LRP1 and its interacting partners in tissue homeostasis

Abstract

Purpose of Review

LDL receptor-related protein 1 (LRP1) is a multi-functional protein with endocytic and signal transduction properties due to its interaction with numerous extracellular ligands and intracellular proteins. This brief review highlights key developments in identifying novel functions of LRP1 in liver, lung, and the central nervous system in disease pathogenesis.

Recent findings

In hepatocytes, LRP1 complexes with phosphatidylinositol 4-phosphate 5-kinase-1 and its related protein to maintain intracellular levels of phosphatidylinositol (4,5) bisphosphate and preserve lysosome and mitochondria integrity. In contrast, in smooth muscle cells, macrophages, and endothelial cells, LRP1 interacts with various different extracellular ligands and intracellular proteins in a tissue- and microenvironment-dependent manner to either enhance or suppress inflammation, disease progression or resolution. Similarly, LRP1 expression in astrocytes and oligodendrocyte progenitor cells regulates cell differentiation and maturation in a developmental-dependent manner to modulate neurogenesis, gliogenesis, and white matter repair after injury.

Summary

LRP1 modulates metabolic disease manifestation, inflammation, and differentiation in a cell-, time-, and tissue-dependent manner. Whether LRP1 expression is protective or pathogenic is dependent on its interaction with specific ligands and intracellular proteins, which in turn is dependent on the cell type and the microenvironment where these cells reside.

INTRODUCTION

Low density lipoprotein receptor-related protein-1 (LRP1) is an ubiquitously-expressed type 1 transmembrane protein with both endocytic and signal transduction functions. The importance of LRP1 functions is highlighted by the embryonic lethality observed in mice with total Lrp1 gene ablation and the relationship between human LRP1 gene polymorphisms with a wide spectrum of diseases, including cardiovascular disease, obesity and diabetes, neurodegenerative disorders, and tumor invasion and metastasis. LRP1 modulates disease manifestation in a tissue-specific manner via its plethora of functions in various cell types. The cell type-specific functions of LRP1 have been reviewed comprehensively in several earlier review articles [1, 2]. Herein we provide an update of the literature, highlighting key developments in identifying novel functions of LRP1 in hepatocytes, alveolar smooth muscle cells and macrophages, and endothelial cells, as well as its role in regulating progenitor cell differentiation.

Hepatocyte LRP1 expression is required for intracellular organelle integrity in the liver

The best-known function of LRP1 in the liver is its role in chylomicron remnant clearance from the plasma circulation. However, the LDL receptor can serve similar functions hence the inactivation of both LDL receptor and LRP1 in the liver is necessary to cause cholesteryl ester-rich remnant lipoprotein accumulation in circulation [3]. Interestingly, hepatocyte-specific LRP1 knockout (hLrp1^−/−) mice were more sensitive than wild type mice to diet-induced hepatosteatosis and steatohepatitis [4, 5]. The accelerated liver disease phenotype in hLrp1^−/− mice did not require LDL receptor gene inactivation, hence suggesting that LRP1 has additional functions that are not shared by LDL receptor. One difference between these two receptors is that hepatic LRP1, but not LDL receptor, is required for maintaining lysosomal integrity. Thus, LRP1 interacts with cathepsin D and serves to complement the mannose 6-phosphate receptor in the liver for the transport of extracellular cathepsin D to the lysosome for prosaposin activation [6]. Lysosomal enzyme deficiency with LRP1 inactivation also increases oxidative stress and sensitivity to palmitate-induced lysosomal permeability and toxicity [7]. Additionally, LRP1 deficiency also impairs autophagosome fusion with lysosomes, thereby promoting intracellular lipid accumulation due to lipophagy defects.

Our more recent data showed that LRP1 inactivation in the liver also has direct impact on mitochondrial functions and integrity. In comparison to mitochondria in wild type mouse livers, the mitochondria isolated from the livers of hLrp1^−/− mice contain less anionic phospholipids, leading to the impairment of mitochondrial fusion and the increase in mitochondrial fragmentation that causes liver mitochondrial dysfunction in hLrp1^−/− mice [8*]. These mitochondrial abnormalities reduce the capacity of the hLrp1^−/− hepatocytes to oxidize glucose and fatty acids, thus providing another mechanism by which LRP1 deficiency in the liver accelerates diet-induced hepatosteatosis and steatohepatitis [5].

The mechanism by which LRP1 deficiency compromises lysosome and mitochondria integrity and functions have not been identified definitively. LRP1 is located in the plasma membrane and in intracellular compartments, primarily endosomes and the endoplasmic reticulum. This receptor is known to interact with numerous cytoplasmic adaptor proteins in regulating cell functions. Hence, it is likely that LRP1 interaction with specific adaptor proteins in the plasma membrane and/or endosomes may be responsible for its maintenance of lysosome and mitochondria integrity in hepatocytes. One LRP1 interacting protein with the potential to modulate mitochondria and lysosome integrity is phosphatidylinositol 4-phosphate 5-kinase like protein-1 (PIP5KL1) originally identified through yeast two-hybrid assays [9]. While this protein does not possess enzyme activity, it heterodimerizes with several phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) proteins to enhance the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisposphate (PI(4,5)P₂) [10]. We found that LRP1 forms a complex with PIP5KL1 and PIP5K1β in liver and the absence of LRP1 lowers the levels of these proteins in the plasma membrane, leading to an overall reduction of PI(4,5)P₂ levels in hepatocytes [8*]. Reduced PI(4,5)P₂ levels have been shown to impair autophagosome-lysosome fusion [11, 12]. The PI(4,5)P₂ cycle also generates cytidine-5’ diphosphate-diacylglycerol, an important substrate for phosphatidylinositol and cardiolipin synthesis. A proposed mechanism by which LRP1 interacts with the inositol lipid kinases to regulate PI(4,5)P₂ levels and intracellular organelle integrity is shown in Figure 1. Whether the reduced PI(4,5)P₂ levels in the livers of hLrp1^−/− mice are directly responsible for the compromised lysosome and mitochondria integrity remains to be determined.

Figure 1. Schematic model depicting the multiple functions of LRP1 in hepatocytes.

LRP1 in hepatocytes serves as an endocytic receptor for clearance of ligands such as apoE-containing lipoproteins from the plasma circulation as well as signaling transduction receptor through its binding to PIP5KL1 and PIP5K1β to generate PI(4,5)P₂ that is important for intracellular organelle maintenance. Note that the sizes of the protein as well as intracellular organelles are not drawn to scale.

LRP1 sustains pulmonary function and modulates the inflammatory response in lung disease

LRP1 is abundantly expressed in smooth muscle cells. Earlier studies showed that LRP1 interacts with PDGF- and TGF-receptors to reduce smooth muscle cell proliferation and migration in protection against vascular occlusive diseases in the vessel wall. A more recent study revealed that LRP1 expression in smooth muscle cells is also important in lung functions. In comparison to wild type mice, smooth muscle cell-specific LRP1 knockout (smLrp1^−/−) mice showed increased lung resistance and reduced compliance [13*]. Airway responsiveness to muscarinic receptor agonist stimulation was also enhanced in smLrp1^−/− mice. The difference in pulmonary function cannot be attributed to differences in immune response between wild type and smLrp1^−/− mice, but the analysis of bronchoalveolar lavage fluid revealed the accumulation of proteins known to affect pulmonary function and asthma in smLrp1^−/− mice [13*]. These include a >20-fold increase in calreticulin and α1-antitrypsin that are known ligands for LRP1-mediated endocytosis clearance. Thus, LRP1-mediated endocytosis plays a key role in airway responsiveness of smooth muscle cells. Interestingly, the increased lung resistance and hyperresponsiveness of smLrp1^−/− mice in response to muscarinic receptor agonists are in striking contrast to the aortic dilatation observed in smLrp1^−/− mice due to attenuated response of LRP1-deficient smooth muscle cells to vasoconstrictor stimulation [14]. The latter phenotype was attributed to modulation of intracellular calcium levels by binding to the voltage-gated Ca²⁺ channel auxiliary subunit α₂δ-1 thereby affecting plasma membrane expression of these calcium channels and defects in calcium release in response to ryanodine receptor activation with LRP1 deficiency [14]. Nevertheless, both studies revealed the importance of LRP1 expressed in smooth muscle cells in limiting disease pathogenesis. Importantly, these contrasting results showed that LRP1 has both endocytic and signaling properties in smooth muscle cells, and protects disease pathogenesis through these mechanisms in a tissue-specific manner. A schematic model depicting the bifunctional properties of LRP1 in smooth muscle cells is shown in Figure 2.

Figure 2. Schematic model depicting the multiple functions of LRP1 in smooth muscle cells.

LRP1 in smooth muscle cells serves as an endocytic receptor for clearance of ligands such as α1-antitrypsin (AAT) to maintain lung compliance and tissue homeostasis, as well as signaling transduction receptor through its function as a co-receptor with tyrosine receptor kinase to modulate contraction, proliferation and migration. Note that the sizes of the protein as well as intracellular organelles are not drawn to scale.

Although innate immunity cannot explain the differences between wild type and smLrp1^−/− mice in airway responsiveness to muscarinic receptor stimulation, LRP1 expressed in alveolar macrophages participates in inflammatory response to infection or injury in the lung. In this tissue, surfactant proteins act as collectins to opsonize and engulf foreign organisms, dead cells, and cellular debris, and the entire complex is recognized by calreticulin and LRP1 for endocytic clearance by macrophages. The binding of surfactant proteins to the calreticulin/LRP1 complex in macrophages induces p38 mitogen-activated protein kinase phosphorylation, NFĸB activation, and the expression of pro-inflammatory cytokines that additionally recruit monocytes and neutrophils to accelerate clearance of infectious agents and dead cells [15]. Hence, LRP1 expressed in alveolar macrophages serves as a pro-inflammatory activator during infection and cell injury to preserve lung functions. Interestingly, LRP1 is also involved with inflammation resolution. The phagocytosis of dead cells via binding of the calreticulin-LRP1 complex to surfactant proteins on the surface of apoptotic cells also activates intramembrane proteolysis, releasing the intracellular domain of LRP1 that translocate to the nucleus to suppress pro-inflammatory cytokine gene expression. Hence, LRP1 serves as a signaling and endocytic receptor in alveolar macrophages to participate in both the inflammation and resolution phase to combat lung disease (Figure 3).

Figure 3. Schematic model depicting the pro- and anti-inflammatory roles of LRP1 in macrophages.

In macrophages, LRP1 can bind to anti-inflammatory proteins such as sFRP5 or pro-inflammatory proteins such as TGFβ-2 and mediates their endocytosis, leading to a pro-inflammatory or anti-inflammatory state, respectively. LRP1 can also be processed and the intracellular domain (ICD) can translocate to the nucleus to limit transcription of pro-inflammatory genes.

Pro- and anti-inflammatory properties of macrophage LRP1 in cardiometabolic disease development

The pro- and anti-inflammatory properties of macrophage-expressed LRP1 in disease pathogenesis and resolution are also evident in cardiometabolic disease modulation. On one hand, macrophage LRP1 deficiency reduces diet-induced adiposity, improves glucose tolerance, and suppresses diet-induced liver steatosis and inflammation in Ldlr^−/− mice [16]. On the other hand, macrophage LRP1 inactivation or dysfunction accelerates diet-induced atherogenesis and injury-induced neointimal hyperplasia in the same Ldlr^−/− mouse model [17–20]. In the context of obesity and diabetic metabolic diseases, macrophage LRP1 promotes diet-induced inflammation by sequestration of secreted frizzled-related protein-5 (sFRP5), an anti-inflammatory adipokine, thereby alleviating its ability to inhibit Wnt signaling and the consequential effect of β-catenin induced inflammatory gene activation [16]. In the context of vascular disease protection, macrophage LRP1 protects the vessel wall through multiple anti-inflammatory mechanisms, including inhibiting cell death, facilitating the clearance of apoptotic cells, and limiting monocytosis in mechanisms related to Akt activation and suppression of TGF-β signaling events. Macrophage LRP1 activates Akt signaling by serving as an anchor for the recruitment of Rab8a and phosphatidylinositol 3-kinase-γ to the macropinosomal membrane [21], while suppressing TGF-β signaling by binding and clearance of TGF-β2 [19]. Additionally, LRP1 also attenuates inflammatory cytokine expression in activated macrophages via its intracellular domain interaction with interferon regulatory factor-3 to promote its nuclear export [22]. These differences reinforce the concept that macrophage LRP1 modulates disease pathogenesis in a tissue-specific manner.

Despite the preponderance of data showing the importance of macrophage LRP1 in suppression of atherogenesis, macrophage LRP1 deletion has also been shown to accelerate regression of pre-existing lesions in ApoE^−/− mice [23]. This observation suggested that macrophage LRP1 may actually promote atherosclerosis at later stages of the disease. The difference between LRP1-repleted and LRP1-depleted macrophages in lesion regression is due to increased expression of the C-C chemokine receptor type 7 (CCR7) with LRP1-deficiency thereby accelerating the egress of macrophage foam cells from the lesion site to the mediastinal lymph node [23]. Additionally, macrophage LRP1 deficiency was also shown to facilitate reverse cholesterol transport in the regression model [23]. How macrophage LRP1 deficiency accelerates reverse cholesterol transport in this model remains unclear since LRP1 dysfunction reduces the expression of the cholesterol exporter ABCA1 [6, 20]. Moreover, since the pro- and anti-inflammatory properties of LRP1 is depending on LRP1 ligands present in the macrophage environment [24], additional studies evaluating the influence of macrophage LRP1 inactivation on atherosclerosis regression in another animal model with physiologically-regulated apoE expression may clarify whether the detrimental effects of macrophage LRP1 on lesion resolution are ligand dependent. Importantly, another caveat to this study is that atherosclerotic lesion regression was examined only in the aortic roots where complex necrotic lesions are limited. In view of the established role of macrophage LRP1 in efferocytosis, examination of a different anatomic region such as the innominate arteries where lesion necrosis is more prominent is clearly worthwhile. Whether macrophage LRP1 participates in the resolution of necrotic lesions that are vulnerable to rupture is an important issue with translational implications.

Opposing roles of endothelial LRP1 in metabolic and neurodegenerative disease modulation

Although LRP1 is expressed at low levels in endothelial cells, studies with endothelial-specific LRP1 knockout (eLrp1^−/−) mice revealed its key role in regulation of metabolic and neurodegenerative diseases. Surprisingly, the eLrp1^−/− mice are protected from diet-induced adiposity and glucose intolerance [25]. Apparently, LRP1 interacts with PPARγ in endothelial cells and promotes its translocation to the nucleus after intramembrane proteolysis to activate PPARγ target gene transcription [25]. One of these target genes is pyruvate dehydrogenase kinase 4, and its activation leads to suppression of pyruvate dehydrogenase activity and the reduction of mitochondrial respiration activities. Hence, LRP1 inactivation lowers pyruvate dehydrogenase kinase 4 levels thereby increasing respiratory activities to protect against diet-induced obesity and glucose intolerance. Whether LRP1-PPARγ interaction is restricted to endothelial cells remains unclear. Nevertheless, the report showing pulmonary arterial hypertension in smLrp1^−/− mice can be reversed by PPARγ activation suggests that LRP1 may also mediate PPARγ activation in smooth muscle cells [26].

In contrast to the metabolic benefits associated with endothelial LRP1 inactivation, LRP1 is required to maintain endothelial function and integrity in the cerebral vascular system (Figure 4). The depletion of LRP1 specifically in endothelial cells results in abnormal angiogenesis in an oxygen-induced retinopathy model as well as impaired clearance of amyloid-β_1–42 across the blood brain barrier [27, 28]. In the retinopathy model, endothelial LRP1 regulates angiogenesis via its interaction with poly(ADP-ribose) polymerase-1 (PARP1) and the subsequent reduction in phosphorylation of retinoblastoma and cyclin-dependent kinase-2 to limit cell cycle progression [27]. Hence, eLrp1^−/− mice displayed significantly more neovascularization with more branch points and angiogenic sprouts at the leading edge of the vasculature under hypoxic stress conditions [27]. In the blood brain barrier, endothelial LRP1 inhibits cyclophilin A activity and the loss of LRP1 in endothelial cells activates a self-autonomous cyclophilin A-matrix metalloproteinase 9 pathway that degrades endothelial tight junction proteins to cause blood brain barrier dysfunction and breakdown [29**]. Importantly, cyclophilin A inhibition restores blood brain barrier integrity and improves cognitive functions in eLrp1^−/− mice [29**]. In view of the progressive loss of endothelial LRP1 with aging and in Alzheimer’s disease, this study identified a potential therapeutic target for neurodegenerative disease management.

Figure 4. Schematic model depicting the multiple functions of LRP1 in endothelial cells.

LRP1 expression in endothelial cells favors diet-induced obesity and diabetes via the interaction of its intracellular domain (ICD) with PPARγ to enhance pyruvate dehydrogenase transcription and the reduction of mitochondrial respiration activities. In contrast, LRP1 in endothelial cells of the cerebral vascular system is required to maintain endothelium integrity by suppressing cyclophilin A (CypA)-MMP9 and PARP signaling.

Role of LRP1 in astroglial and oligodendrocyte differentiation is developmental stage-dependent

LRP1 expressed in other cell types in the central nervous system also plays key role in tissue homeostasis. The loss of LRP1 in the forebrain radial glia cells impairs neurogenesis and gliogenesis during the prenatal period, leading to severe neurological symptoms with expanded ventricles and seizure [30**]. In contrast, deletion of LRP1 in astroglia cells postnatally after the completion of neurogenesis only delays astrocyte maturation during early development but the number of mature astrocytes and neuronal activities in the hippocampus were found to be comparable to wild type mice with ongoing development [31**]. Reduction in Erk kinase signaling cascade was found to be responsible for the delay in maturation of LRLP1-deficient astroglial cells [31**].

The role of LRP1 in oligodendrocyte differentiation and maturation is also dependent on the stage of development. In the developing nerve and injured white matter, LRP1 deletion in oligodendrocyte progenitor cells reduces the number of oligodendrocytes and leads to prenatal hypomyelination and compromised white matter repair [32]. This defect can be reversed by a combination of cholesterol supplementation and pioglitazone, thus indicating LRP1 participates in peroxisomal biogenesis and lipid homeostasis pathways during oligodendrocyte progenitor cell differentiation [32]. In contrast, the deletion of LRP1 from adult oligodendrocyte progenitor cells increases the number of mature oligodendrocytes and enhances myelin repair in response to demyelination [33*]. The mechanism by which LRP1 expression suppresses differentiation of adult oligodendrocyte progenitor cells has not be revealed. An intriguing possibility is that LRP1 may coordinate with bone morphogenetic proteins (BMPs) in suppressing oligodendrocyte progenitor cell differentiation while promoting the differentiation of astroglial cells. The opposing effects of BMPs in astrocyte and oligodendrocyte differentiation have been reported previously [34]. In view of studies showing that LRP1 is a co-receptor for BMP-4 and regulates its signaling in endothelial cells during vascular development [35], it is worthwhile to test the relationship between LRP1 and BMPs in regulating cell differentiation in the central nervous system.

Conclusion

LRP1 is a multi-functional protein that modulates metabolic disease manifestation, inflammation, and central nervous system development and injury repair. Whether LRP1 expression is protective or pathogenic is dependent on its interaction with specific extracellular ligands and intracellular adaptor proteins, which in turn is dependent on the various cell types, the developmental stage, and the microenvironment of each tissue. The development of any therapeutics that target LRP1 modulation needs to be cell type specific and be delivered to specific tissues for effective disease management.

Key points:

• LRP1 expression in hepatocytes is necessary to maintain intracellular organelle integrity.

• LRP1 expression in smooth muscle cells sustains pulmonary function and vascular reactivity in the vessel wall.

• LRP1 expression in macrophages has pro- and anti-inflammatory properties and modulates disease pathogenesis in a tissue-dependent manner.

• LRP1 inactivation in endothelial cells has metabolic benefits but causes neurodegenerative diseases.

• LRP1 modulates differentiation of glial stem cells in the central nervous system in a developmental stage-dependent manner.