Takayuki Yoshizaki

Identification and Characterization of a Novel Human Collectin CL‐K1

Collectins are a family of C‐type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host‐defense. Here we report the cloning and characterization of a novel collectin CL‐K1. RT‐PCR analyses showed CL‐K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL‐K1 cDNA expressing CHO cells revealed that CL‐K1 is expressed as a secreted protein. CL‐K1 is found in blood by immunoblotting and partial amino acid analyses. CL‐K1 showed Ca2+‐dependent sugar binding activity of fucose and weakly mannose but not N‐acetyl‐galactosamine, N‐acetyl‐glucosamine, or maltose, though mannose‐binding lectin (MBL) containing similar amino acid motif. CL‐K1 can recognize specially several bacterial saccharides due to specific sugar‐binding character. Elucidation of the role of two ancestor collectins of CL‐K1 and CL‐L1 could lead to see the biological function of collectin family.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Comparison of human blood concentrations of collectin kidney 1 and mannan-binding lectin.

Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 ± 0.13 µg/ml and that in MBL was 1.72 ± 1.51 µg/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.

Immunolocalization of a Novel Collectin CL-K1 in Murine Tissues

We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central veins in liver, which suggests that murine CL-K1 may be mainly produced in the liver and secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.

A Jak2 inhibitor, AG490, reverses lipin-1 suppression by TNF-α in 3T3-L1 adipocytes

Lipin-1 is a multifunctional metabolic regulator, involving in triacylglycerol and bioactive glycerolipids synthesis as an enzyme, transcriptional regulation as a coactivator, and adipogenesis. In obesity, adipose lipin-1 expression is decreased. Although lipin-1 is implicated in the pathogenesis of obesity, the mechanism is still not clear. Since TNF-alpha is deeply involved in the pathogenesis of obesity, insulin resistance, and diabetes, here we investigated the role of TNF-alpha on lipin-1 expression in adipocytes. Quantitative PCR studies showed that TNF-alpha suppressed both lipin-1A and -1B isoform expression in time- and dose-dependent manners in mature 3T3-L1 adpocytes. A Jak2 inhibitor, AG490, reversed the suppressive effect of TNF-alpha on both lipin-1A and -1B. In contrast, NF-kappaB, MAPKs, ceramide, and beta-catenin pathway tested were not involved in the mechanism. These results suggest that TNF-alpha could be involved in obesity-induced lipin-1 suppression in adipocytes and Jak2 may play an important role in the mechanism.

Lipopolysaccharide induces adipose differentiation-related protein expression and lipid accumulation in the liver through inhibition of fatty acid oxidation in mice

BackgroundIn the present study, we examined the effect of lipopolysaccharide (LPS) on liver histopathology with special reference to lipid metabolism in mice.MethodsMice were injected with LPS intraperitoneally, and its effect on the liver was investigated pathologically and biochemically.ResultsOil-red O staining and adipose differentiation-related protein (ADRP) immunohistochemistry demonstrated that injection of LPS transiently induced lipid accumulation and ADRP expression in hepatocytes, especially around the portal vein. Microscopic observation revealed that lipid accumulation started 12 h after LPS injection. Time-course studies showed that LPS rapidly, within 2 h, decreased hepatic expression of nuclear hormone receptors, including peroxisome proliferator-activated receptor (PPAR) α. LPS inhibited the expression of PPARα-target genes involved in fatty acid oxidation in the liver such as those coding for enoyl-CoA hydratase, acyl-CoA dehydrogenase, and carnitine palmitoyl transferase-1, whereas LPS also suppressed the expression of genes related to fatty acid synthesis such as those for fatty acid synthase, stearoyl-CoA desaturase, and acetyl-CoA carboxylase α.ConclusionsLPS induces transient lipid accumulation and expression of ADRP in the liver through inhibition of fatty acid oxidation by downregulation of the PPARα-related transcriptional mechanism.

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome

BackgroundCollectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals.ResultsIn this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca2+ binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca2+ during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder.ConclusionsWe have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca2+ binding during biosynthesis.

Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1

BACKGROUND Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. METHODS We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). RESULTS Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. CONCLUSIONS In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. GENERAL SIGNIFICANCE These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.

Troglitazone increases expression of E-cadherin and claudin 4 in human pancreatic cancer cells.

We examined the effects of troglitazone on expression of E-cadherin and claudin 4 in human pancreatic cancer cells. Troglitazone dose-dependently increased expression of E-cadherin and claudin 4 mRNA and protein in PK-1 cells. Snail, Slug and ZEB1, mRNAs were not changed by troglitazone, indicating that these three transcriptional repressors would not play a role in the induction of E-cadherin by troglitazone. GW9662, a PPARgamma antagonist, failed to block the increased expression of E-cadherin or claudin 4 mRNA, suggesting a PPARgamma-independent pathway. A MEK inhibitor, U0126, increased E-cadherin or claudin 4 mRNA and protein expression, and potently inhibited cell invasion. Because troglitazone down-regulates MEK-ERK signaling and inhibit cell invasion in PK-1 as shown in our previous study, these results suggest that troglitazone increases expression of E-cadherin and claudin 4 possibly through inhibition of MEK-ERK signaling in pancreatic cancer cells, which might be involved in the troglitazone-induced inhibition of cell invasive activity.

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes.

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.