The laminin receptor is a receptor for Micropterus salmoides rhabdovirus.

Micropterus salmoides rhabdovirus (MSRV) is an important pathogen of largemouth bass. Despite extensive research, the functional receptors of MSRV remained unknown. This study identified the host protein, laminin receptor (LamR), as a cellular receptor facilitating MSRV entry into host cells. Our results demonstrated that LamR directly interacts with MSRV G protein, playing a pivotal role in the attachment and internalization processes of MSRV. Knockdown of LamR with siRNA, blocking cells with LamR antibody, or incubating MSRV virions with soluble LamR protein significantly reduced MSRV entry. Notably, we found that LamR mediated MSRV entry via clathrin-mediated endocytosis. Additionally, our findings revealed that MSRV G and LamR were internalized into cells and co-localized in the early and late endosomes. These findings highlight the significance of LamR as a cellular receptor facilitating MSRV binding and entry into target cells through interaction with the MSRV G protein. IMPORTANCE: Despite the serious epidemic caused by Micropterus salmoides rhabdovirus (MSRV) in largemouth bass, the precise mechanism by which it invades host cells remains unclear. Here, we determined that laminin receptor (LamR) is a novel target of MSRV, that interacts with its G protein and is involved in viral attachment and internalization, transporting with MSRV together in early and late endosomes. This is the first report demonstrating that LamR is a cellular receptor in the MSRV life cycle, thus contributing new insights into host-pathogen interactions.

PEDF-34 attenuates neurological deficit and suppresses astrocyte-dependent neuroinflammation by modulating astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic-lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH. METHODS: A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro. RESULTS: Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1ß at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines. CONCLUSION: PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.

PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

Identification and functional characterization of laminin receptor in the mud crab, Scylla paramamosain, in response to MCDV-1 challenge.

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5' untranslated region (UTR) of 66 bp and a 3' UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs.

Flavonoid quercetin and its glucuronide and sulfate conjugates bind to 67-kDa laminin receptor and prevent neuronal cell death induced by serum starvation.

Quercetin, a dietary flavonoid, has been shown to protect against various neurodegenerative diseases with mechanisms largely unknown. After oral administration, quercetin is rapidly conjugated, and the aglycone is not detectable in the plasma and brain. However, its glucuronide and sulfate conjugates are present only at low nanomolar concentrations in the brain. Since quercetin and its conjugates have limited antioxidant capability at low nanomolar concentrations, it is crucial to determine whether they induce neuroprotection by binding to high-affinity receptors. Previously we found that (-)-epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, induces neuroprotection by binding to the 67-kDa laminin receptor (67LR). Therefore, in this study, we determined whether quercetin and its conjugates bind 67LR to induce neuroprotection and compared their ability with EGCG. Based on the quenching of intrinsic tryptophan fluorescence of peptide G (residues 161-180 in 67LR), we found quercetin, quercetin-3-O-glucuronide, and quercetin-3-O-sulfate bind to this peptide with a high affinity comparable to EGCG. Molecular docking using the crystal structure of 37-kDa laminin receptor precursor supported the high-affinity binding of all these ligands to the site corresponding to peptide G. A pretreatment with quercetin (1-1000 nM) did not effectively protect Neuroscreen-1 cells from death induced by serum starvation. Contrarily, a pretreatment with low concentrations (1-10 nM) of quercetin conjugates better protected these cells than quercetin and EGCG. The 67LR-blocking antibody substantially prevented neuroprotection by all these agents, suggesting the role of 67LR in this process. Collectively, these studies reveal that quercetin induces neuroprotection primarily through its conjugates via high affinity binding to 67LR.

Host cell factors involved in classical swine fever virus entry.

Classical swine fever virus (CSFV) is an ancient pathogen that continues to pose a threat to animal agriculture worldwide. The virus belongs to the genus Pestivirus and the family Flaviviridae. It causes a multisystemic disease that affects only pigs and is responsible for significant economic losses. CSFV infection is probably a multistep process that involves the proteins in the virus envelope and more than one receptor in the membrane of permissive cells. To date, the cellular receptors essential for CSFV entry and their detailed functions during this process remains unknown. All the viral envelope proteins Erns, E1 and E2 are involved in the entry process to some extent and the experimental approaches conducted until now have helped to unveil their contributions. This review aims to provide an overview of current knowledge on cellular molecules described to be involved in CSFV entry, including complement regulatory protein 46 (CD46), heparan sulphate (HS), Laminin receptor, Integrin ß3, Annexin II, MERKT and ADAM17. This knowledge would not only help to understand the molecular mechanisms involved in pestivirus infection, but also provide a rational basis for the development of nonvaccinal alternatives for CSFV control.

Expression and significance of laminin receptor in squamous cell carcinoma of the tongue.

BACKGROUND: Laminin receptor is a non-integrin cell-surface receptor that binds laminin present on the basement membrane. It has been reported to be associated with infiltration and metastasis of various malignant tumors. However, no studies regarding tongue cancer have been reported. This study aimed to clarify the role of laminin receptor in squamous cell carcinoma of the tongue. METHODS: We performed immunohistochemical staining of specimens from 66 patients with squamous cell carcinoma of the tongue and assessed laminin receptor expression and clinicopathological factors. As epithelial-mesenchymal transition has been shown to be associated with infiltration and metastasis of malignant tumors, staining for E-cadherin, vimentin, and N-cadherin were also performed. RESULTS: Of 20 patients with postoperative recurrence, 14 exhibited high laminin receptor expression (p = 0.0025). Kaplan-Meier analysis revealed a significantly shorter time to postoperative recurrence for the high laminin receptor expression group than that for the low laminin receptor expression group (p = 0.0008). Based on multivariate analyses for postoperative recurrence, high laminin receptor expression was associated with poor prognosis (high expression vs. low expression; HR =3.19, 95% CI =0.92-11.08; p = 0.0682). There was a correlation between laminin receptor and N-cadherin (p = 0.0089) but not between laminin receptor and E-cadherin (p = 0.369) or vimentin (p = 0.4221). CONCLUSION: These results suggest that high laminin receptor expression is a useful prognostic factor for postoperative recurrence and may be a target for molecular therapy to treat squamous cell carcinoma of the tongue.

The Role of Diacylglycerol Kinase in the Amelioration of Diabetic Nephropathy.

The drastic increase in the number of patients with diabetes and its complications is a global issue. Diabetic nephropathy, the leading cause of chronic kidney disease, significantly affects patients' quality of life and medical expenses. Furthermore, there are limited drugs for treating diabetic nephropathy patients. Impaired lipid signaling, especially abnormal protein kinase C (PKC) activation by de novo-synthesized diacylglycerol (DG) under high blood glucose, is one of the causes of diabetic nephropathy. DG kinase (DGK) is an enzyme that phosphorylates DG and generates phosphatidic acid, i.e., DGK can inhibit PKC activation under diabetic conditions. Indeed, it has been proven that DGK activation ameliorates diabetic nephropathy. In this review, we summarize the involvement of PKC and DGK in diabetic nephropathy as therapeutic targets, and its mechanisms, by referring to our recent study.

Knock-down of LRP/LR influences signalling pathways in late-stage colorectal carcinoma cells.

BACKGROUND: The 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in several tumourigenic-promoting processes including cellular viability maintenance and apoptotic evasion. Thus, the aim of this study was to assess the molecular mechanism of LRP/LR on apoptotic pathways in late stage (DLD-1) colorectal cancer cells upon siRNA-mediated down-regulation of LRP/LR. METHODS: siRNAs were used to down-regulate the expression of LRP/LR in DLD-1 cells which was assessed using western blotting and qPCR. To evaluate the mechanistic role of LRP/LR, proteomic analysis of pathways involved in proliferation and apoptosis were investigated. The data from the study was analysed using a one-way ANOVA, followed by a two-tailed student's t-test with a confidence interval of 95%. RESULTS: Here we show that knock-down of LRP/LR led to significant changes in the proteome of DLD-1 cells, exposing new roles of the protein. Moreover, analysis showed that LRP/LR may alter components of the MAPK, p53-apoptotic and autophagic signalling pathways to aid colorectal cancer cells in continuous growth and survival. Knock-down of LRP/LR also resulted in significant decreases in telomerase activity and telomerase-related proteins in the DLD-1 cells. CONCLUSIONS: These findings show that LRP/LR is critically implicated in apoptosis and cell viability maintenance and suggest that siRNA-mediated knock-down of LRP/LR may be a possible therapeutic strategy for the treatment of colorectal cancer.

Exploration of novel anti-angiogenic PEDF-derived peptides with improved activitives by inhibiting proliferation, suppressing migration, and inducing 67LR internalization.

Diabetic retinopathy (DR) remains high incidence and accounts for severe impact on vision in diabetics, but its mechanism is still poorly understood. Abnormal migration and proliferation of endothelial cells (ECs) drive neovascular retinopathies, which has an important role in promoting the occurrence and development of DR. In this study, we designed and synthesized a series of PEDF-derived peptides as angiogenesis inhibitors. Especially, compound G24 significantly inhibited the cell proliferation in VEGF-activated human umbilical vein endothelial cells (HUVECs) with IC50 values of 2.88 ± 0.19 µM. Further biological evaluation demonstrated that compound G24 exhibited strong inducing-effects on cell apoptosis and internalization of 67LR, and advanced inhibitory potency in cell migration and angiogenesis formed by HUVECs in vitro. In summary, the optimal compound G24 as a novel angiogenesis inhibitor showed the potentiality in the further research for the treatment for DR.

Modified Gold Nanoparticles for Efficient Delivery of Betulinic Acid to Cancer Cell Mitochondria.

Advances in nanomedicine have seen the adaptation of nanoparticles (NPs) for subcellular delivery for enhanced therapeutic impact and reduced side effects. The pivotal role of the mitochondria in apoptosis and their potential as a target in cancers enables selective induction of cancer cell death. In this study, we examined the mitochondrial targeted delivery of betulinic acid (BA) by the mitochondriotropic TPP+-functionalized epigallocatechin gallate (EGCG)-capped gold NPs (AuNPs), comparing the impact of polyethylene glycol (PEG) and poly-L-lysine-graft-polyethylene glycol (PLL-g-PEG) copolymer on delivery efficacy. This included the assessment of their cellular uptake, mitochondrial localization and efficacy as therapeutic delivery platforms for BA in the human Caco-2, HeLa and MCF-7 cancer cell lines. These mitochondrial-targeted nanocomplexes demonstrated significant inhibition of cancer cell growth, with targeted nanocomplexes recording IC50 values in the range of 3.12-13.2 µM compared to that of the free BA (9.74-36.31 µM) in vitro, demonstrating the merit of mitochondrial targeting. Their mechanisms of action implicated high amplitude mitochondrial depolarization, caspases 3/7 activation, with an associated arrest at the G0/G1 phase of the cell cycle. This nano-delivery system is a potentially viable platform for mitochondrial-targeted delivery of BA and highlights mitochondrial targeting as an option in cancer therapy.

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aß) production and the amyloidogenic pathway, generating Aß, whose accumulation characterizes Alzheimer's disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aß and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aß, may mediate Aß-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3ß.

Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization.

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 µg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 µM, PEDF 336 (100 µM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

An adiponectin paralog protein, CTRP6 decreased the proliferation and invasion activity of oral squamous cell carcinoma cells: possible interaction with laminin receptor pathway.

C1q/tumor necrosis factor-related protein-6 (CTRP6), also known as CTRP6 is identified adiponectin paralog. Although recent studies have revealed that adiponectin has an inhibitory role in carcinogenesis, the role of CTRP6 in carcinogenesis remains unclear. In this study, we found that eukaryotic recombinant CTRP6 protein bound to the cell surface membrane of cultured oral squamous cell carcinoma cells by immunofluorescence staining. Screening of CTRP6 binding protein in expression library followed by co-immunoprecipitation assay revealed that CTRP6 bound to the precursor of laminin receptor. CTRP6 disturbed the binding of laminin to the laminin receptor. Interestingly, the eukaryotic recombinant CTRP6 protein significantly suppressed the proliferation and Matrigel invasion activity of oral squamous cell carcinoma SAS cells in a dose-dependent manner. Moreover, administration of CTRP6 significantly attenuated the growth of SAS cells in xenoplant mice model. Laminin and laminin receptor are known to be overexpressed and promote the tumor growth in OSCC. Combined together, the present findings suggest that CTRP6 could repress progression of oral squamous cell carcinoma cells, putatively through disrupting the laminin-laminin receptor axis.

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. IMPLICATIONS: siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis.

Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.

Epigallocatechin-3-Gallate Regulates Anti-Inflammatory Action Through 67-kDa Laminin Receptor-Mediated Tollip Signaling Induction in Lipopolysaccharide-Stimulated Human Intestinal Epithelial Cells.

BACKGROUND/AIMS: Inflammatory bowel disease (IBD) is a condition that involves chronic inflammation in all or part of the digestive tract. Often painful and debilitating, IBD can lead to life-threatening complications and increase the risk for colon cancer. In this study, we investigated the epigallocatechin-3-gallate (EGCG) mediated anti-inflammation response in lipopolysaccharide (LPS)-stimulated human colorectal cells through the negative regulator of Toll-like receptor (TLR) signaling. METHODS: human intestinal epithelial cells (HT-29) were used in all experiments. Cell cytotoxicity and nitric oxide (NO) were evaluated by WST-1 and the Griess reagent. Western blot analysis and ELISA were used to determine inflammatory mediators and 67-kDa laminin receptor (67LR)-mediated Tollip signaling pathways. RESULTS: Treatment of EGCG and LPS did not affect the cytotoxicity in HT-29 cells. LPS treatment dose-dependently increased the pro-inflammatory cytokine, such as interleukin (IL)-8, whereas EGCG significantly reduced the LPS-stimulated IL-8 production. Additionally, EGCG treatment markedly increased the Toll-interacting protein (Tollip) expression, which negatively regulates the TLR signaling in a dose and time-dependent manner. In particular, in the result from an RNA interference-mediated assay, our finding showed that silencing of Tollip resulted in abrogation of the inhibitory action of EGCG on LPS-induced production of pro-inflammatory mediators (inducible nitric oxide synthase-mediated NO/COX2, and IL-8) and activation of MAPKs and NF-κB signaling pathways. Interestingly, we also found that Tollip expression induced by EGCG could be modulated through 67LR expressed on the surface of HT-29 cells. CONCLUSIONS: Our novel finding indicates that 67LR and Tollip signaling activated by EGCG treatment is essential for inhibition of inflammation in human intestinal epithelial cells.

Laminin-1 induces endocytosis of 67KDa laminin receptor and protects Neuroscreen-1 cells against death induced by serum withdrawal.

Although the function of laminin in the basement membrane is known, the function of soluble "neuronal" laminin is unknown. Since laminin is neuroprotective, we determined whether the soluble laminin-1 induces signaling for neuroprotection via its 67KDa laminin-1 receptor (67LR). Treatment of Neuroscreen-1 (NS-1) cells with laminin-1 or YIGSR peptide, which corresponds to a sequence in laminin-1 ß1 chain that binds to 67LR, induced a decrease in the cell-surface expression of 67LR and caused its internalization. Furthermore, intracellular cAMP-elevating agents, dibutyryl-cAMP, forskolin, and rolipram, also induced this internalization. Both soluble laminin-1 and YIGSR induced a sustained elevation of intracellular cAMP under defined conditions, suggesting a causal role of cAMP in the endocytosis of 67LR. This endocytosis was not observed in cells deficient in protein kinase A (PKA) nor in cells treated with either SQ 22536, an inhibitor for adenylyl cyclase, or ESI-09, an inhibitor for the exchange protein directly activated by cAMP (Epac). In addition, when internalization occurred in NS-1 cells, 67LR and adenylyl cyclase were localized in early endosomes. Under conditions in which endocytosis had occurred, both laminin-1 and YIGSR protected NS-1 cells from cell death induced by serum withdrawal. However, under conditions in which endocytosis did not occur, neither laminin-1 nor YIGSR protected these cells. Conceivably, the binding of laminin-1 to 67LR causes initial signaling through PKA and Epac, which causes the internalization of 67LR, along with signaling enzymes, such as adenylyl cyclase, into early endosomes. This causes sustained signaling for protection against cell death induced by serum withdrawal.

Knock-down of LRP/LR promotes apoptosis in early and late stage colorectal carcinoma cells via caspase activation.

BACKGROUND: Cancer remains one of the leading causes of death around the world, where incidence and mortality rates are at a constant increase. Tumourigenic cells are characteristically seen to over-express the 37 kDa/67 kDa laminin receptor (LRP/LR) compared to their normal cell counterparts. This receptor has numerous roles in tumourigenesis including metastasis, angiogenic enhancement, telomerase activation, cell viability and apoptotic evasion. This study aimed to expose the role of LRP/LR on the cellular viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells. METHODS: siRNA were used to down-regulate the expression of LRP/LR in SW-480 and DLD-1 cells which was assessed using western blotting. Subsequently, cell survival was evaluated using the MTT cell survival assay and confocal microscopy. Thereafter, Annexin V-FITC/PI staining and caspase activity assays were used to investigate the mechanism underlying the cell death observed upon LRP/LR knockdown. The data was analysed using Student's t-test with a confidence interval of 95%, with p-values of less than 0.05 seen as significant. RESULTS: Here we reveal that siRNA-mediated knock-down of LRP led to notable decreases in cell viability through increased levels of apoptosis, apparent by compromised membrane integrity and significantly high caspase-3 activity. Down-regulated LRP resulted in a significant increase in caspase-8 and -9 activity in both cell lines. CONCLUSIONS: These findings show that the receptor is critically implicated in apoptosis and that LRP/LR down-regulation induces apoptosis in early and late stage colorectal cancer cells through both apoptotic pathways. Thus, this study suggests that siRNA-mediated knock-down of LRP could be a possible therapeutic strategy for the treatment of early and late stage colorectal carcinoma.

Innate differences and colostrum-induced alterations of jejunal mucosal proteins in piglets with intra-uterine growth restriction.

Mammalian neonates undergo rapid transitions from a sterile uterine environment with a continuous intravenous supply of nutrients to a microbe-rich environment with intermittent ingesting of colostrum/milk via the gut. Currently, little is known about the colostrum-induced alterations of intestinal mucosal proteins in piglets with intra-uterine growth restriction (IUGR). In this study, we sought to investigate the innate differences and effects of colostrum on alterations in small-intestinal proteomes of IUGR piglets. Two IUGR (approximately 0·9 kg) and two normal-birth weight (NBW; approximately 1·3 kg) piglets were obtained from each of six sows at birth. One half (n 12; 6 IUGR v. 6 NBW) of the selected newborn piglets were killed to obtain jejunum samples, and the other half (n 12; 6 IUGR v. 6 NBW) of the newborn piglets were allowed to suckle colostrum from their own mothers for 24 h before jejunum sample collection. On the basis of proteomic analysis, we identified thirty-one differentially expressed proteins in the jejunal mucosa between IUGR and normal neonates before or after colostrum consumption. The intestinal proteins altered by colostrum feeding play important roles in the following: (1) increasing intestinal integrity, transport of nutrients, energy metabolism, protein synthesis, immune response and, therefore, cell proliferation; and (2) decreasing oxidative stress, and therefore cell apoptosis, in IUGR neonates. However, colostrum only partially ameliorated the inferior status of the jejunal mucosa in IUGR neonates. These findings provide the first evidence in intestinal protein alterations of IUGR neonates in response to colostrum ingestion, and thus render new insights into the mechanisms responsible for impaired growth in IUGR neonates and into new nutritional intervention strategies.