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Endocytosis of hyaluronan in rat Kupffer cells.

ABSTRACT: The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

SUBMITTER: Alston-Smith J

PROVIDER: S-EPMC1132928 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Project description:We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5'-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig kappa-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of (125)I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the approximately 300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.

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Project description:Background & aimsComplex communications between hepatocytes and Kupffer cells (KCs) are known to drive or suppress hepatocarcinogenesis, with controversial data in the literature. In previous experiments that aimed to decipher hepatocyte/KC interactions, we unexpectedly unveiled a tumor-suppressing effect of polyinosinic-polycytidylic acid, a widely used inducer of MX dynamin like GTPase 1 (Mx1)-cre expression, which questioned a theory of interleukin 1a/6 cytokine circuit in hepatocyte/KC communication. The goal of this study was to clarify the controversy and decipher unique functions of KCs and non-KC macrophages in liver tumorigenesis.MethodsWe used the C-type lectin domain family 4 member F (Clec4f)-cre system to delete Src-homology 2 domain-containing tyrosine phosphatase 2 (Shp2)/protein tyrosine phosphatase nonreceptor 11 (Ptpn11) in KCs, and a combination of Clec4f-cre and adeno-associated virus-cre to delete Shp2 in KCs and hepatocytes to investigate the effects on hepatocellular carcinoma development and immune cell compositions/activities.ResultsAblating Shp2 in KCs generated a tumor-promoting niche, which was exacerbated further by concurrent removal of Shp2 in both KCs and hepatocytes. Shp2 deficiency induced KC apoptosis and decreased its numbers, which induced compensatory recruitment of bone marrow-derived monocytes into liver. These newly recruited monocytes differentiated into non-KC macrophages with tumor-associated macrophage function, leading to aggravated tumor progression through down-regulation of CD8 T cells. Tumor-associated macrophage blockade by anti-chemokine (C-C motif) ligand 2 (CCL2) antibody inhibited hepatocellular carcinoma progression, while depletion of all macrophages had a tumor-promoting effect by increasing myeloid-derived suppressor cells (M-MDSCs) and decreasing CD8 T cells.ConclusionsShp2 loss in KCs or hepatocytes generated a protumorigenic microenvironment, which was exacerbated by its removal in both cell types. These results show the complexity of intercellular signaling events in liver tumorigenesis and raises caution on the use of specific Shp2 inhibitor in liver cancer therapy. Transcript profiling: RNA sequencing data are available at Gene Expression Omnibus (GSE222594).

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Endocytosis of hyaluronan in rat Kupffer cells.

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