Low-density Lipoprotein Receptor-related Protein 1 Protein Research Articles

Jun-APOE-LRP1 axis promotes tumor metastasis in colorectal cancer.

Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains largely unexplored. This study aimed to investigate the role of apoE in CRC metastasis and to identify the transcription factor and receptor of apoE involved in regulation of CRC metastasis. Bioinformatic analyses were conducted to examine the expression pattern and prognosis of apolipoproteins. APOE-overexpressing cell lines were utilized to explore the effects of apoE on proliferation, migration and invasion of CRC cells. Additionally, the transcription factor and receptor of apoE were screened via bioinformatics, and further validated through knockdown experiments. We discovered that the mRNA levels of APOC1, APOC2, APOD and APOE were higher in lymphatic invasion group, and a higher apoE level indicated poorer overall survival and progression-free interval. In vitro studies demonstrated that APOE-overexpression did not affect proliferation but promoted the migration and invasion of CRC cells. We also reported that APOE-expression was modulated by the transcription factor Jun by activating the proximal promoter region of APOE, and APOE-overexpression reversed the metastasis suppression of JUN knockdown. Furthermore, bioinformatics analysis suggested an interaction between apoE and low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 was highly expressed in both the lymphatic invasion group and the APOEHigh group. Additionally, we found that APOE-overexpression upregulated LRP1 protein levels, and LRP1 knockdown attenuated the metastasis-promoting function of APOE. Overall, our study suggests that the Jun-APOE-LRP1 axis contributes to tumor metastasis in CRC.

Fusion of uPA to Anti-aIIbb3 Scfv Facilitates Its Uptake By in Vitro-Generated CD34+ Megakaryocytes (MKs) to Promote Targeted Fibrinolysis

We wish to develop a clinically relevant strategy to deliver anti-thrombotic urokinase plasminogen activator (uPA)-carrying platelets (uPA-PLTs) as a targeted thromboprophylaxis that can be administered to patients with a concurrent high risk of bleeding. This intent was based on our previous data showing that transgenic mice, ectopically expressing uPA in megakaryocytes (MKs), released PLTs, containing uPA in α granules, that were effective as a thromboprophylaxis agent (PMID12689937). We faced two major challenges in further developing uPA-containing PLTs for clinical application: (1) How to get uPA into PLTs as human Plts do not take up uPA, and (2) α-granular uPA led to proteolysis of other α-granule proteins. To diminish degradation of α-granular proteins by uPA, we generated a plasmin-insensitive, but thrombin-activatable, truncated uPA mutant (uPA-T, bottom, Fig. 2). We found that both native single-chain uPA (scuPA) and uPA-T can be taken up by human MKs into the same α granules whereby they co-localize with endocytosed Factor V (FV) and/or fibrinogen (FBGN), as evidenced by confocal microscopy, suggesting that the uPA variants use the same endocytic receptor pathway, also utilized by FV and FBGN (Fig. 1). To delineate what receptor system(s) mediate uPA uptake by CD34+ MK, we found that FV (known to be partially internalized via low-density lipoprotein receptor-related protein 1 (LRP1) competed with both uPA variants for uptake, and that uptake of uPA variants and FV was partially inhibited by the LRP1 antagonist, receptor-associated protein (RAP), further supporting that LRP1 mediates internalization of the uPAs. We also found that uPA and FBGN uptake was partially inhibited by αIIbβ3 integrin antagonist ReoPro. Since we observed that LRP1 protein expression and extent of uPA uptake by MKs declines upon maturation, whereas αIIbβ3 expression does not, we tested if targeting of uPA to αIIbβ3 would facilitate its uptake by CD34+ MKs. We generated a chimeric prodrug composed of a single-chain version of the variable region (scFv) of an anti-αIIbβ3 monoclonal antibody fused to uPA-T (PLT/uPA-T, bottom Fig. 2) which binds human αIIbβ3 on both quiescent and activated PLTs, and is enzymatically activated specifically by thrombin. We have previously shown that in two murine injury models, PLT/uPA-T did not lyse preexisting clots, while it concurrently prevented the development of nascent thrombi (PMC4731161). However, direct infusion of PLT/uPA-T in mice caused significant thrombocytopenia. Now, we found that CD34+ MK internalized PLT/uPA-T into the same α granules of MKs, but with significantly higher efficiency than uPA-T protein (top Fig. 2) which will optimize loading of MKs by uPA-T for subsequent use as a source of uPA-PLTs. We found that scuPA-loaded platelets released from infused MKs prevent thrombus formation in a Rose Bengal photochemical injury model. These studies were done using immunocompromised NOD-scid IL2rγnull (NSG) mice homozygous for VWFR1326H (a VWF mutation switching binding specificity from mouse to human GPIb/IX). NSG/VWFR1326H mice had impaired clotting after vascular injury compared to NSG mice unless infused with human PLTs or MKs. We found that MKs that endocytosed uPA-T can also prevent thrombi formation in this model. Future studies will test whether PLTs released from PLT/uPA-T-loaded MKs would further enhance efficacy of this thromboprophylactic strategy, while avoiding thrombocytopenia, and can become a point-of-care thromboprophylaxis therapeutic in the clinical setting of high-risk of developing thrombi with concurrent concerns of inducing significant bleeding. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The protective role of hippocampal LRP1 knockdown involves synaptic plasticity through the promoting microtubule dynamics and activation of Akt/GSK-3β pathway in depressive rats

BackgroundThe mechanism by which synaptic plasticity mediates the occurrence of depression is unknown. Low-density lipoprotein receptor-related protein 1 (LRP1) affects axon growth and neurogenesis in the brain, but its role in depressive-like behaviors is poorly understood. MethodsAdeno-associated virus-mediated small interfering RNA was injected into the bilateral hippocampus 14 days before chronic unpredicted mild stress (CUMS). Behavior performance was assessed for depressive-like behaviors. Western blot was conducted to detect levels of LRP1, neurogenesis-related proteins, synaptic markers, microtubule system molecules and Akt/GSK-3β signaling-related proteins. Immunohistochemical staining was performed for LRP1 protein, immunofluorescence staining was conducted to determine the Sox2 protein, Nissl's staining and transmission electron microscope staining were used to observe hippocampal morphological features. ResultsThe expression of hippocampal LRP1 was positively correlated with depressive-like behaviors. Treatment with iAAV-LRP1 exerted protective effects on depressive-like behaviors. LRP1 Knockdown relieved the inhibition of synaptic plasticity induced by CUMS. Expression of Sox2, GluR2 and SYP was significantly increased in iAAV-LRP1 CUMS rats. LRP1 knockdown reduced the p-tau (Ser262 and Thr404) and Acet-tubule levels in depressed rats. Finally, we found that LRP1 knockdown activated the PI3K/Akt pathway and inhibited GSK-3β signal transduction. LimitationsMore neurogenesis markers would be considered, and stereotactic injection into hippocampal DG region could be performed to investigate the effects of LRP1. ConclusionsThese findings indicated that hippocampal LRP1 deficiency in stressed rats plays an important protective role in depressive-like behavior by increasing synaptic plasticity mediated by microtubule dynamic and activating Akt/GSK-3β signaling pathway. Therefore, LRP1 may represent a potential therapeutic target for depression.

Decreased low-density lipoprotein receptor-related protein 1 expression in pro-inflammatory monocytes is associated with subclinical atherosclerosis.

Subclinical atherosclerosis (SCA) occurs in asymptomatic individuals. Blood peripheral monocytes are involved in the development of atherosclerosis. Circulating monocytes acquire pro-inflammatory profiles, and they are involved in the early stages of atherosclerosis development. Low-density lipoprotein Receptor-related Protein 1 (LRP1) is expressed in monocytes, mainly in classical and intermediate subsets. Although LRP1 is highly expressed in macrophages and vascular smooth muscle cells (VSMCs) in atherosclerotic plaque formation, its expression in circulating monocytes has not been studied in SCA. The aim of this study was to characterize the LRP1 expression level in circulating monocytes of individuals with SCA and compared with individuals with low (LR) and intermediate (IR) risk of cardiovascular diseases, both without evidence of atherosclerotic lesions in carotid and coronary arteries. LRP1 and additional markers (CD11b, CD11c, and CD36) at cell surface of monocytes were analyzed by flow cytometry assays, whereas LRP1 and pro-inflammatory factors gene expressions were measured in isolated monocytes by quantitative RT-PCRs. Both LRP1 protein and LRP1 mRNA were significantly reduced in monocytes in SCA and IR respect to LR. Conversely, CD36, CD11b, and CD11c monocytic markers showed no significant changes between the different study groups. Finally, increased gene expressions of TNF-α and IL-1β were detected in monocytes of SCA, which were associated with decreased LRP1 expression at the cell surface in total monocytes. In summary, we propose that the decreased LRP1 expression at cell surface in total monocytes with pro-inflammatory profile is associated with the development of atherosclerosis in asymptomatic individuals.

Inflammatory Cytokine IL-1β Downregulates Endothelial LRP1 via MicroRNA-mediated Gene Silencing

Effective clearance of neurotoxic amyloid-beta (Aβ) from the brain is a critical process to prevent Alzheimer’s disease (AD). One major clearance mechanism is Aβ transcytosis mediated by low-density lipoprotein receptor-related protein 1 (LRP1) in capillary endothelial cells. A marked loss of endothelial LRP1 is found in AD brains and is believed to significantly impair Aβ clearance. Recently, we demonstrated that pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, significantly down-regulated LRP1 in human primary microvascular endothelial cells (MVECs). In this study, we sought to determine the underlying molecular mechanism by which IL-1β led to LRP1 loss in MVECs. Reduced LRP1 protein and transcript were detected up to 24 h post-exposure and returned to the baseline levels after 48 h post-exposure with 1 ng/ml IL-1β. This reduction was in part mediated by microRNA-205-5p, -200b-3p, and -200c-3p, as these microRNAs were concomitantly upregulated in MVECs exposed to IL-1β. Synthetic microRNA-205-5p, -200b-3p, and -200c-3p mimics recapitulated LRP1 loss in MVECs without IL-1β, and their synthetic antagomirs effectively reversed IL-1β-mediated LRP1 loss. Importantly, we found that the expression of these three microRNAs was controlled by NF-κB as pharmacological NF-κB inhibitor, BMS-345541, inhibited the IL-1β-mediated upregulation of these microRNAs and rescued LRP1 expression. siRNA-mediated silencing of IκB in MVECs elevated microRNA-200b-3p and decreased LRP1 transcript, partially confirming our overall findings. In conclusion, our study provides a mechanism by which pro-inflammatory IL-1β instigates the suppression of LRP1 expression in MVECs. Our findings could implicate spatiotemporal loss of LRP1 and impairment of the LRP1-mediated clearance mechanism by endothelial cells.

Chinese medicine Tongxinluo capsule protects against blood-brain barrier disruption after ischemic stroke by inhibiting the low-density lipoprotein receptor-related protein 1 pathway in mice

BackgroundChinese medicine Tongxinluo capsule (TXL) has been extensively used to treat ischemic stroke in China, and one of its mechanisms is to protect against blood brain barrier (BBB) disruption after stroke. However, the underlying protective mechanisms are not fully illuminated. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP-1) is involved in BBB disruption after brain ischemia. In this study, we explored whether TXL could downregulate LRP-1 expression and subsequently protect against BBB disruption after stroke using permanent middle cerebral artery occlusion (pMCAO) in mice. MethodsThe animal model of ischemic stroke was induced by pMCAO in male adult C57BL/6J mice. The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 h after pMCAO. Meanwhile, the LRP-1 antagonist receptor associated protein (RAP) was intracerebroventricularly injected at 1 and 21 h after stroke. We measured the following parameters at 6 and 24 h: LRP-1 protein level, BBB leakage, and the expression of tight junction (TJ) proteins including occludin, claudin‐5 and zonula occludens‐1 (ZO‐1). ResultsOur results showed that TXL downregulated LRP-1 level, upregulated these TJ proteins level, and reduced BBB leakage in peri-infarct regions after pMCAO. Further study found that the inhibitor RAP played the same role as did TXL in upregulating these TJ proteins level and reducing BBB leakage after stroke. ConclusionOur study demonstrates that TXL protects against BBB disruption after stroke via inhibiting the LRP-1 pathway.

Effect of moxibustion on expression of RAGE and LRP-1 and neuronal ultrastructure of frontal cortex and hippocampus in vascular dementia rats

To explore the effect of moxibustion of acupoints of Governor Vessel on learning-memory ability and expression of the receptor for advanced glycation end products (RAGE), low density lipoprotein (LDL) receptor related protein-1 (LRP-1), β-amyloid precursor protein (APP) and neuronal ultrastructure of the frontal cortex and hippocampus in vascular dementia (VD) rats, so as to investigate its underlying mechanisms in relieving VD. A total of 24 male SD rats were randomized into normal, model, and moxibustion groups (n＝8 rats in each group). The VD model was established by occlusion of the bilateral common carotid arteries for 20 min and reperfusion for 10 min which was repeated for 3 times. Moxibustion was applied to "Baihui" (GV20), "Dazhui"(GV14) and "Fengfu"(GV16)for 30 min, once daily for 4 weeks. The learning-memory ability was evaluated by using Morris water maze tests. At the end of experiments, the frontal lobe of cerebral cortex and hippocampus tissues were collected for detecting the expression of RAGE and LRP-1 proteins with Western blot, and RAGE, LRP-1 and APP mRNAs with quantitative real-time PCR, respectively. The ultrastructure of neurons in the frontal cortex and hippocampal CA1 region was observed with transmission electron microscopy. Following modeling, Morris water maze tests showed that the average escape latency of the model group and moxibustion group was significantly prolonged on the 4th day of modeling (P<0.01). The expression levels of RAGE protein and mRNA and APP mRNA were significantly increased (P<0.01), while those of LRP-1 protein and mRNA in the cortex and hippocampus remarkably decreased in the model group relevant to the normal group (P<0.01). Following moxibustion, modeling-induced increase of expression of RAGE protein and mRNA and APP mRNA, and down-regulation of expression of LRP-1 protein and mRNA were reversed in the moxibustion group relevant to the model group (P<0.01). Results of electron microscopy showed dilation of the mitochondria with disappearance of the cristae, partial vacuolar degeneration or dissolved external membrane, and cytoplasmic edema with basic disappearance of the rough endoplasmic reticulum and glycogen particles in neurons of the frontal cortex and hippocampal CA1 region after modeling, which was relatively milder in the moxibustion group. Moxibustion of acupoints of the Governor Vessel may reduce the content of APP in the frontal cortex and hippocampus by regulating the expression of RAGE and LRP-1 proteins and mRNA in VD rats.

Dengue virus reduces expression of low-density lipoprotein receptor-related protein 1 to facilitate replication in Aedes aegypti

Aedes aegypti is the primary vector of a number of viruses pathogenic to humans including dengue virus (DENV). DENV infection leads to widespread transcriptomic and proteomic alterations in mosquito cells. Here we identified alterations to the mosquito cell secretome during DENV infection by performing liquid chromatography tandem mass spectrometry. We found that an extracellular fragment of low-density lipoprotein receptor-related protein 1 (LRP-1) was present during infection. Previous literature suggests that LRP-1 regulates cholesterol homeostasis. Therefore, we hypothesized that DENV modifies LRP-1 protein expression to maintain host-derived intracellular cholesterol, which would facilitate virus replication within membrane-associated replication compartments. Accordingly, stimuli that are present during flavivirus infection reduced LRP-1 protein expression. We also found that dsRNA knockdown of LRP-1 increased intracellular cholesterol and DENV viral RNA. Further, depletion of intracellular lipids reduced infection. Together, these data suggest that DENV reduces LRP-1 protein expression, possibly through regulated intramembrane proteolysis (RIP), to increase intracellular cholesterol and facilitate replication in Ae. aegypti.

P53 Regulates the Expression of LRP1 and Apoptosis through a Stress Intensity-Dependent MicroRNA Feedback Loop

SUMMARYUnderstanding how p53 activates certain gene programs and not others is critical. Here, we identify low-density lipoprotein receptor-related protein 1 (LRP1), a transmembrane endocytic receptor, as a p53 target gene. We show that, although LRP1 transcript expression is upregulated in response to both sub-lethal and lethal doses of p53-activating stress, LRP1 protein is only upregulated in response to sub-lethal stress. Interestingly, lethal doses of p53-activating stress inhibit LRP1 de novo translation through an miRNA-based translational repression mechanism. We show that the p53-regulated miRNAs miR-103 and miR-107 are significantly upregulated by lethal doses of stress, resulting in suppression of LRP1 translation and cell death. Our results define a negative feedback loop involving the p53-regulated coding gene LRP1 and p53-regulated miRNA genes. These findings provide mechanistic insight into the selective expression of p53 target genes in response to different stress intensities to elicit either cell survival or cell death.

Treadmill Exercise Ameliorates Spatial Learning and Memory Deficits Through Improving the Clearance of Peripheral and Central Amyloid-Beta Levels.

Aggregated amyloid beta (Aβ) peptides are believed to play a decisive role in the pathology of Alzheimer's disease (AD). Previous evidence suggested that exercise contributes to the improvement of cognitive decline and slows down pathogenesis of AD; however, the exact mechanisms for this have not been fully understood. Here, we evaluated the effect of a 4-week moderate treadmill exercise on spatial memory via central and peripheral Aβ clearance mechanisms following developed AD-like neuropathology induced by intra-hippocampal Aβ1-42 injection in male Wistar rats. We found Aβ1-42-treated animals showed spatial learning and memory impairment which was accompanied by increased levels of amyloid plaque load and soluble Aβ1-42 (sAβ1-42), decreased mRNA and protein expression of neprilysin (NEP), insulin degrading enzyme (IDE) and low-density lipoprotein receptor-related protein-1 (LRP-1) in the hippocampus. Aβ1-42-treated animals also exhibited a higher level of sAβ1-42 and a lower level of soluble LRP-1 (sLRP-1) in plasma, as well as a decreased level of LRP-1 mRNA and protein content in the liver. However, exercise training improved the spatial learning and memory deficits, reduced both plaque load and sAβ1-42 levels, and up-regulated expression of NEP, IDE, and LRP-1 in the hippocampus of Aβ1-42-treated animals. Aβ1-42-treated animals subjected to treadmill exercise also revealed decreased levels of sAβ1-42 and increased levels of sLRP-1 in plasma, as well as increased levels of LRP-1 mRNA and protein in the liver. In conclusion, our findings suggest that exercise-induced improvement in both of central and peripheral Aβ clearance are likely involved in ameliorating spatial learning and memory deficits in an animal model of AD. Future studies need to determine their relative contribution.

Abstract 114: Smooth Muscle Origin-specific Effects of LRP1 Deletion on Angiotensin II-induced Ascending Aortic Aneurysm

Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) plays a critical role in maintaining aortic wall integrity. LRP1 deletion in smooth muscle cells (SMCs) augments angiotensin II (AngII)-induced ascending aortic aneurysms. SMCs in the ascending aorta originate from both the second heart field (SHF) and cardiac neural crest (CNC). The purpose of this study was to determine whether LRP1 depletion in these two SMC origins has differential effects on AngII-induced ascending aortic aneurysm. Methods and Results: Mef2c-Cre was used to delete LRP1 in SMCs of SHF origin; while Wnt-1-Cre was utilized to delete LRP1 in SMCs of CNC origin in mice. Saline or AngII (1,000 ng/kg/min) was infused for 28 days into 12 - 14 week-old male mice with LRP1 depletion in either SHF or CNC origin as well as their wild type littermates. No mice died in saline-infused groups of either genotype. In the AngII-infused mouse group, LRP1 depletion in SMCs of SHF origin led to 38% death due to ascending aortic rupture, compared to a 4% rupture rate in wild type littermates (p = 0.002). In the survivors of AngII-infused group, LRP1 deletion in SMCs of SHF origin resulted in larger ascending aortic diameter compared to wild type littermates (1.6 ± 0.1 vs 2.0 ± 0.1 mm, p &lt; 0.05), as measured by ultrasonography. In contrast to the increases of aortic rupture and luminal dilation in mice with LRP1 depletion in SMCs of SHF origin, LRP1 deletion in SMCs of CNC origin did not affect AngII-induced aortic rupture rate or luminal dilation, compared to wild type littermates. To explore potential signaling mechanisms on how LRP1 depletion in SMCs of SHF origin augments AngII-induced ascending aortic aneurysm, mice were infused with either saline or AngII for 24 hours, and ascending aortic tissues were harvested for Western blot analyses. Aortic LRP1 protein abundance was decreased in mice with LRP1 depletion in SMCs of SHF origin regardless of infusion. Although SMAD2 and ERK signaling contribute to aortic wall integrity, this short interval of AngII infusion did not change these activities in the aorta of SHF-SMC specific LRP1 deleted mice. Conclusion: LRP1 expression in SHF, but not in CNC, -derived SMCs exerts a critical role in the augmentation of AngII-induced ascending aortic aneurysm.

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 10h 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 40min post-addition but had entered the nuclei by 2h, while dox entered the nuclei by 40min. 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.

Low‐Density Lipoprotein Receptor‐Related Protein 1 (LRP1) Is Degraded Primarily by The Lysosomal Degradation System in Hepatocellular Carcinoma (HepG2) Cells

LRP1 is a transmembrane protein responsible for binding and internalizing at least 50 different ligands, one of which is amyloid beta (Aβ). LRP1 has a role in clearance of brain and systemic Aβ. Understanding LRP1's role in Aβ degradation could have important implications for understanding Alzheimer's Disease (AD) pathogenesis. Since LRP1 in the liver is primarily responsible for systemic clearance of Aβ, its degradation could have important implications for AD pathogenesis.Normal degradation of proteins typically occurs through the ubiquitin proteasome system (UPS) and/or the lysosomal degradation system. To study LRP1 degradation through either of these two processes, one system was inhibited, then subsequent LRP1 output was analyzed. By inhibiting one degradation system at a time, we can analyze the protein output to quantify how much protein is degraded by each system. Bafilomycin A1 was used to inhibit the lysosomal degradation system, and MG132 was used to inhibit UPS. Finally, cycloheximide, a protein synthesis inhibitor, was used to determine LRP1 half‐life and to determine whether protein output is significantly affected by protein synthesis during inhibition of the protein degradation system (cycloheximide chase analysis).HepG2 cells were used to study LRP1 degradation in these experiments. The cells were treated with Bafilomycin A1, MG132, or dimethyl sulfoxide (DMSO; control group) and lysed at time points 0, 8, 16, and 24 hours to analyze LRP1 protein levels over time. Experiments with Bafilomycin A1 showed increasing levels of LRP1 over time, whereas experiments with MG132 showed unchanged, then decreasing levels of LRP1 over time. Cycloheximide was tested to determine the optimal concentration for HepG2 cells, which was found to be 30 μM. Then, experiments were performed with either cycloheximide and DMSO (as control), cycloheximide and Bafilomycin A1, or cycloheximide and MG132 at time points 0, 8, 16, and 24 hours to determine the half‐life of LRP1. Our results showed an extension of LRP1 half‐life by inhibition of lysosomal degradation system when using Bafilomycin A1, but half‐life was not extended when UPS was inhibited by MG132. Due to its significant increase in half‐life with Bafilomycin A1, but not with MG132, it's concluded that LRP1's degradation is primarily produced through the lysosomal degradation system.Support or Funding InformationMCPHS UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Verification and characterization of an alternative low density lipoprotein receptor-related protein 1 splice variant.

BackgroundLow density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a ubiquitously expressed multi-ligand endocytosis receptor implicated in a wide range of signalling, among others in tumour biology. Tumour-associated genomic mutations of the LRP1 gene are described, but nothing is known about cancer-associated expression of LRP1 splice variants Therefore, the focus of this study was on an annotated truncated LRP1 splice variant (BC072015.1; NCBI GenBank), referred to as smLRP1, which was initially identified in prostate and lung carcinoma.MethodsUsing PCR and quantitative PCR, the expression of LRP1 and smLRP1 in different human tissues and tumour cell lines was screened and compared on tumour biopsies of head and neck squamous cell carcinoma (HNSCC). Using a recently developed anti-smLRP1 antibody, the expression of the putative LRP1 protein isoform in tumour cell lines in Western blot and immunofluorescence staining was further investigated.ResultsThe alternative transcript smLRP1 is ubiquitously expressed in 12 human cell lines of different origin and 22 tissues which is similar to LRP1. A shift in expression of smLRP1 relative to LRP1 towards smLRP1 was observed in most tumour cell lines compared to healthy tissue. The expression of LRP1 as well as smLRP1 is decreased in HNSCC cell lines in comparison to healthy mucosa. In vitro results were checked using primary HNSCC. Furthermore, the expression of the protein isoform smLRP1 (32 kDa) was confirmed in human tumour cell lines.ConclusionsSimilar to LRP1, the truncated splice variant smLRP1 is ubiquitously expressed in healthy human tissues, but altered in tumours pointing to a potential role of smLRP1 in cancer. Comparative results suggest a shift in expression in favour of smLRP1 in tumour cells that warrant further evaluation. The protein isoform is suggested to be secreted.

Abstract 128: LRP1 Deletion in Smooth Muscle Cells of the Outer Aortic Media Promotes Angiotensin II-induced Thoracic Aortic Aneurysm

Objective: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional protein that is linked to several vascular pathologies. LRP1 deletion in smooth muscle cells (SMCs) accelerates angiotensin II (AngII)-induced thoracic aortic aneurysm (TAA). In association with TAA formation, there is medial thickening that is characterized by a transmural gradient in which pathology progressively increases from lumen to adventitial aspect. We hypothesized that deletion of LRP1 in the outer medial layers of the proximal thoracic aorta has a pivotal role in the pathogenesis of TAA. The aim of this study was to determine whether LRP1 deletion in the outer media accelerates AngII-induced TAA formation. Methods and Results: SMCs in the outer media of the ascending aorta are derived from the second heart field, as demonstrated by lineage tracing studies using Cre under the control of Mef2c. Therefore, we used Mef2c-driven Cre to delete LRP1 in SMCs of the outer medial layers. Female LRP1 flox/flox mice were bred to male Mef2c-Cre1/0 mice to generate study mice. We first confirmed LRP1 deletion in Cre1/0 mice by both immunostaining and Western blot. LRP1 was expressed ubiquitously across smooth muscle cells of all aortic medial layers in Cre 0/0 mice. In mice expressing Mef2c-Cre, aortic LRP1 protein was detected only in SMCs of the inner laminar medial layers. Western blotting demonstrated LRP1 protein abundance in Cre expressing mice was reduced by 43%. Saline or AngII (1,000 ng/kg/min) was infused by subcutaneous osmotic pumps for 28 days into 12 - 14 week-old male Cre0/0 and 1/0 mice. As expected, systolic blood pressure increased similarly in both AngII-infused Cre 0/0 and 1/0 mice compared to saline-infused mice. Aortic rupture occurred within 3 to 10 days after AngII infusion in 17% of AngII-infused Cre 0/0 mice, while LRP1 deletion in Cre 1/0 mice increased aortic rupture to 27%. Aortic diameter in the survivors was significantly increased in Cre1/0 mice compared to Cre0/0 mice. Histologically, elastin fragmentation was detected in the aorta of AngII-infused Cre 0/0 mice and greater in Cre1/0 mice. Conclusion: LRP1 in second heart field-derived SMCs of the outer media may play a critical role in the pathogenesis of TAA.

Relationship among LRP1 expression, Pyk2 phosphorylation and MMP-9 activation in left ventricular remodelling after myocardial infarction.

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low‐density lipoprotein receptor‐related protein 1 (LRP1) and MMP‐9 and MMP‐2 spatiotemporal expression after MI. Real‐time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri‐infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri‐infarct and infarct areas. LRP1 also colocalized with proline‐rich tyrosine kinase 2 (pPyk2) and MMP‐9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP‐9 activity in fibroblasts, without significant changes in MMP‐2 activity. MMP‐9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP‐9 up‐regulation associated with ventricular remodelling after MI.

High Concentration of Rosiglitazone Reduces Low‐density Lipoprotein Receptor‐Related Protein‐1 (LRP1) Levels Via Lysosomal Degradation in HepG2 Cells

LRP1 is a transmembrane receptor involved in the internalization of molecules by either transcellular transport or lysosomal degradation via clathrin. PPARγ is a nuclear receptor that transcribes several target genes involved in cell differentiation, lipid metabolism and glucose homeostasis. PPARγ activation has been shown to upregulate LRP1 mRNA and protein levels in HepG2 cells. However, overactivation of this nuclear receptor has exhibited a reduction in LRP1 protein levels. The aim of this research was to evaluate the effect of rosiglitazone (RGZ), a known PPARγ agonist, on LRP1 protein levels in HepG2 cells. Additionally, the LRP1 protein reduction mechanism in RGZ treated‐cells studying PPARγ activation, ubiquitin proteasome system and lysosomal degradation were evaluated.HepG2 cells were treated with RGZ (0.003 – 30 μM) to show no increase on LRP1 protein levels as reported in the literature. However, cells incubated for 24 and 48 hours with 30 μM of RGZ reduced LRP1 protein levels by 40 and 90%, respectively. PPARγ antagonism and ubiquitin proteasome system inhibition were studied in RGZ treated‐cells demonstrating no effect on LRP1 reduction. Bafilomycin A1, a lysosome degradation inhibitor, increased LRP1 protein levels up to 4‐fold in RGZ treated‐cells.These results suggest that overactivation of PPARγ reduces LRP1 protein levels via lysosome degradation. Further experiments are required to evaluate the mechanism involved in the effect of RGZ on LRP1 protein expression.

Low-density lipoprotein receptor-related protein-1 facilitates heme scavenging after intracerebral hemorrhage in mice

Heme-degradation after erythrocyte lysis plays an important role in the pathophysiology of intracerebral hemorrhage. Low-density lipoprotein receptor-related protein-1 is a receptor expressed predominately at the neurovascular interface, which facilitates the clearance of the hemopexin and heme complex. In the present study, we investigated the role of low-density lipoprotein receptor-related protein-1 in heme removal and neuroprotection in a mouse model of intracerebral hemorrhage. Endogenous low-density lipoprotein receptor-related protein-1 and hemopexin were increased in ipsilateral brain after intracerebral hemorrhage, accompanied by increased hemoglobin levels, brain water content, blood–brain barrier permeability and neurological deficits. Exogenous human recombinant low-density lipoprotein receptor-related protein-1 protein reduced hematoma volume, brain water content surrounding hematoma, blood–brain barrier permeability and improved neurological function three days after intracerebral hemorrhage. The expression of malondialdehyde, fluoro-Jade C positive cells and cleaved caspase 3 was increased three days after intracerebral hemorrhage in the ipsilateral brain tissues and decreased with recombinant low-density lipoprotein receptor-related protein-1. Intracerebral hemorrhage decreased and recombinant low-density lipoprotein receptor-related protein-1 increased the levels of superoxide dismutase 1. Low-density lipoprotein receptor-related protein-1 siRNA reduced the effect of human recombinant low-density lipoprotein receptor-related protein-1 on all outcomes measured. Collectively, our findings suggest that low-density lipoprotein receptor-related protein-1 contributed to heme clearance and blood–brain barrier protection after intracerebral hemorrhage. The use of low-density lipoprotein receptor-related protein-1 as supplement provides a novel approach to ameliorating intracerebral hemorrhage brain injury via its pleiotropic neuroprotective effects.

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo.

Clearance of matrix metalloproteinase-9 is dependent on low-density lipoprotein receptor-related protein-1 expression downregulated by microRNA-205 in human abdominal aortic aneurysm

Low-density lipoprotein receptor-related protein-1 (LRP1) has been suggested to be a crucial regulator in the pathogenesis of abdominal aortic aneurysm (AAA) from previous genome association and animal studies. Our prior study using human aortic samples has further revealed a significant reduction of LRP1 protein expression associated with AAA. However, the downregulation of LRP1 in the pathophysiology of AAA remained unresolved. We hypothesized that LRP1 downregulation may be mediated by microRNA (miR) and that LRP1 may function as a scavenger of matrix metalloproteinase-9 (MMP-9), a well-known protease for degradation of extracellular matrix proteins at the aortic wall for AAA pathogenesis. This study investigated the cause of LRP1 downregulation and its potential effect on AAA pathogenesis. An observational study of LRP1 protein, LRP1 messenger RNA (mRNA), and its three predicted miR candidates (miR-205, miR-338-5p, and miR-545-3p) was first performed in AAA compared with nonaneurysmal tissues from humans, followed by a functional study testing the effect on LRP1 expression of miR-205 overexpression and knockdown in human vascular smooth muscle cells (VSMCs) explant cultured from human abdominal aortic tissues. Lastly, another functional study was performed to test for the clearance of exogenous MMP-9 upon silencing of LRP1 in human VSMCs. From the observational study, significantly higher miR-205 (P< .001) and lower LRP1 protein (P< .001) expressions were found in human AAA tissues compared with nonaneurysmal aortic tissues, and no significant difference in LRP1 mRNA expression was observed. Further statistical analysis showed a significant negative correlation between miR-205 and LRP1 protein expressions (r=-0.65; P< .01). For the functional study, a significant downregulation of LRP1 protein expression was shown in miR-205-overexpressing VSMCs (P< .05), without any alteration in LRP1 mRNA expression. Moreover, a significantly reduced clearance of exogenous MMP-9 was observed in LRP1-silenced VSMCs (P< .05), and this difference in MMP-9 clearance was completely abolished with a pretreatment of anti-LRP1 antibody. Our study revealed the downregulation of LRP1 protein expression may be tightly regulated by miR-205 through translational inhibition in human VSMCs. Also, such LRP1 down-regulation in VSMCs may hinder the removal of pericellular MMP-9, leading to excess MMP-9 remaining in the extracellular matrix. Hence, the integrity of the vascular wall may be disrupted, promoting AAA formation.