Influence of PDZK1 on lipoprotein metabolism and atherosclerosis

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.     

Identification of small PDZK1-associated protein,DD96/MAP17, as a regulator of PDZK1 and plasma high density lipoprotein levels

Scavenger receptor class B, type I (SR-BI) is the high density lipoprotein (HDL) receptor essential for hepatic uptake of HDL cholesterol. SR-BI was shown to impact plasma HDL levels and be anti-atherogenic. Thus, the ability to regulate hepatic SR-BI may allow for the modulation of plasma HDL cholesterol and progression of atherosclerosis. However, regulation of SR-BI in liver is not well understood. Recently, the PDZ domain containing protein PDZK1 was shown to interact with SR-BI and may serve an essential role in SR-BI cell surface expression. Here we identify an in vivo PDZK1-interacting protein that we named small PDZK1-associated protein (SPAP; also known as DD96/MAP17). Unexpectedly, we found that hepatic overexpression of SPAP in mice resulted in liver deficiency of PDZK1. The absence of PDZK1 in SPAP transgenic mice resulted in a deficiency of SR-BI in liver and markedly increased plasma HDL. Metabolic labeling experiments showed that the proteasome plays a role in the turnover of newly synthesized PDZK1, but that SPAP overexpression in liver increased PDZK1 turnover in an alternate, proteasome-independent pathway. Thus, SPAP may be an endogenous regulator of cellular PDZK1 levels by regulating PDZK1 turnover.     

Fenofibrate induces a novel degradation pathway for scavenger receptor B-I independent of PDZK1

Fibrate drugs improve cardiovascular health by lowering plasma triglycerides, normalize low density lipoprotein levels, and raise high density lipoprotein (HDL) levels in patients with dyslipidemias. The HDL-raising effect of fibrates has been shown to be due in part to an increase in human apolipoprotein AI gene expression. However, it has recently been shown that fibrates can affect HDL metabolism in mouse by significantly decreasing hepatic levels of the HDL receptor scavenger receptor B-I (SR-BI) and the PDZ domain containing protein PDZK1. PDZK1 is essential for maintaining hepatic SR-BI levels. Therefore, decreased SR-BI might be secondary to decreased PDZK1, but the mechanism by which fibrates lower SR-BI has not been elucidated. Here we show that feeding PDZK1-deficient mice fenofibrate resulted in the near absence of SR-BI in liver, definitively demonstrating that the effect of fenofibrate on SR-BI is PDZK1-independent. Metabolic labeling experiments in primary hepatocytes from fenofibrate-fed mice demonstrated that fenofibrate enhanced the degradation of SR-BI in a post-endoplasmic reticulum compartment. Moreover, fenofibrate-induced degradation of SR-BI was independent of the proteasome, calpain protease, or the lysosome, and antioxidants did not inhibit fenofibrate-induced degradation of SR-BI. Using metabolic labeling coupled with cell surface biotinylation assays, fenofibrate did not inhibit SR-BI trafficking to the plasma membrane. Together, the data support a model in which fenofibrate enhances the degradation of SR-BI in a post-ER, post-plasma membrane compartment. The further elucidation of this novel degradation pathway may provide new insights into the physiological and pathophysiological regulation of hepatic SR-BI.     

Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice

Background

PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (∼95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI.

Principal Findings

Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle.

Conclusions

These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.     

Cholesteryl ester transfer protein (CETP) expression enhances HDL cholesteryl ester liver delivery, which is independent of scavenger receptor BI, LDL receptor related protein and possibly LDL receptor

Cholesteryl ester transfer protein (CETP) is a hydrophobic plasma glycoprotein that mediates the transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between plasma lipoproteins, and also plays an important role in HDL metabolism. Previous studies have indicated that, compared to wild type mice, human CETP transgenic mice had significantly lower plasma HDL CE levels, which was associated with enhancement of HDL CE uptake by the liver. However, the mechanism of this process is still unknown. To evaluate the possibility that this might be directly mediated by CETP, we utilized CETP transgenic (CETPTg) mice with liver scavenger receptor BI (SR-BI) deficiency [i.e., PDZK1 gene knockout (PDZK1O)], and with receptor associated protein (RAP) overexpression, to block LDL receptor-related protein (LRP) and LDL receptor (LDLR). We found that (1) CETPTg/PDZK1O mice have significantly lower HDL-C than that of PDZK1 KO mice (36%, p<0.01); (2) CETPTg and CETPTg/PDZK1O mice have same HDL-C levels; (3) CETPTg/PDZK1O/RAP mice had significant lower plasma HDL-C levels than that of PDZK1O/RAP ones (50%, p<0.001); (4) there is no incremental transfer of HDL CE radioactivity to the apoB-containing lipoprotein fraction in mice expressing CETP; and (5) CETPTg/PDZK1O/RAP mice had significant higher plasma and liver [(3)H]CEt-HDL turnover rates than that of PDZK1O/RAP ones (50% and 53%, p<0.01, respectively). These results suggest that CETP expression in mouse increases direct removal of HDL CE in the liver and this process is independent of SR-BI, LRP, and possibly LDLR.     

A carboxyl-terminal PDZ-interacting domain of scavenger receptor B,type I is essential for cell surface expression in liver

Scavenger receptor B, type I (SR-BI) was recently shown to interact with a PDZ domain-containing protein, PDZK1 (CLAMP/Diphor-1/CAP70/NaPi-Cap1), but the importance of this interaction in vivo in terms of SR-BI function has not been determined. In an effort to elucidate the role of this interaction in vivo, the PDZK1-interacting domain of SR-BI was identified and mutated and expressed liver-specifically in mice. The PDZKI-interacting domain on SR-BI was identified as the last three carboxyl-terminal amino acids, Arg-Lys-Leu. A mutant SR-BI (SR-BIdel509) that lacked only the leucine in the PDZ-interacting domain failed to interact with PDZK1 in vitro, while showing normal selective uptake function in nonpolarized cells. Transgenic mice with liver overexpression of SR-BIdel509 showed marked accumulation of SR-BI mRNA with only a moderate increase in SR-BI protein in liver, with no reduction in plasma cholesterol levels. Measurement of cell surface SR-BI levels and HDL cholesteryl ester-selective uptake in primary hepatocytes from transgenic mice revealed that SR-BIdel509 was not expressed at the plasma membrane correlating with normal levels of selective uptake compared with hepatocytes from nontransgenic littermates. This study indicates that the PDZK1-interacting domain of SR-BI is essential for cell surface expression of SR-BI in liver and suggests that PDZK1 or other PDZ domain proteins may play an important role in regulating SR-BI cell surface expression and hence reverse cholesterol transport.     

Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice

Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.     

Influence of the HDL receptor SR-BI on atherosclerosis

The scavenger receptor class B, type I (SR-BI) is an HDL receptor that mediates selective cholesterol uptake from HDL to cells. In rodents, SR-BI has a critical influence on plasma HDL-cholesterol concentration and structure, the delivery of cholesterol to steroidogenic tissues, female fertility, and biliary cholesterol concentration. SR-BI can also serve as a receptor for non-HDL lipoproteins and appears to play an important role in reverse cholesterol transport. Recent studies involving the manipulation of SR-BI expression in mice, either using adenovirus-mediated or transgenic hepatic overexpression or using homologous recombination for complete functional ablation, indicate that the expression of SR-BI protects against atherosclerosis. If SR-BI has a similar activity in humans, it may become an attractive target for therapeutic intervention.     

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B,Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys¹⁴-Xaa₄-Asn¹⁹-Tyr-Gly-Phe-Phe-Leu²⁴), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (⁵⁰³VLQEAKL⁵⁰⁹). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K_d) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (⁵⁰⁵QEAKL⁵⁰⁹) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.     

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B,Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.     

The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism

The HDL receptor scavenger receptor class B type I (SR-BI), which mediates selective HDL cholesterol uptake, plays a role in murine HDL metabolism, reverse cholesterol transport and whole-body cholesterol homeostasis. SR-BI is found in the liver, where its expression is regulated by estrogen, dietary cholesterol and fat, and controls murine plasma HDL cholesterol levels and bile cholesterol secretion. SR-BI is also highly expressed in rodent steroidogenic cells, where it facilitates cholesterol uptake for storage or steroid hormone synthesis and where its expression is regulated by trophic hormones. The detailed mechanism(s) underlying SR-BI-mediated selective cholesterol uptake have not yet been elucidated. Further analysis of the molecular and cellular bases of SR-BI regulation and function should provide new insights into the physiology and pathophysiology of cholesterol metabolism.     

Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“target peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr²⁰ → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K_d = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr²⁵³ → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide (⁵⁰⁵QEAKL⁵⁰⁹) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr²⁵³ → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.     

Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile

The clearance of free cholesterol from plasma lipoproteins by tissues is of major quantitative importance, but it is not known whether this is passive or receptor-mediated. Based on our finding that scavenger receptor BI (SR-BI) promotes free cholesterol (FC) exchange between high density lipoprotein (HDL) and cells, we tested whether SR-BI would effect FC movement in vivo using [(14)C]FC- and [(3)H]cholesteryl ester (CE)-labeled HDL in mice with increased (SR-BI transgenic (Tg)) or decreased (SR-BI attenuated (att)) hepatic SR-BI expression. The initial clearance of HDL FC was increased in SR-BI Tg mice by 72% and decreased in SR-BI att mice by 53%, but was unchanged in apoA-I knockout mice compared with wild-type mice. Transfer of FC to non-HDL and esterification of FC were minor and could not explain differences. The hepatic uptake of FC was increased in SR-BI Tg mice by 34% and decreased in SR-BI att mice by 22%. CE clearance and uptake gave similar results, but with much slower rates. The uptake of HDL FC and CE by SR-BI Tg primary hepatocytes was increased by 2.2- and 2.6-fold (1-h incubation), respectively, compared with control hepatocytes. In SR-BI Tg mice, the initial biliary secretion of [(14)C]FC was markedly increased, whereas increased [(3)H]FC appeared after a slight delay. Thus, in the mouse, a major portion of the clearance of HDL FC from plasma is mediated by SR-BI.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.