Turnover Rates of the Low-Density Lipoprotein Receptor and PCSK9: Added Dimension to the Cholesterol Homeostasis Model.

OBJECTIVE: We measured the turnover rates of the LDLR (low-density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type 9) in mice by metabolic labeling with heavy water and mass spectrometry. Approach and Results: In liver of mice fed high-cholesterol diets, LDLR mRNA levels and synthesis rates were markedly lower with complete suppression of cholesterol synthesis and higher cholesterol content, consistent with the Brown-Goldstein model of tissue cholesterol homeostasis. We observed markedly lower PCSK9 mRNA levels and synthesis rates in liver and lower concentrations and synthesis rates in plasma. Hepatic LDLR half-life (t½) was prolonged, consistent with an effect of reduced PCSK9, and resulted in no reduction in hepatic LDLR content despite reduced mRNA levels and LDLR synthesis rates. These changes in PCSK9 synthesis complement and expand the well-established model of tissue cholesterol homeostasis in mouse liver, in that reduced synthesis and levels of PCSK9 counterbalance lower LDLR synthesis by promoting less LDLR catabolism, thereby maintaining uptake of LDL cholesterol into liver despite high intracellular cholesterol concentrations. CONCLUSIONS: Lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. The physiological and therapeutic implications of these opposing control mechanisms over liver LDLR are of interest and may reflect subservience of hepatic cholesterol homeostasis to whole body cholesterol needs.

Low density lipoprotein receptor-related protein 1 regulates cardiac hypertrophy induced by pressure overload.

BACKGROUND: Cardiac hypertrophy is associated with functional changes in cardiomyocytes, which often results in heart failure. The low-density lipoprotein receptor-related protein 1 (LRP1) is a large multifunctional endocytic receptor involved in many physiological and pathological processes. However, its function in the development of cardiac hypertrophy remains largely unclear. METHODS: Adenoviral constructs were used for either overexpression or silencing of LRP1 in both in vitro and in vivo experiments. Cardiac function was measured using the Millar catheter. RESULTS: LRP1 expression was upregulated in both transverse aortic constriction (TAC)-induced hypertrophic myocardium and catecholamine (phenylephrine (PE) and norepinephrine (NE))- and angiotensin II (AngII)-induced hypertrophic cardiomyocytes. In addition, cell surface area, protein/DNA ratio, and the mRNA levels of hypertrophic markers were significantly increased in LRP1-overexpressing cardiomyocytes without catecholamine stimulation. Conversely, LRP1 inhibition by LRP1-specific siRNA or a specific ligand-binding antagonist (RAP) significantly rescued hypertrophic effects in PE, NE, or AngII-induced cardiomyocytes. LRP1 overexpression induced PKCα, then activated ERK, resulting in cardiac hypertrophy with the downregulation of SERCA2a and calcium accumulation, which was successfully restored in both LRP1-silenced cardiomyocytes and TAC-induced hearts. CONCLUSIONS: LRP1 regulates cardiac hypertrophy via the PKCα-ERK dependent signaling pathway resulting in the alteration of intracellular calcium levels, demonstrating that LRP1 might be a potential therapeutic target for cardiac hypertrophy.

Overexpression of low density lipoprotein receptor-related protein 1 (LRP1) is associated with worsened prognosis and decreased cancer immunity in clear-cell renal cell carcinoma.

AIM: Clear-cell renal cell carcinoma (ccRCC) is characterized with underlying genetic disorders and the role of low density lipoprotein receptor-related protein 1 (LRP1) in ccRCC is unknown. METHOD: An in silico exploratory analysis using multiple public genetic datasets was used to establish association between LRP1 expression and clinicopathological parameters. Associations of interest were validated using 155 ccRCC samples using immunohistochemistry. RESULTS: LRP1 was overexpressed in tumor compared with normal kidney tissue. Increased LRP1 expression in ccRCC was associated with advanced stage, grade and worsened overall survival and progression-free survival. Functional annotation indicated an immune-modulatory role of LRP1 in ccRCC. LRP1 expression was significantly correlated with expressions of PBRM1, SETD2, and KDM5C. Positive correlations between LRP1 and pro-angiogenic factors ERAP1, SCG2, STAB1, and RUNX1 were observed. LRP1 expression was positively correlated with PD-L2 level. Negative correlations between LRP1 and anti-angiogenic factors EMCN and IL18 were observed. LRP1 expression was not associated with microvessel density (MVD) yet was negatively correlated with tumor-infiltrating lymphocytes (TIL). CONCLUSION: LRP1 is associated with worsened prognosis in ccRCC and is related to cancer immune modulation. LRP1-targeted therapy can be of therapeutic potential.

Effects of increased accumulation of doxorubicin due to emodin on efflux transporter and LRP1 expression in lung adenocarcinoma and colorectal carcinoma cells.

Treatment with doxorubicin (dox) and emodin, separately and together, under normoxic and hypoxia-like conditions induced by CoCl2, led to greater intracellular compound accumulation over 10 h post-addition in the presence of CoCl2 in lung adenocarcinoma (A549) and colorectal carcinoma (HCT-15) cell lines. Confocal microscopy revealed that emodin, by itself, showed high cytosolic distribution in both cell lines, at 40 min post-addition but had entered the nuclei by 2 h, while dox entered the nuclei by 40 min. Both compounds modulated the expression of the efflux transporters (PgP, ABCG2, or MRP1-4) and the endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), to different extents under the study conditions. Efflux transporter upregulation was linked to lower intracellular compound levels due to greater efflux. Increased dox accumulation was accompanied by unaltered expression or upregulation of LRP1 in A549 cells. In both cell lines, increased accumulation of dox and emodin was observed whenever LRP1 and the efflux transporters known to transport dox and emodin were all up- or downregulated concomitantly. Increased growth inhibition was linked to co-treatment with dox and emodin and with increased ligand accumulation. The results presented in this study raise the hypothesis that higher production of LRP1 protein may be associated with higher endocytosis of upregulated transporter proteins at the cell surface, and hence, increased dox and emodin accumulation and growth inhibition. If so, elevation of LRP1 expression may be a useful target for interventions to promote the efficacy of these and other anticancer drugs.

Microbiome-host systems interactions: protective effects of propionate upon the blood-brain barrier.

BACKGROUND: Gut microbiota composition and function are symbiotically linked with host health and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognised mechanisms exist by which the microbiome influences the gut-brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesised interactions between circulating gut-derived microbial metabolites, and the blood-brain barrier (BBB) also contribute to the gut-brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed. RESULTS: After demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 µM) on BBB properties in vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signalling. CONCLUSIONS: Together, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut-brain axis that warrants further attention.

Sulfated Hyaluronan Alters the Interaction Profile of TIMP-3 with the Endocytic Receptor LRP-1 Clusters II and IV and Increases the Extracellular TIMP-3 Level of Human Bone Marrow Stromal Cells.

Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.

Study of Valproic Acid-Enhanced Hepatocyte Steatosis.

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1.

T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130,000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

Regulation of LRP-1 expression: make the point.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a membrane receptor displaying both scavenging and signaling functions. The wide variety of extracellular ligands and of cytoplasmic scaffolding and signaling proteins interacting with LRP-1 gives it a major role not only in physiological processes, such as embryogenesis and development, but also in critical pathological situations, including cancer and neurological disorders. In this review, we describe the molecular mechanisms involved at distinct levels in the regulation of LRP-1, from its expression to the proper location and stability at the cell surface.

Standardized flow cytometry assay for identification of human monocytic heterogeneity and LRP1 expression in monocyte subpopulations: decreased expression of this receptor in nonclassical monocytes.

In this article, we present a flow cytometry assay by which human blood monocyte subpopulations-classical (CD14(++) CD16(-)), intermediate (CD14(++) CD16(+)), and nonclassical (CD14(+) CD16(++)) monocytes-can be determined. Monocytic cells were selected from CD45(+) leukocyte subsets by differential staining of the low-density lipoprotein receptor-related protein 1 (LRP1), which allows reducing the spill-over of natural killer cells and granulocytes into the CD16(+) monocyte gate. Percentages of monocyte subpopulations established by this procedure were significantly comparable with those obtained by a well-standardized flow cytometry assay based on the HLA-DR monocyte-gating strategy. We also demonstrated that LRP1 is differentially expressed at cell surface of monocyte subpopulations, being significantly lower in nonclassical monocytes than in classical and intermediate monocytes. Cell surface expression of LRP1 accounts for only 20% of the total cellular content in each monocyte subpopulation. Finally, we established the within-individual biological variation (bCV%) of circulating monocyte subpopulations in healthy donors, obtaining values of 21%, 20%, and 17% for nonclassical, intermediate, and classical monocytes, respectively. Similar values of bCV% for LRP1 measured in each monocyte subpopulation were also obtained, suggesting that its variability is mainly influenced by the intrinsic biological variation of circulating monocytes. Thus, we conclude that LRP1 can be used as a third pan-monocytic marker together with CD14 and CD16 to properly identify monocyte subpopulations. The combined determination of monocyte subpopulations and LRP1 monocytic expression may be relevant for clinical studies of inflammatory processes, with special interest in atherosclerosis and cardiovascular disease.

Molecular and functional characterization of LRP1 promoter polymorphism c.1-25 C>G (rs138854007).

The transcription of the Low-density lipoprotein receptor-related protein (LRP1) is upregulated by aggregated LDL (agLDL) and angiotensin II (AngII) in human vascular smooth muscle cells (hVSMC). The polymorphism c.1-25C>G creates a new GC-box in the LRP1 promoter recognized by Sp1/Sp3 transcription factors. The aims of this study were 1) to evaluate the impact of c.1-25C>G polymorphism on LRP1 transcriptional activity and expression, and 2) to examine the response of c.1-25C>G LRP1 promoter to LDL and AngII. EMSA and Luciferase assays in HeLa cells showed that -25G promoter has enhanced basal transcriptional activity and specific Sp1/Sp3 binding. hVSMC with GG genotype (GG-hVSMC) had higher LRP1 mRNA and protein levels, respectively than CC genotype (CC-hVSMC). EMSA assays showed that the polymorphism determines scarce amount of SRE-B/SREBP-2 complex formation and the failure of agLDL to further reduce these SRE-B/SREBP-2 complexes. Taken together, these results suggest that c.1-25C>G, by difficulting SREBP-2 binding, prevents SREBP-2 displacement required for LRP1 promoter response to LDL. In contrast, c.1-25C>G strongly favours Sp1/Sp3 binding and AngII-induced activity in Sp1/Sp3 dependent manner in GG-hVSMC. This increase is functionally translated into a higher capacity of GG-hVSMC to become foam cells from agLDL in presence of AngII. These results suggest that c.1-25C>G determines a lack of response to agLDL and an exacerbated response to AngII. It is thus conceivable that the presence of the polymorphism would be easily translated to vascular alterations in the presence of the pro-hypertensive autacoid, AngII.

Endoplasmic reticulum resident heat shock protein-gp96 as morphogenetic and immunoregulatory factor in syngeneic pregnancy.

The severe remodeling of endometrial stroma during blastocyst adhesion and trophoblast invasion initiates at maternal-fetal interface the reaction of evolutionary old heat shock response, in which heat shock proteins, as molecular chaperons, monitor the configurations of newly synthesized proteins and prevent the formation of functionless aggregates of misfolded proteins, targeting them to degradation by a the ubiquitin-proteasome system. In addition, the endoplasmic reticulum (ER)-resident HSPs, such as gp96/GRP94 may, after binding to CD91 and TLRs, elicit antigen-specific and antigen-unspecific immune responses, owing to its peptide-chaperoning capacity and ability to activate APCs. Considering these properties, we examined tissue expression of gp96 at the maternal-fetal interface and in the maternal liver and spleen on the 16th day of undisturbed syngeneic pregnancy and after the treatment with peptidoglycan monomer linked with zinc (PGM-Zn). The data showed that in undisturbed pregnancy the gp96, CD91 and TLR2 were markedly expressed on extravillous and villous trophoblast. PGM-Zn enhanced these findings, as well as the number of uterine natural killer cells and local NFκB immunoreactivity. Gp96 expression arose also in the maternal spleen and liver, where an accumulation of NKT cells or γδT lymphocytes was seen. The data point to roles of gp96 in maintenance of proteostasis and local and systemic immune balance in pregnancy complicated by infection.

Sevoflurane alters the expression of receptors and enzymes involved in Aß clearance in rats.

BACKGROUND: Patients with Alzheimer's disease (AD) exhibit a failure in the clearance of amyloid ß peptides (Aß) from the central nervous system. Previous studies have suggested an association between anesthesia and the occurrence of AD. The aim of the present report was to further explore this possibility. METHODS: Animals were administered sevoflurane for 2 h. We performed immunohistochemistry and real-time polymerase chain reaction to assess the levels of low-density lipoprotein receptor-related protein 1 (LRP-1), the receptor for advanced glycation end products (RAGE) protein, insulin-degrading enzyme (IDE), and neprilysin (NEP) in aged and young rat's brain. RESULT: Levels of LRP-1 were significantly decreased, while those of RAGE increased in the aged and young groups. Immunoreactivity for IDE was significantly decreased at 3 and increased at 15 days in the young group. In contrast, immunoreactivity for NEP was significantly increased at 1 but decreased at 15 days in aged rats. Levels of IDE messenger RNA (mRNA) were significantly decreased at 3 and 7 days in the aged group but was consistently decreased at 1, 3, 7, and 15 days in the young group. Levels of NEP mRNA were significantly decreased in the aged group but increased in the young group at 1, 3, 7, and 15 days. CONCLUSION: Sevoflurane leads to a reduction in the levels of LRP-1, while increasing RAGE and decreasing IDE and NEP in both aged and, to a lesser extent, young rat's brain. These receptor and enzymatic changes may promote the accumulation of Aß in brain tissues and thus exacerbate Alzheimer's-like pathology.

Lipopolysaccharide downregulates CD91/low-density lipoprotein receptor-related protein 1 expression through SREBP-1 overexpression in human macrophages.

Sterol regulatory element-binding proteins (SREBPs) negatively modulate the expression of the CD91/low-density lipoprotein receptor-related protein (LRP1), a carrier and signaling receptor that mediates the endocytosis of more than 40 structurally and functionally distinct ligands. The aim of this work was to analyze whether lipopolysaccharide (LPS) can regulate LRP1 expression through SREBPs in human monocyte-derived macrophages (HMDM). LPS led to LRP1 mRNA and protein inhibition in a dose- and time-dependent manner. Concomitantly, a strong upregulation of SREBP-1 mRNA and SREBP-1 nuclear protein levels was observed in LPS-treated HMDM. The specific silencing of SREBP-1 efficiently prevented LRP1 reduction caused by LPS. SREBP-1 mRNA and nuclear protein levels remained high in HMDM treated with LPS unexposed or exposed to LDL. Native (nLDL) or aggregated LDL (agLDL) per se downregulated SREBP-2 expression levels and increased LRP1 expression. However, lipoproteins did not significantly alter the effect of LPS on SREBP-1 and LRP1 expression. Collectively, these data support that lipoproteins and LPS exert their modulatory effect on LRP1 expression through different SREBP isoforms, SREBP-2 and SREBP-1, respectively. These results highlight a crucial role of SREBP-1 as a mediator of the downregulatory effects of LPS on LRP1 expression in human macrophages, independently of the absence or presence of modified lipoproteins.

Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.

When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver.

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of ß-amyloid peptides (Aß) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma Aß and a decrease in brain Aß monomer after 7 d, indicating increased transport of Aß from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the Aß-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain Aß levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain Aß. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain Aß, indicating that increase in liver LRP and sLRP occurring independent of Aß concentration could result in clearance of Aß. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for Aß clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

The serine protease inhibitor neuroserpin regulates the growth and maturation of hippocampal neurons through a non-inhibitory mechanism.

Neuroserpin is a brain-specific serine protease inhibitor that is expressed in the developing and adult nervous system. Its expression profile led to suggestions that it played roles in neuronal growth and connectivity. In this study, we provide direct evidence to support a role for neuroserpin in axon and dendritic growth. We report that axon growth is enhanced while axon and dendrite diameter are reduced following neuroserpin treatment of hippocampal neurons. More complex effects are seen on dendritic growth and branching with neuroserpin-stimulating dendritic growth and branching in young neurons but switching to an inhibitory response in older neurons. The protease inhibitory activity of neuroserpin is not required to activate changes in neuronal morphology and a proportion of responses are modulated by an antagonist to the LRP1 receptor. Collectively, these findings support a key role for neuroserpin as a regulator of neuronal development through a non-inhibitory mechanism and suggest a basis for neuroserpin's effects on complex emotional behaviours and recent link to schizophrenia.

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1.

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.