Lipolysis-Stimulated Lipoprotein Receptor in Proximal Tubule, BMP-SMAD Signaling, and Kidney Disease.

BACKGROUND: Lipolysis-stimulated lipoprotein receptor (LSR) is a single-pass membrane protein which plays essential roles in tricellular tight junction organization in epithelium and endothelium, but its function in kidney physiology and disease development remains unknown. METHODS: Conditional Lsr deletion mice were generated and analyzed to investigate function of LSR in proximal tubule. Unilateral ischemia-reperfusion was used as injury model to investigate the role of LSR in acute kidney injury (AKI) and the progression to chronic kidney disease (CKD). Detailed mechanistic analyses were conducted using whole-transcriptome RNA sequencing, immunofluorescence, dual-luciferase reporter gene assay, coimmunoprecipitation, RNA immunoprecipitation, and adeno-associated virus-mediated gene overexpression and knockdown. RESULTS: The nuclear localization of LSR was found in the kidney. Proximal tubule-specific Lsr knockout mice exhibited alleviated kidney damage and fibrosis than those in wildtype mice in response unilateral ischemia-reperfusion injury. Loss of LSR resulted in downregulation of Chrdl1 and activation of BMP-SMAD signaling in proximal tubules. Treatment with CHRDL1 counteracted the protective effect of LSR deletion in the unilaterally ischemic injured kidney. Additionally, systemic delivery of Chrdl1 shRNA attenuated injury-induced kidney fibrosis. LSR formed a complex with 14-3-3θ in the nucleus of proximal tubular cells, thereby reducing the interaction between human antigen R and 14-3-3θ, consequently leading to the translocation of unbound human antigen R to the cytoplasm. The absence of LSR promoted the association of 14-3-3θ with human antigen R, potentially resulting in decreased human antigen R levels in the cytoplasm. Reduced human antigen R levels impaired Chrdl1 mRNA stability, subsequently leading to the activation of BMP-SMAD signaling. CONCLUSIONS: Deletion of LSR in proximal tubule deregulated Chrdl1 to activate BMP-SMAD signaling and ameliorated kidney disease.

LSR targets YAP to modulate intestinal Paneth cell differentiation.

Lipolysis-stimulated lipoprotein receptor (LSR) is a multi-functional protein that is best known for its roles in assembly of epithelial tricellular tight junctions and hepatic clearance of lipoproteins. Here, we investigated whether LSR contributes to intestinal epithelium homeostasis and pathogenesis of intestinal disease. By using multiple conditional deletion mouse models and ex vivo cultured organoids, we find that LSR elimination in intestinal stem cells results in the disappearance of Paneth cells without affecting the differentiation of other cell lineages. Mechanistic studies reveal that LSR deficiency increases abundance of YAP by modulating its phosphorylation and proteasomal degradation. Using gain- and loss-of-function studies, we show that LSR protects against necrotizing enterocolitis through enhancement of Paneth cell differentiation in small-intestinal epithelium. Thus, this study identifies LSR as an upstream negative regulator of YAP activity, an essential factor for Paneth cell differentiation, and a potential therapeutic target for necrotizing enterocolitis.

MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin.

Age-related hearing loss (ARHL) is the most common sensory degenerative disease and can significantly impact the quality of life in elderly people. A previous study using GeneChip miRNA microarray assays showed that the expression of miR-29a changes with age, however, its role in hearing loss is still unclear. In this study, we characterized the cochlear phenotype of miR-29a knockout (miR-29a-/-) mice and found that miR-29a-deficient mice had a rapid progressive elevation of the hearing threshold from 2 to 5 months of age compared with littermate controls as measured by the auditory brainstem response. Stereocilia degeneration, hair cell loss and abnormal stria vascularis (SV) were observed in miR-29a-/- mice at 4 months of age. Transcriptome sequencing results showed elevated extracellular matrix (ECM) gene expression in miR-29a-/- mice. Both Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the key differences were closely related to ECM. Further examination with a transmission electron microscope showed thickening of the basilar membrane in the cochlea of miR-29a-/- mice. Five Col4a genes (Col4a1-a5) and two laminin genes (Lamb2 and Lamc1) were validated as miR-29a direct targets by dual luciferase assays and miR-29a inhibition assays with a miR-29a inhibitor. Consistent with the target gene validation results, the expression of these genes was significantly increased in the cochlea of miR-29a-/- mice, as shown by RT-PCR and Western blot. These findings suggest that miR-29a plays an important role in maintaining cochlear structure and function by regulating the expression of collagen and laminin and that the disturbance of its expression could be a cause of progressive hearing loss.

Loss of CLDN5 in podocytes deregulates WIF1 to activate WNT signaling and contributes to kidney disease.

Although mature podocytes lack tight junctions, tight junction integral membrane protein claudin-5 (CLDN5) is predominantly expressed on plasma membranes of podocytes under normal conditions. Using podocyte-specific Cldn5 knockout mice, we identify CLDN5 as a crucial regulator of podocyte function and reveal that Cldn5 deletion exacerbates podocyte injury and proteinuria in a diabetic nephropathy mouse model. Mechanistically, CLDN5 deletion reduces ZO1 expression and induces nuclear translocation of ZONAB, followed by transcriptional downregulation of WNT inhibitory factor-1 (WIF1) expression, which leads to activation of WNT signaling pathway. Podocyte-derived WIF1 also plays paracrine roles in tubular epithelial cells, as evidenced by the finding that animals with podocyte-specific deletion of Cldn5 or Wif1 have worse kidney fibrosis after unilateral ureteral obstruction than littermate controls. Systemic delivery of WIF1 suppresses the progression of diabetic nephropathy and ureteral obstruction-induced renal fibrosis. These findings establish a function for podocyte CLDN5 in restricting WNT signaling in kidney.

Experimental Study on the Lateral Seepage Characteristics in the Tension Saturated Zone.

To study the lateral seepage field in the tension saturated zone (TSZ), an experiment with no evaporation and precipitation infiltration was carried out in a self-made seepage tank filled up with fine sand. Based on the data and plots obtained, the lateral seepage field distribution features in the TSZ can be divided into three area for discussion: ascending area, descending area, and the nearly horizontal flow area. In the ascending and descending area, the total water potential gradient diminished from the recharge area to the discharge area and the seepage velocity was faster. In the nearly horizontal flow area, the total water potential gradient was lower and the seepage velocity was slower. The pressure potential gradually decreased horizontally from the recharge area to the discharge area, while in the vertical profile, it gradually decreased from the bottom to the top in the whole seepage area. In the absence of evaporation, the vertical water exchange among the saturated zone, TSZ, and unsaturated zone in nearly horizontal flow area is weak. Contrarily, in the ascending area and descending area, vertical water flows through both the phreatic surface and the upper interface of the TSZ. When there is lateral seepage in the TSZ, the thickness of the TSZ generally increases from the ascending area to the nearly horizontal area and then to the descending area. It should be pointed out that in the nearly horizontal area, the TSZ thickness is approximately equal to the height of the water column. Overall, the lateral seepage in the TSZ can be regarded as a stable siphon process, hence the siphon tube model can be further used to depict this lateral seepage.

In-situ SEM observations of ultrasonic cavitation erosion behavior of HVOF-sprayed coatings.

Several typical high-velocity oxy-fuel (HVOF)-sprayed coatings, including WC-10Co4Cr coatings, Co-based coatings, WC-10Co4Cr/Co-based composite coatings, and Fe-based amorphous/nanocrystalline coatings were fabricated, and their cavitation behavior was evaluated in deionized water. Further, in-situ SEM surface observations were used to understand the microstructure of tested coatings. The results show that cavitation erosion initially occurred at pre-existing defects in the coatings. Meanwhile, it was found that cavitation erosion damage of the WC-10Co4Cr/Co-based composite coating, which contained a hard reinforcing phase (WC-10Co4Cr phase) and a soft matrix phase (Co-based phase), preferentially occurred at or around pores and microcracks in the reinforcement, rather than in the defect free matrix. This suggested that defects were a critical contributing factor to cavitation damage of the composite coatings. Furthermore, a mechanism was suggested to explicate the cavitation behavior of composite coatings. The approach of using in-situ SEM surface observations proved to be useful for the analysis of the cavitation mechanism of engineering materials and protective coatings.

Quantitative Visualization of Nanoscale Ion Transport.

Understanding ion transport properties at various interfaces, especially at small length scales, is critical in advancing our knowledge of membrane materials and cell biology. Recently, we described potentiometric-scanning ion conductance microscopy (P-SICM) for ion-conductance measurement in polymer membranes and epithelial cell monolayers at discrete points in a sample. Here, we combine hopping mode techniques with P-SICM to allow simultaneous nanometer-scale conductance and topography mapping. First validated with standard synthetic membranes and then demonstrated in living epithelial cell monolayers under physiological conditions, this new method allows direct visualization of heterogeneous ion transport of biological samples for the first time. These advances provide a noncontact local probe, require no labeling, and present a new tool for quantifying intrinsic transport properties of a variety of biological samples.

ILDR1 is important for paracellular water transport and urine concentration mechanism.

Whether the tight junction is permeable to water remains highly controversial. Here, we provide evidence that the tricellular tight junction is important for paracellular water permeation and that Ig-like domain containing receptor 1 (ILDR1) regulates its permeability. In the mouse kidney, ILDR1 is localized to tricellular tight junctions of the distal tubules. Genetic knockout of Ildr1 in the mouse kidney causes polyuria and polydipsia due to renal concentrating defects. Microperfusion of live renal distal tubules reveals that they are impermeable to water in normal animals but become highly permeable to water in Ildr1 knockout animals whereas paracellular ionic permeabilities in the Ildr1 knockout mouse renal tubules are not affected. Vasopressin cannot correct paracellular water loss in Ildr1 knockout animals despite normal effects on the transcellular aquaporin-2-dependent pathway. In cultured renal epithelial cells normally lacking the expression of Ildr1, overexpression of Ildr1 significantly reduces the paracellular water permeability. Together, our study provides a mechanism of how cells transport water and shows how such a mechanism may be exploited as a therapeutic approach to maintain water homeostasis.

Virus entry and replication in the brain precedes blood-brain barrier disruption during intranasal alphavirus infection.

Viral infections of the central nervous system (CNS) are often associated with blood-brain barrier (BBB) disruption, yet the impact of virus replication and immune cell recruitment on BBB integrity are incompletely understood. Using two-photon microscopy, we demonstrate that Venezuelan equine encephalitis virus (VEEV) strain TC83-GFP, a GFP expressing, attenuated strain with a G3A mutation within the 5' UTR that is associated with increased sensitivity to type I interferons (IFNs), does not directly impact BBB permeability. Following intranasal infection of both wild-type and IFN-induced protein with tetratricopeptide repeats 1 (IFIT1)-deficient mice, which fail to block TC83-specific RNA translation, virus spreads to the olfactory bulb and cortex via migration along axonal tracts of neurons originating from the olfactory neuroepithelium. Global dissemination of virus in the CNS by 2days post-infection (dpi) was associated with increased BBB permeability in the olfactory bulb, but not in the cortex or hindbrain, where permeability only increased after the recruitment of CX3CR1+ and CCR2+ mononuclear cells on 6 dpi, which corresponded with tight junction loss and claudin 5 redistribution. Importantly, despite higher levels of viral replication, similar results were obtained in IFIT1-deficient mice. These findings indicate that TC83 gains CNS access via anterograde axonal migration without directly altering BBB function and that mononuclear and endothelial cell interactions may underlie BBB disruption during alphavirus encephalitis.

Claudins in barrier and transport function-the kidney.

Claudins are discovered to be key players in renal epithelial physiology. They are involved in developmental, physiological, and pathophysiological differentiation. In the glomerular podocytes, claudin-1 is an important determinant of cell junction fate. In the proximal tubule, claudin-2 plays important roles in paracellular salt reabsorption. In the thick ascending limb, claudin-14, -16, and -19 regulate the paracellular reabsorption of calcium and magnesium. Recessive mutations in claudin-16 or -19 cause an inherited calcium and magnesium losing disease. Synonymous variants in claudin-14 have been associated with hypercalciuric nephrolithiasis by genome-wide association studies (GWASs). More importantly, claudin-14 gene expression can be regulated by extracellular calcium levels via the calcium sensing receptor. In the distal tubules, claudin-4 and -8 form paracellular chloride pathway to facilitate electrogenic sodium reabsorption. Aldosterone, WNK4, Cap1, and KLHL3 are powerful regulators of claudin and the paracellular chloride permeability. The lessons learned on claudins from the kidney will have a broader impact on tight junction biology in other epithelia and endothelia.

Inducible Expression of Claudin-1 in Glomerular Podocytes Generates Aberrant Tight Junctions and Proteinuria through Slit Diaphragm Destabilization.

The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.

Capturing Rare Conductance in Epithelia with Potentiometric-Scanning Ion Conductance Microscopy.

Tight junctions (TJs) are barrier forming structures of epithelia and can be described as tightly sealed intercellular spaces. Transport properties have been extensively studied for bicellular TJs (bTJs). Knowledge of the barrier functions of tricellular junctions (tTJs) are less well understood, due largely to a lack of proper techniques to locally measure discrete tTJ properties within a much larger area of epithelium. In this study, we use a nanoscale pipet to precisely locate tTJs within epithelia and measure the apparent local conductance of tTJs with a technique termed potentiometric scanning ion conductance microscopy (P-SICM). P-SICM shows the ability to differentiate transport through tTJs and bTJs, which was not possible with previous techniques and assays. We describe P-SICM investigations of both wild type and tricellulin overexpression Madin-Darby Canine Kidney (strain II, MDCKII) cells.

Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization.

The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision.

KLHL3 regulates paracellular chloride transport in the kidney by ubiquitination of claudin-8.

A rare Mendelian syndrome--pseudohypoaldosteronism type II (PHA-II)--features hypertension, hyperkalemia, and metabolic acidosis. Genetic linkage studies and exome sequencing have identified four genes--with no lysine kinase 1 (wnk1), wnk4, Kelch-like 3 (KLHL3), and Cullin 3 (Cul3)--mutations of which all caused PHA-II phenotypes. The previous hypothesis was that the KLHL3-Cul3 ubiquitin complex acted on the wnk4-wnk1 kinase complex to regulate Na(+)/Cl(-) cotransporter (NCC) mediated salt reabsorption in the distal tubules of the kidney. Here, we report the identification of claudin-8 as a previously unidentified physiologic target for KLHL3 and provide an alternative explanation for the collecting duct's role in PHA-II. Using a tissue-specific KO approach, we have found that deletion of claudin-8 in the collecting duct of mouse kidney caused hypotension, hypokalemia, and metabolic alkalosis, an exact mirror image of PHA-II. Mechanistically, the phenotypes in claudin-8 KO animals were caused by disruption of the claudin-8 interaction with claudin-4, the paracellular chloride channel, and delocalization of claudin-4 from the tight junction. In mouse collecting duct cells, knockdown of KLHL3 profoundly increased the paracellular chloride permeability. Mechanistically, KLHL3 was directly bound to claudin-8, and this binding led to the ubiquitination and degradation of claudin-8. The dominant PHA-II mutation in KLHL3 impaired claudin-8 binding, ubiquitination, and degradation. These findings have attested to the concept that the paracellular pathway is physiologically regulated through the ubiquitination pathway, and its deregulation may lead to diseases of electrolyte and blood pressure imbalances.

Epigenetic regulation of microRNAs controlling CLDN14 expression as a mechanism for renal calcium handling.

The kidney has a major role in extracellular calcium homeostasis. Multiple genetic linkage and association studies identified three tight junction genes from the kidney--claudin-14, -16, and -19--as critical for calcium imbalance diseases. Despite the compelling biologic evidence that the claudin-14/16/19 proteins form a regulated paracellular pathway for calcium reabsorption, approaches to regulate this transport pathway are largely unavailable, hindering the development of therapies to correct calcium transport abnormalities. Here, we report that treatment with histone deacetylase (HDAC) inhibitors downregulates renal CLDN14 mRNA and dramatically reduces urinary calcium excretion in mice. Furthermore, treatment of mice with HDAC inhibitors stimulated the transcription of renal microRNA-9 (miR-9) and miR-374 genes, which have been shown to repress the expression of claudin-14, the negative regulator of the paracellular pathway. With renal clearance and tubule perfusion techniques, we showed that HDAC inhibitors transiently increase the paracellular cation conductance in the thick ascending limb. Genetic ablation of claudin-14 or the use of a loop diuretic in mice abrogated HDAC inhibitor-induced hypocalciuria. The genetic mutations in the calcium-sensing receptor from patients with autosomal dominant hypocalcemia (ADH) repressed the transcription of miR-9 and miR-374 genes, and treatment with an HDAC inhibitor rescued the phenotypes of cell and animal models of ADH. Furthermore, systemic treatment of mice with antagomiRs against these miRs relieved claudin-14 gene silencing and caused an ADH-like phenotype. Together, our findings provide proof of concept for a novel therapeutic principle on the basis of epigenetic regulation of renal miRs to treat hypercalciuric diseases.

The Cap1-claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation.

The paracellular pathway through the tight junction provides an important route for transepithelial chloride reabsorption in the kidney, which regulates extracellular salt content and blood pressure. Defects in paracellular chloride reabsorption may in theory cause deregulation of blood pressure. However, there is no evidence to prove this theory or to demonstrate the in vivo role of the paracellular pathway in renal chloride handling. Here, using a tissue-specific KO approach, we have revealed a chloride transport pathway in the kidney that requires the tight junction molecule claudin-4. The collecting duct-specific claudin-4 KO animals developed hypotension, hypochloremia, and metabolic alkalosis due to profound renal wasting of chloride. The claudin-4-mediated chloride conductance can be regulated endogenously by a protease-channel-activating protease 1 (cap1). Mechanistically, cap1 regulates claudin-4 intercellular interaction and membrane stability. A putative cap1 cleavage site has been identified in the second extracellular loop of claudin-4, mutation of which abolished its regulation by cap1. The cap1 effects on paracellular chloride permeation can be extended to other proteases such as trypsin, suggesting a general mechanism may also exist for proteases to regulate the tight junction permeabilities. Together, we have discovered a theory that paracellular chloride permeability is physiologically regulated and essential to renal salt homeostasis and blood pressure control.

Claudin-14 underlies Ca⁺⁺-sensing receptor-mediated Ca⁺⁺ metabolism via NFAT-microRNA-based mechanisms.

Pathologic dysregulation of extracellular calcium metabolism is difficult to correct. The extracellular Ca(++)-sensing receptor (CaSR), a G protein-coupled receptor that regulates renal Ca(++) handling through changes in paracellular channel permeability in the thick ascending limb, has emerged as an effective pharmacological candidate for managing calcium metabolism. However, manipulation of CaSR at the systemic level causes promiscuous effects in the parathyroid glands, kidneys, and other tissues, and the mechanisms by which CaSR regulates paracellular transport in the kidney remain unknown. Here, we describe a CaSR-NFATc1-microRNA-claudin-14 signaling pathway in the kidney that underlies paracellular Ca(++) reabsorption through the tight junction. With CaSR-specific pharmacological reagents, we show that the in vivo gene expression of claudin-14 is regulated through a transcriptional mechanism mediated by NFATc1-microRNA and associated chromatin remodeling. Transgenic knockout and overexpression approaches showed that claudin-14 is required for CaSR-regulated renal Ca(++) metabolism. Together, our results define an important signaling cascade that, when dysregulated, may mediate Ca(++) imbalance through changes in tight junction permeability.

Claudin-14 regulates renal Ca⁺⁺ transport in response to CaSR signalling via a novel microRNA pathway.

The paracellular claudin channel of the thick ascending limb (TAL) of Henle is critical for Ca(++) reabsorption in the kidney. Genome-wide association studies (GWASs) have identified claudin-14 associated with hypercalciuric nephrolithiasis. Here, we show that claudin-14 promoter activity and transcript are exclusively localized in the TAL. Under normal dietary condition, claudin-14 proteins are suppressed by two microRNA molecules (miR-9 and miR-374). Both microRNAs directly target the 3'-UTR of claudin-14 mRNA; induce its mRNA decay and translational repression in a synergistic manner. Through physical interaction, claudin-14 blocks the paracellular cation channel made of claudin-16 and -19, critical for Ca(++) reabsorption in the TAL. The transcript and protein levels of claudin-14 are upregulated by high Ca(++) diet, while downregulated by low Ca(++) diet. Claudin-14 knockout animals develop hypermagnesaemia, hypomagnesiuria, and hypocalciuria under high Ca(++) dietary condition. MiR-9 and miR-374 transcript levels are regulated by extracellular Ca(++) in a reciprocal manner as claudin-14. The Ca(++) sensing receptor (CaSR) acts upstream of the microRNA-claudin-14 axis. Together, these data have established a key regulatory role for claudin-14 in renal Ca(++) homeostasis.

The DC-SIGN of zebrafish: insights into the existence of a CD209 homologue in a lower vertebrate and its involvement in adaptive immunity.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN/CD209) has become hot topic in recent studies because of its important roles in immune responses and immune escape. CD209 has been well characterized in humans and several other mammals, but little documentation exists about it in lower vertebrates. This is the first report on the identification and functional characterization of a fish DC-SIGN/CD209 molecule. The zebrafish DC-SIGN/CD209 cDNA translates into 343 aa organized into three domains structurally conserved among vertebrates. An EPN motif essential for interacting with Ca(2+) and for recognizing mannose-containing motifs has been identified. Several conserved motifs crucial for internalization and signal transduction are also present within the cytoplasmic tail. Phylogenetic analysis supports the hypothesis that CD209 family members diverged from a common ancestor. The expression of DC-SIGN/CD209 in immune-related tissues can be significantly up-regulated by exogenous Ags and IL-4. This molecule associates with various APCs, including macrophages, B lymphocytes, and a possible dendritic cell-like (CD83(+)/CD80(+)CD209(+)) population. Functionally, T cell activation, Ab (IgM) production, and bacterial vaccination-elicited immunoprotection can be dramatically inhibited by a CD209 blockade after stimulation with keyhole limpet hemocyanin (KLH) in vivo or challenged with Aeromonas hydrophila, suggesting that DC-SIGN/CD209 in zebrafish is crucial for the initiation and development of adaptive immunity. Phagocytosis analysis showed that DC-SIGN/CD209 does not participate in the uptake of KLH Ag, suggesting that other mechanisms might exist that underlie DC-SIGN/CD209 involvement. We hope that the present study will contribute to a better cross-species understanding of the evolutionary history of the DC-SIGN/CD209 family.

CD154-CD40 interactions are essential for thymus-dependent antibody production in zebrafish: insights into the origin of costimulatory pathway in helper T cell-regulated adaptive immunity in early vertebrates.

The CD154-CD40-mediated costimulatory pathway is critical for T-B cell cooperation in thymus-dependent (TD) immune response in mammals. However, little is known about its existence and occurrence in lower vertebrates. Here, we report on the identification and functional characterization of CD154 and CD40 homologs from the zebrafish (Danio rerio) model. Zebrafish CD154 is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, whose tertiary structure is a sandwich containing two stacked sheets with "jelly roll" topology, just as the human TNF members do. The zebrafish CD40 is a type I membrane-bound protein with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The consensus TNFR-associated factor (TRAF)2- and TRAF6-binding motifs in mammalian CD40 are found in the cytoplasmic tail of zebrafish CD40, which indicates similar signal transduction mechanisms to higher vertebrates. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by thymus-dependent Ag. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and it was enhanced by soluble CD154 or CD154-encoding plasmid in vivo. Thymus-dependent Ag-induced CD154 expression was inhibited by cyclosporin A, suggesting that CD154 functionally associates with T cells. Double immunofluorescence staining showed that CD40 and membrane IgM colocalized in B cells. CD154-CD40 binding assays showed that CD154 specifically binds to CD40 at homodimeric form. Our results provide the first evidence for the existence of the functional CD154-CD40-mediated costimulatory pathway and helper T cell regulatory mechanism underlying adaptive immunity in a fish species.