RESUMO
Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.
Assuntos
Hiperferritinemia , Sobrecarga de Ferro , Humanos , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/diagnóstico , Ferritinas/genética , Macrófagos , AlelosRESUMO
Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response. Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation.
Assuntos
Infecções por Coxsackievirus , Miocardite , Viroses , Animais , Moléculas de Adesão Celular Neuronais , Modelos Animais de Doenças , Enterovirus Humano B , Fibronectinas , Macrófagos , Camundongos , Monócitos/patologia , NecroseRESUMO
Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Endotélio/metabolismo , Nanopartículas , Proteínas de Peixe-Zebra/fisiologia , Animais , Ânions , Proteínas de Ligação ao Cálcio/genética , Técnicas de Silenciamento de Genes , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
PURPOSE: Emerging evidence has shown that macrophages (Mφs) at different tumor sites have diverse clinical attributes. Stabilin-1 is a multi-functional scavenger marker for specialized tumor-associated Mφs. This study investigates the relationship between the density and microlocalization of stabilin-1+ Mφs within tumors and the clinical outcomes of patients with urothelial carcinoma of the bladder (UCB). METHODS: In this retrospective study, 283 UCB patients who received radical cystectomy or transurethral resection were examined. Immunohistochemistry and immunofluorescence analyses were used to colocalize the expression of stabilin-1 with other markers for Mφs (CD14, CD68, CD163, and CD206). Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS) and recurrence-free survival (RFS). RESULTS: In UCB tissues, stabilin-1 was primarily expressed on Mφs, as evident from triple immunofluorescence staining for stabilin-1 and Mφ markers. Stabilin-1+ Mφs were often more prominent in stromal regions rather than intratumoral regions in UCB tissues (P < 0.0001). After dichotomization at the median cell density for stabilin-1+ Mφs, only intratumoral stabilin-1+ Mφ density was a predictor of poor OS (P < 0.001) and RFS (P = 0.026). Moreover, intratumoral stabilin-1+ Mφ density was positively associated with tumor stage (P < 0.01) and histological grade (P < 0.01), and emerged as an independent prognostic factor for OS (HR 2.371; P < 0.0001), but not for RFS (HR 1.491; P = 0.061). CONCLUSIONS: Our findings indicate that intratumoral stabilin-1+ Mφs could potentially be used as a pro-tumoral prognostic marker for UCB patients.
Assuntos
Carcinoma de Células de Transição/patologia , Macrófagos Associados a Tumor/patologia , Neoplasias da Bexiga Urinária/patologia , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Carcinoma de Células de Transição/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Receptores de Superfície Celular/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida , Microambiente Tumoral , Macrófagos Associados a Tumor/citologia , Macrófagos Associados a Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismoRESUMO
The placenta undergoes reconstruction at different times during fetal development to supply oxygen and nutrients required throughout pregnancy. To accommodate the rapid growth of the fetus, small spiral arteries undergo remodeling in the placenta. This remodeling includes apoptosis of endothelial cells that line spiral arteries, which are replaced by trophoblasts of fetal origin. Removal of dead cells is critical during this process. Stabilin-1 (Stab1) and stabilin-2 (Stab2) are important receptors expressed on scavenger cells that absorb and degrade apoptotic cells, and Stab1 is expressed in specific cells of the placenta. However, the role of Stab1 and Stab2 in placental development and maintenance remain unclear. In this study, we assessed Stab1 and Stab2 expression in the placenta and examined the reproductive capacity and placental development using a double-knockout mouse strain lacking both Stab1 and Stab2 (Stab1/2 dKO mice). Most pregnant Stab1/2 dKO female mice did not produce offspring and exhibited placental defects, including decidual hemorrhage and necrosis. Findings of this study offer the first description of the phenotypic characteristics of placentas and embryos of Stab1/2 dKO females during pregnancy, suggesting that Stab1 and Stab2 are involved in placental development and maintenance.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Placenta/metabolismo , Placentação/genética , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Desenvolvimento Fetal/genética , Humanos , Camundongos , Camundongos Knockout , Oxigênio/metabolismo , Placenta/patologia , Gravidez , Reprodução/genética , Trofoblastos/metabolismo , Trofoblastos/patologia , Remodelação Vascular/genéticaRESUMO
Stabilin-2/HARE is the primary clearance receptor for circulating hyaluronan (HA), a polysaccharide found in the extracellular matrix (ECM) of metazoans. HA has many biological functions including joint lubrication, ocular turgor pressure, skin elasticity and hydration, cell motility, and intercellular signaling, among many others. The regulatory system for HA content in the tissues, lymphatics, and circulatory systems is due, in part, to Stabilin-2/HARE. The activity of this receptor was discovered about 40 years ago (early 1980s), cloned in the mid-1990s, and has been characterized since then. Here, we discuss the overall domain organization of this receptor and how it correlates to ligand binding, cellular signaling, and its role in known physiological disorders such as cancer.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Doença , Saúde , Receptores de Hialuronatos/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular Neuronais/química , Humanos , Receptores de Hialuronatos/química , Ligantes , Neoplasias/patologia , Domínios ProteicosRESUMO
Stabilin-1 is a transmembrane receptor that regulates molecule recycling and cell homeostasis by controlling the intracellular trafficking and participates in cell-cell adhesion and transmigration. Stabilin-1 expression is observed in various organs, including bones; however, its function and regulatory mechanisms in the bone remain unclear. In this study, we evaluated the physiological function of stabilin-1 in bone cells and tissue using a stabilin-1 knockout (Stab1 KO) mouse model. In wild-type (WT) mice, stabilin-1 was expressed in osteoblasts and osteoclasts, and its expression was maintained during osteoblast differentiation but significantly decreased after osteoclast differentiation. There was no difference in osteoblast differentiation and function, or the expression of osteoblast differentiation markers between mesenchymal stem cells isolated from Stab1 KO and WT mice. However, osteoclast differentiation marker levels demonstrated a non-significant increase and bone-resorbing activity was significantly increased in vitro in RANKL-induced osteoclasts from Stab1-deficient bone marrow macrophages (BMMs) compared with those of WT BMMs. Microcomputed tomography showed a negligible difference between WT and Stab1 KO mice in bone volume and trabecular thickness and number. Moreover, no in vivo functional defect in bone formation by osteoblasts was observed in the Stab1 KO mice. The osteoclast surface and number showed an increased tendency in Stab1 KO mice compared to WT mice in vivo, but this difference was not statistically significant. Overall, these results indicate that Stab1 does not play an essential role in in vivo bone development and bone cell function, but it does affect in vitro osteoclast maturation and function for bone resorption.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Osteoclastos/citologia , Animais , Células da Medula Óssea/citologia , Reabsorção Óssea , Osso e Ossos , Adesão Celular , Diferenciação Celular , Linhagem Celular , Movimento Celular , Feminino , Genótipo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteócitos/citologia , Osteogênese , Microtomografia por Raio-XRESUMO
Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/complicações , Homeostase , Cirrose Hepática/prevenção & controle , Macrófagos/fisiologia , Animais , Tetracloreto de Carbono , Quimiocina CCL3/fisiologia , Deficiência de Colina/complicações , Humanos , Lipoproteínas LDL/metabolismo , Malondialdeído/análogos & derivados , Malondialdeído/metabolismo , CamundongosRESUMO
Overexpression of sarcolipin (SLN), a regulator of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), stimulates calcineurin signaling to enhance skeletal muscle oxidative capacity. Some studies have shown that calcineurin may also control skeletal muscle mass and remodeling in response to functional overload and unload stimuli by increasing myofiber size and the proportion of slow fibers. To examine whether SLN might mediate these adaptive responses, we performed soleus and gastrocnemius tenotomy in wild-type (WT) and Sln-null (Sln-/-) mice and examined the overloaded plantaris and unloaded/tenotomized soleus muscles. In the WT overloaded plantaris, we observed ectopic expression of SLN, myofiber hypertrophy, increased fiber number, and a fast-to-slow fiber type shift, which were associated with increased calcineurin signaling (NFAT dephosphorylation and increased stabilin-2 protein content) and reduced SERCA activity. In the WT tenotomized soleus, we observed a 14-fold increase in SLN protein, myofiber atrophy, decreased fiber number, and a slow-to-fast fiber type shift, which were also associated with increased calcineurin signaling and reduced SERCA activity. Genetic deletion of Sln altered these physiological outcomes, with the overloaded plantaris myofibers failing to grow in size and number, and transition towards the slow fiber type, while the unloaded soleus muscles exhibited greater reductions in fiber size and number, and an accelerated slow-to-fast fiber type shift. In both the Sln-/- overloaded and unloaded muscles, these findings were associated with elevated SERCA activity and blunted calcineurin signaling. Thus, SLN plays an important role in adaptive muscle remodeling potentially through calcineurin stimulation, which could have important implications for other muscle diseases and conditions.
Assuntos
Calcineurina/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteolipídeos/genética , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Músculo Esquelético/cirurgia , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , TenotomiaRESUMO
The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions.
Assuntos
Separação Celular , Células Endoteliais/citologia , Endotélio/citologia , Hepatócitos/citologia , Fígado/citologia , Animais , Separação Celular/métodos , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura/métodosRESUMO
The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn(2280)), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991-1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756-1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/metabolismo , Ácido Hialurônico/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Substituição de Aminoácidos , Asparagina/química , Transporte Biológico Ativo , Moléculas de Adesão Celular Neuronais/genética , Dermatan Sulfato/metabolismo , Endocitose , Expressão Gênica , Células HEK293 , Heparina/metabolismo , Humanos , Ligantes , Lipoproteínas LDL/metabolismo , Mutagênese Sítio-Dirigida , Polissacarídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by â¼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Endocitose/fisiologia , Regulação da Expressão Gênica/fisiologia , Ácido Hialurônico/metabolismo , Lipoproteínas LDL/metabolismo , NF-kappa B/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Sulfatos de Condroitina/genética , Dermatan Sulfato/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Ácido Hialurônico/genética , Lipoproteínas LDL/genética , NF-kappa B/genética , Transdução de Sinais/fisiologiaRESUMO
The hyaluronan receptor for endocytosis (HARE), or Stabilin-2, is the mammalian endocytic clearance receptor for HA, heparin, advanced glycation end-products, acetylated and oxidized low-density lipoproteins and collagen N-terminal propeptides. This large 2551 amino acid receptor is encoded by a gene that covers over 180 kbp on human chromosome 12 and is predicted to be composed of 69 exons. Due to the expression profile of this gene and the number of exons it contains, we hypothesized that splice variants of stab2 are encoded in these tissues. In addition, a correlation between alternative splice variants and cancer progression has been shown in other HA receptors such as RHAMM and CD42. In this study, two methods were utilized in identifying and/or isolating the HARE splice variants. The first method used primer sets to amplify the 190-HARE encoding region that could contain splice junctions; therefore, they were purified from agarose gels and sequenced. Five splice variants were detected in that manner. In the second approach, the entire open reading frame of HARE was amplified. This allowed four splice variants with extensive exon splicing to be isolated. After the splice variants were sequenced, three were cloned into a mammalian expression vector. Next, stable cell lines expressing the variants were created in order to determine stable protein expression. In this study, the splice variants were found to be tissue specific in most cases. This suggests that tissue specific regulatory splicing mechanisms may lead to differences in functionality between the splice variants.
Assuntos
Processamento Alternativo , Medula Óssea/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linfonodos/metabolismo , Baço/metabolismo , Colágeno/metabolismo , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Éxons , Células HEK293 , Humanos , Fases de Leitura Aberta , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de ProteínaRESUMO
The hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047-15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HA(TM) for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20-50 nM with an apparent K(m) ~10 nM, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Regulação da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , NF-kappa B/metabolismo , Animais , Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Ligantes , Ligação Proteica , Ratos , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Profound edema or formation of a pseudocyst containing plasma proteins is a prominent characteristic of nasal polyps (NP). However, the mechanisms underlying NP retention of plasma proteins in the submucosa remain unclear. Recently, we reported that impairment of fibrinolysis causes excessive fibrin deposition in NP and this might be involved in the retention of plasma proteins. Although the coagulation cascade plays a critical role in fibrin clot formation at extravascular sites, the expression and role of coagulation factors in NP remain unclear. OBJECTIVE: The objective of this study was to investigate the expression of coagulation factors in patients with chronic rhinosinusitis (CRS). METHODS: Sinonasal tissues were collected from patients with CRS and control subjects. We assayed mRNA for factor XIII-A (FXIII-A) by using real-time PCR and measured FXIII-A protein by means of ELISA, immunohistochemistry, and immunofluorescence. RESULTS: FXIII-A mRNA levels were significantly increased in NP tissue from patients with CRS with NP (P < .001) compared with uncinate tissue from patients with CRS or control subjects. Similarly, FXIII-A protein levels were increased in NP. Immunofluorescence analysis revealed that FXIII-A expression in inflammatory cells and FXIII-A(+) cell numbers were significantly increased in NP. Most FXIII-A staining was observed within CD68(+)/CD163(+) M2 macrophages in NP. Levels of FXIII-A correlated with markers of M2 macrophages, suggesting that M2 macrophages are major FXIIIA-producing cells in NP. CONCLUSION: Overproduction of FXIII-A by M2 macrophages might contribute to the excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with NP.
Assuntos
Fator XIIIa/biossíntese , Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Adolescente , Adulto , Idoso , Doença Crônica , Fator XIIIa/genética , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Regulação para Cima , Adulto JovemRESUMO
Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are scavenger receptors expressed by liver sinusoidal endothelial cells (LSECs). The Stabilin-mediated scavenging function is responsible for regulating the molecular composition of circulating blood in mammals. Stab1 and Stab2 have been shown to influence fibrosis in liver and kidneys and to modulate inflammation in atherosclerosis. In this context, circulating and localized TGFBi and POSTN are differentially controlled by the Stabilins as their receptors. To assess Stab1 and Stab2 functions in inflammatory and fibrotic skin disease, topical Imiquimod (IMQ) was used to induce psoriasis-like skin lesions in mice and Bleomycin (BLM) was applied subcutaneously to induce scleroderma-like effects in the skin. The topical treatment with IMQ, as expected, led to psoriasis-like changes in the skin of mice, including increased epidermal thickness and significant weight loss. Clinical severity was reduced in Stab2-deficient compared to Stab1-deficient mice. We did not observe differential effects in the skin of Stabilin-deficient mice after bleomycin injection. Interestingly, treatment with IMQ led to a significant increase of Stabilin ligand TGFBi plasma levels in Stab2-/- mice, treatment with BLM resulted in a significant decrease in TGFBi levels in Stab1-/- mice. Overall, Stab1 and Stab2 deficiency resulted in minor alterations of the disease phenotypes accompanied by alterations of circulating ligands in the blood in response to the disease models. Stabilin-mediated clearance of TGFBi was altered in these disease processes. Taken together our results suggest that Stabilin deficiency-associated plasma alterations may interfere with preclinical disease severity and treatment responses in patients.
RESUMO
Apoptotic cell death occurs under normal physiological conditions, such as development, tissue remodeling, and inflammation. Appropriate removal of apoptotic cells by phagocytes and the secretion of anti-inflammatory cytokines such as IL-10 are important mechanisms for maintaining tissue homeostasis. Apoptotic cell phagocytosis is mediated by several phosphatidylserine recognition receptors on non-professional or professional phagocytes, such as neighboring epithelial cells or macrophages. Stabilin-2 is reported as a phosphatidylserine recognition receptor for apoptotic cell phagocytosis, and its downstream signaling pathway for cytoskeletal rearrangement for phagocytosis is well known. However, the mechanisms for stabilin-2-mediated IL-10 production has not yet been reported. In this study, we aimed to investigate stabilin-2 receptor-mediated IL-10 transcription regulation signaling pathway.
Assuntos
Moléculas de Adesão Celular Neuronais , Interleucina-10 , Fagocitose , Transdução de Sinais , Animais , Humanos , Camundongos , Apoptose , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Interleucina-10/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a malignant tumour that is characterized by extensive vascular remodelling and responsiveness to treatment with the anti-angiogenic multikinase inhibitor sorafenib. The aim was to study endothelial remodelling in HCC. METHODS: The murine inducible albumin-SV40-large T-antigen model and two tissue microarrays (TMA) with 295 tumourous and 83 peri-tumourous samples of 296 patients with HCC were analysed for expression of liver sinusoidal endothelial cell (LSEC)-specific marker proteins, stabilin-1 and stabilin-2, LYVE-1 and CD32b. RESULTS: LSEC marker proteins were sequentially lost during HCC progression in the murine HCC model being absent from tumour nodules larger than 800 µm in diameter. Similarly, the TMA analysis of human HCCs revealed loss of all four marker proteins in the majority of tumourous tissue samples. Preservation of LYVE-1 expression showed a significant correlation with low grading (G1). In corresponding peri-tumourous liver tissue, loss of all marker proteins was seen in a minor proportion of cases (34%) while the majority of cases retained expression of at least one of the marker proteins. Loss of stabilin-2 expression in peri-tumourous liver tissue of patients with HCC was significantly less likely to occur (38%) than loss of the other marker proteins (63-95%) and it was associated with significantly longer tumour-specific (P = 0.0523) and overall (P = 0.0338) survival. Loss of stabilin-2 may enhance survival in HCC by preventing endothelial-tumour cell adhesive interactions and microvascular invasion. CONCLUSIONS: In summary, endothelial transdifferentiation is a major pathogenic event in HCC development indicating a switch from vessel co-option/intussusceptive angiogenesis to sprouting angiogenesis.
Assuntos
Biomarcadores/metabolismo , Carcinoma Hepatocelular/fisiopatologia , Moléculas de Adesão Celular Neuronais/metabolismo , Transdiferenciação Celular/fisiologia , Células Endoteliais/fisiologia , Neoplasias Hepáticas/fisiopatologia , Neovascularização Patológica/fisiopatologia , Animais , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Microscopia de Fluorescência , Receptores de IgG/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Non-DNA-binding Stabilin-2/HARE receptors expressed on liver sinusoidal endothelial cells specifically bind to and internalize several classes of phosphorothioate antisense oligonucleotides (PS-ASOs). After Stabilin-mediated uptake, PS-ASOs are trafficked within endosomes (>97%-99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes lowers therapeutic efficacy, thereby increasing the overall dose for patients. Here, we use confocal microscopy to characterize the intracellular route transverse by PS-ASOs after Stabilin receptor-mediated uptake in stable recombinant Stabilin-1 and -2 cell lines. We found that PS-ASOs as well as the Stabilin-2 receptor transverse the classic path: clathrin-coated vesicle-early endosome-late endosome-lysosome. Chloroquine exposure facilitated endosomal escape of PS-ASOs leading to target knockdown by more than 50% as compared to untreated cells, resulting in increased PS-ASO efficacy. We also characterize cytosolic galectins as novel contributor for PS-ASO escape. Galectins knockdown enhances ASO efficacy by more than 60% by modulating EEA1, Rab5C, and Rab7A mRNA expression, leading to a delay in the endosomal vesicle maturation process. Collectively, our results provide additional insight for increasing PS-ASO efficacy by enhancing endosomal escape, which can further be utilized for other nucleic acid-based modalities.
RESUMO
Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified. Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer. Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.