholar, 9Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar, 17Kocher O. Cheresh P. Lee S.W. Am. J. Pathol. 1996; 149: 493-500PubMed Google Scholar, 18Silver D.L. Wang N. Vogel S. J. Biol. Chem. 2003; 278: 28528-28532Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, PDZK1 KO mice differ from SR-BI KO mice in a number of ways. First, in contrast to the presence of excess unesterified cholesterol in the plasma of SR-BI KO mice (42Braun A. Zhang S. Miettinen H. Ebrahim S. Holm T. Vasile E. Post M.J. Yoerger D. Picard M. Krieger J. Andrews N. Simons M. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7283-7288Crossref PubMed Scopus (119) Google Scholar), the unesterified-to-total cholesterol ratio was normal in PDZK1 KO mice. Second, the steroidogenic tissues of PDZK1 KO mice appeared to contain normal cholesteryl ester stores, presumably because these tissues expressed normal amounts of SR-BI protein. Finally, female PDZK1 KO mice, but not SR-BI KO females, are fertile (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar, 15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar). Thus, PDZK1 joins ARH in what is likely to be a growing family of adaptors that control the tissue-specific expression and activity of cell surface receptors. Our findings also indicate that PDZK1 plays a key role in the control of HDL metabolism through its interaction with SR-BI in the liver and suggest that PDZK1 KO mice might be useful surrogates for mice with liver-specific (e.g. cre/lox) ablation of SR-BI expression for the analysis of the role of hepatic SR-BI expression in lipoprotein metabolism. Animals—PDZK1 knock-out and wild-type mice (129SvEv background) were maintained on a normal chow diet (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar) and were ∼6–12 weeks old at the time of the experiments. All procedures were performed in accordance with the Beth Israel Deaconess Medical Center guidelines. Immunoblot Analysis, Immunoperoxidase, and Oil Red O Studies— Antibodies against SR-BI, SR-BII, and actin were purchased from Novus Biologicals (Littleton, CO) and Sigma or prepared as previously described (19Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2006) Google Scholar, 20Gu X. Kozarsky K. Krieger M. J. Biol. Chem. 2000; 275: 29993-30001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Anti-apolipoprotein E (apoE) antiserum was a gift from R. L. Raffai and K. H. Weisgraber (University of California San Francisco, CA), and antiserum against apolipoprotein A-I (apoA-I) was purchased from Biodesign (Saco, ME). Karen Kozarsky (GlaxoSmithKline) provided the KKB-1 antibody that recognizes the extracellular domains of both SR-BI and SR-BII. For immunoblot analysis protein extracts (50 μg/lane) were fractionated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, transferred to nitrocellulose or polyvinylidene difluoride membranes, and incubated with appropriate antisera and subsequently with an anti-rabbit IgG conjugated to horseradish peroxidase at a dilution of 1/10,000 (Invitrogen). Bound antibodies were visualized using ECL chemiluminescence reaction and Super Signal West Pico Luminal reagents (Pierce). To quantitate the relative amounts of SR-BI in tissues, tissue lysates were serially diluted and subjected to electrophoresis/immunoblotting, and the relative intensities of the signals were compared by visual examination. For immunoperoxidase studies tissues were fixed for 4 h in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, at 4 °C and then transferred to 30% sucrose in phosphate-buffered saline, pH 7.4, overnight at 4 °C. Tissues were then frozen in OCT compound (Miles Diagnostics, Elkhart, IN) and stored in liquid nitrogen. Immunoperoxidase studies were performed on 5-μm fixed-frozen tissue sections using primary antibodies against SR-BI. Normal rabbit IgG were used as negative controls. The sections were then incubated with a biotinylated anti-rabbit IgG using a 1/200 dilution (Vector, Burlingame, CA) and subsequently treated with the Vectastain ABC reagents (Vector) and diaminobenzidine (Research Genetics, Inc., Huntsville, AL), according to the manufacturer's protocol. Oil Red O and hematoxylin stainings were performed on 5-μm unfixed frozen tissue sections as previously described (21Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9322-9327Crossref PubMed Scopus (442) Google Scholar). RNA Isolation—Tissues were surgically removed from PDZK1 knock-out and wild-type mice, frozen on dry ice, and stored at –80 °C until use. Frozen tissue samples were simultaneously disrupted and homogenized for up to 60 s using a Polytron (Kinematica, Lucerne, Switzerland) in lysis buffer RLT (Qiagen, Valencia, CA) containing 145 mm β-mercaptoethanol. Samples were homogenized further, and total RNA was purified according to the manufacturer's protocol for the Qiagen RNeasy mini kit. Reverse Transcription—Total RNA was reverse-transcribed using the ABI Prism 7700 sequence detection system and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Each reaction contained 1× reverse transcription buffer, 5.5 mm MgCl2, 500 μm each dNTP, 2.5 μm random hexamer, 0.4 μl of RNase inhibitor, 1.25 units of multiscribe reverse transcriptase, 0.1 μg of total RNA, and RNase-free distilled H2O to a total volume of 10 μl per reaction. Reverse transcription reaction cycle parameters were as follows: 10 min at 25 °C, 30 min at 48 °C, and 5 min at 95 °C. SYBR Green Real-time Quantitative PCR—Oligonucleotide primers were designed using Primer Express software version 1.5 based on gene sequences obtained from the National Center for Biotechnology Information (NCBI) data base. All reactions were performed using an ABI Prism 7700 sequence detection system (Applied Biosystems). Reactions were carried out in duplicate in a 50-μl reaction volume containing reverse-transcribed cDNAs, 25 μl of 2× SYBR Green Master Mix, and each forward and reverse primer at a concentration of 50 nm. Conditions for all SYBR Green PCR reactions were 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 95 °C for 10 s and 60 °C for 1 min. Data were gathered and analyzed using the SDS 1.9 software on a G4 Power Macintosh computer (Apple Computer, Cupertino, CA). Results were normalized with respect to glyceraldehyde-3-phosphate dehydrogenase expression. Threshold cycle (Ct) values were exported into a Microsoft Excel worksheet for calculation of gene expression according to the ΔΔCt method (Applied Biosystems). Levels of gene expression in knockout mice were represented with respect to those of wild-type animals as knock-out/wild-type ratios. Plasma Cholesterol and Lipoprotein Analysis—Plasma was collected from PDZK1 knock-out and wild-type mice after a 4-h fast. Total cholesterol, unesterified ("free") cholesterol, triglycerides, and phospholipids were measured using kits from Wako (Richmond, VA). Plasma was fractionated by fast protein liquid chromatography, and the cholesterol and apolipoprotein compositions of the fractions were determined by enzymatic kit and immunoblotting, respectively (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar). Statistical Analysis—Data are shown as the means ± S.E. Statistically significant differences were determined by pairwise comparisons of each value from knock-out mice with wild-type controls by using unpaired Student's t test. p values < 0.05 were considered to be statistically significant for differences between experimental groups. We have previously reported that PDZK1 KO mice exhibit an ∼1.8-fold elevation in plasma total cholesterol concentration (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar), and others have suggested that PDZK1 interactions with SR-BI in the liver may play an important role in controlling SR-BI expression and function and, thus, HDL metabolism (7Ikemoto M. Arai H. Feng D. Tanaka K. Aoki J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar, 8Silver D.L. J. Biol. Chem. 2002; 277: 34042-34047Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Mardones P. Pilon A. Bouly M. Duran D. Nishimoto T. Arai H. Kozarsky K.F. Altoyo M. Miquel J. Luc G. Clavey V. Staels B. Rigotti A. J. Biol. Chem. 2003; 278: 7884-7890Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Silver D.L. Wang N. Vogel S. J. Biol. Chem. 2003; 278: 28528-28532Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because the loss of SR-BI expression in SR-BI KO mice results in an ∼2-fold elevation in plasma cholesterol (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar), it seemed possible that the increased plasma cholesterol in PDZK1 KO mice might have been due to reduced hepatic SR-BI protein levels in these mice. We therefore used immunoblot and immunohistochemical analyses to measure SR-BI protein expression in PDZK1 KO mice. The immunoblot analysis of SR-BI expression (Fig. 1 and data not shown) demonstrates that there was an ∼95% reduction in SR-BI protein expression in the livers of PDZK1 KO mice relative to wild-type controls. In contrast, there was only a modest reduction (50%) in the much lower expression levels of SR-BI protein in the small intestine and no detectable change in the adrenal gland, which exhibits especially high SR-BI levels (19Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2006) Google Scholar). The results of immunoblotting were supported by immunohistochemical (immunoperoxidase) analysis of SR-BI expression (Fig. 2) in the liver (hepatocytes), small intestine (epithelial cells), adrenal gland (cortex), testis (Leydig cells), and ovary (stroma and corpus luteum) in control wild-type (left) and PDZK1 KO (right) mice. The cell type and intracellular patterns of SR-BI expression in these organs were similar to those reported previously (19Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2006) Google Scholar, 22Landschulz K.T. Pathak R. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (469) Google Scholar, 23Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. Williams D.L. J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The intensity of SR-BI staining in PDZK1 KO mice relative to wild-type mice was markedly reduced in liver, moderately reduced in small intestinal mucosa, and not changed in adrenal cortex, ovary, or testis. The ∼50% reduction in SR-BI protein expression in the small intestine (Fig. 1) was apparently due to a homogenous decrease in all SR-BI-expressing epithelial cells of the mucosa (Fig. 2) rather than to varying extents of altered SR-BI expression in different subtypes of cells within this organ.Fig. 2Immunohistochemical analysis of SR-BI expression in tissues from wild-type and PDZK1 knock-out mice. Immunoperoxidase staining of liver, small intestine, adrenal, testis, and ovary harvested from wild-type (WT; left) and PDZK1 KO (right) mice was performed as described under "Experimental Procedures." Note the typical staining pattern of SR-BI in hepatocytes, small intestinal mucosa, adrenal cortex, testicular Leydig cells, ovarian corpus luteum, and stroma. The intensity of staining was markedly reduced in liver, moderately reduced in the epithelial cells of the small intestine, and not changed in the adrenal glands, testes, and ovaries of PDZK1 knock-out compared with wild-type mice. Magnification, ×300.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The expression of SR-BI in steroidogenic tissues has been shown to be essential for the normal accumulation of stored cholesteryl esters in cytoplasmic lipid droplets (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar, 21Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9322-9327Crossref PubMed Scopus (442) Google Scholar). When we visualized the neutral lipid stores in the adrenals by staining tissue sections with the dye Oil Red O we saw essentially no differences in staining between the wild-type and PDZK1 KO samples (Fig. 3). Thus, in steroidogenic tissues, in which PDZK1 expression is low (7Ikemoto M. Arai H. Feng D. Tanaka K. Aoki J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar, 9Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar) and SR-BI expression is normally high (19Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2006) Google Scholar), SR-BI protein was expressed in normal amounts and distribution and apparently functioned properly in providing substrate for cholesteryl ester storage even in the absence of a functional PDZK1 gene. The expression of SR-BI in the liver is generally thought to be responsible for SR-BI marked influence on the structure and abundance of HDL in murine plasma (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar). In homozygous null SR-BI KO relative to wild-type mice, HDL cholesterol levels and the sizes of HDL particles are increased based on gel filtration chromatography analysis. The abnormally large HDL particles in SR-BI KO mice also carry increased amounts of apoE (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar). Thus, if the loss of hepatic SR-BI protein expression in PDZK1 KO mice is responsible for their hypercholesterolemia, we would expect that the plasma lipoprotein profiles in these mutant mice might in some ways resemble the abnormal lipoproteins found in SR-BI KO mice. Table I shows the lipid composition of plasma from wild-type and PDZK1 KO male and female mice. The effects of the mutation on the plasma lipids of males and females were generally similar, and the pooled results will be considered below. As previously reported (14Kocher O. Pal R. Roberts M. Cirovic C. Gilchrist A. Mol. Cell. Biol. 2003; 23: 1175-1180Crossref PubMed Scopus (101) Google Scholar), the PDZK1 KO mice were hypercholesterolemic. The total plasma cholesterol was ∼1.7-fold higher in the PDZK1 KO mice; the fold increases in unesterified and esterified cholesterol were 1.9 and 1.6, respectively. The increases in plasma cholesterol were accompanied by increases in phospholipids and triglycerides. To further assess the structures of the lipoproteins, plasma samples from individual mice were size-fractionated by Sepharose 4B fast protein liquid chromatography size exclusion chromatography. The lipoprotein total cholesterol profiles of representative animals are shown in Fig. 4A.Table IEffects of disruption of the PDZK1 gene on plasma lipid concentrationsGenderGenotypeTCap < 0.001 vs. the corresponding wild-type group.UCbp < 0.001 vs. the corresponding wild-type group.UC/TCTGPLcp < 0.01 vs. the corresponding wild-type group.Sample sizemg/dlmg/dlmg/dlmg/dlMalesWT91 ± 722 ± 10.24 ± 0.0132 ± 7164 ± 9n = 7PDZK1 KO144 ± 838 ± 20.27 ± 0.0136 ± 5212 ± 12n = 6FemalesWT78 ± 418 ± 10.23 ± 0.0116 ± 1131 ± 6n = 8PDZK1 KO139 ± 634 ± 10.24 ± 0.00425 ± 3dp < 0.01 vs. the corresponding wild-type group.188 ± 5n = 8M + FWT84 ± 419 ± 10.23 ± 0.0124 ± 4146 ± 7n = 15PDZK1 KO141 ± 536 ± 10.25 ± 0.0130 ± 3198 ± 6n = 14a p < 0.001 vs. the corresponding wild-type group.b p < 0.001 vs. the corresponding wild-type group.c p < 0.01 vs. the corresponding wild-type group.d p < 0.01 vs. the corresponding wild-type group. Open table in a new tab As previously reported, most of the plasma cholesterol in wild-type mice was in HDL-size particles centered at fraction 34 (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar). The main protein component of HDL, apoA-I, was found in this HDL peak (Fig. 4B, upper panels). Some apoE was found in this peak, but the majority of it was associated with larger particles (lower fraction numbers). In the PDZK1 KO mice (Fig. 4B, lower panels), the peak of HDL cholesterol and apoA-I was shifted to the left, indicating that the HDL particles were larger in the mutant mice. There was also more apoE in the PDZK1 KO plasma distributed into bigger particles within the large HDL- and IDL/LDL-size ranges. The changes in the shape of the lipoprotein profile of the PDZK1 KO mice compared with wild-type controls (Fig. 4A) were similar to those seen in SR-BI KO mice (15Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (758) Google Scholar) and in wild-type mice in which hepatic SR-BI protein expression was abolished by treatment with the peroxisome proliferator-activated receptor-α agonist ciprofibrate (16Mardones P. Pilon A. Bouly M. Duran D. Nishimoto T. Arai H. Kozarsky K.F. Altoyo M. Miquel J. Luc G. Clavey V. Staels B. Rigotti A. J. Biol. Chem. 2003; 278: 7884-7890Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Therefore, we conclude that the loss of hepatic SR-BI protein expression is the most likely cause of the hypercholesterolemia in PDZK1 KO mice. We observed two striking unanticipated differences between PDZK1 KO and SR-BI KO mice. In wild-type mice on a mixed C57/Bl6:129 genetic background, the unesterified to total cholesterol ratio is about 0.26–0.31, whereas that for SR-BI KO mice is about 0.5 (24Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Circ. Res. 2002; 90: 270-276Crossref PubMed Sco