Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10(-9) to 10(-6) M LPA and by 30-300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [(3)H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.     

Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19. Evidence for a role as a potent basement membrane degrading enzyme

We have recently cloned MMP-19, a novel matrix metalloproteinase, which, due to unique structural features, was proposed to represent the first member of a new MMP subfamily (Pendás, A. M., Kn?uper, V. , Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and López-Otin, C. (1997) J. Biol. Chem. 272, 4281-4286). A recombinant COOH-terminal deletion mutant of MMP-19 (proDelta(260-508)MMP-19), comprising the propeptide and the catalytic domain, was expressed in Escherichia coli, refolded, and purified. Interestingly, we found that proDelta(260-508)MMP-19 has the tendency to autoactivate, whereby the Lys(97)-Tyr(98) peptide bond is hydrolyzed, resulting in free catalytic domain. Mutation of two residues (Glu(88) --> Pro and Pro(90) --> Val) within the propeptide latency motif did not prevent autoactivation but the autolysis rate was somewhat reduced. Analysis of the substrate specificity revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH(2) and, with higher efficiency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2). Kinetic analysis of the interactions of the catalytic domain of MMP-19 with the natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-based compounds efficiently inhibited the enzyme. The catalytic domain of MMP-19 was able to hydrolyze the basement membrane components type IV collagen, laminin, and nidogen, as well as the large tenascin-C isoform, fibronectin, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteinase capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrade triple-helical collagen. Finally, and in contrast to studies with other MMPs, MMP-19 catalytic domain was not able to activate any of the latent MMPs tested in vitro.     

基质金属蛋白酶在乳腺癌上皮间质转化中的作用

基质金属蛋白酶(matrix metalloproteinase,MMP)能够分解并修饰细胞外基质及细胞连接,促进上皮细胞从周围组织中分离出来。在乳腺癌中MMP表达量升高,刺激肿瘤发生,引起癌症细胞的入侵和转移。上皮细胞-间质细胞转化(epithelial-mesenchymal transition,EMT)的激活与肿瘤的发生也有关。最近的研究表明MMP在乳腺的发育和致病的EMT过程中充当促进因子和介质的角色。本文主要概括最新的关于MMP是如何调节乳腺癌细胞的运动、入侵和EMT所驱动的乳腺癌发育的研究,为更好地理解MMP在乳腺癌发病过程中的作用提供依据。     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.     

Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1–32], thus revealing a self-amplifying loop for ET-1[1–32] generation. ET-1[1–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work. Published: October 28, 2002     

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). How ever, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions. © 1993 John Wiley & Sons, Inc.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

An acellular human amnionic membrane model for in vitro culture of type ii pneumocytes: The role of the basement membrane in cell morphology and function

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with ³H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8–10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48–60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7–8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of ³H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.     

Observation of production of immunoactive prolactin by normal human connective tissue in cell culture

Summary Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak of first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P≤0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO ⊂) 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg·cell⁻¹·3 d⁻¹ feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. these in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia). A portion of this paper was presented at the 33rd Annual Meeting of the Society for Gynecologic investigation, Toronto, Ontario, Canada, 19–22 March 1986. This work was supported in part by grant HD21883 (to D. H. R.) from the National Institutes of Health, Bethesda, MD.     

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

Evidence for a cooperative role of gelatinase A and membrane type-1 matrix metalloproteinase during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a large family of extracellular or membrane-bound proteases. Their ability to cleave extracellular matrix (ECM) proteins has implicated a role in ECM remodeling to affect cell fate and behavior during development and in pathogenesis. We have shown previously that membrane-type 1 (MT1)-MMP [corrected] is coexpressed temporally and spatially with the MMP gelatinase A (GelA) in all cell types of the intestine and tail where GelA is expressed during Xenopus laevis metamorphosis, suggesting a cooperative role of these MMPs in development. Here, we show that Xenopus GelA and MT1-MMP interact with each other in vivo and that overexpression of MT1-MMP and GelA together in Xenopus embryos leads to the activation of pro-GelA. We further show that both MMPs are expressed during Xenopus embryogenesis, although MT1-MMP gene is expressed earlier than the GelA gene. To investigate whether the embryonic MMPs play a role in development, we have studied whether precocious expression of these MMPs alters development. Our results show that overexpression of both MMPs causes developmental abnormalities and embryonic death by a mechanism that requires the catalytic activity of the MMPs. More importantly, we show that coexpression of wild type MT1-MMP and GelA leads to a cooperative effect on embryonic development and that this cooperative effect is abolished when the catalytic activity of either MMP is eliminated through a point mutation in the catalytic domain. Thus, our studies support a cooperative role of these MMPs in embryonic development, likely through the activation of pro-GelA by MT1-MMP.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Significant role of laminin-1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme-free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin-1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1-nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt-like matrix was formed on the epithelial surface in the LN1-nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV-including peptide (2097-2108), AG10 (2183-2194), AG32 (2370-2381) or AG73 (2719-2730) from the alpha1 chain, or YIGSR-including peptide (926-933) from the beta1 chain. Only AG73 (RKRLQVQLSIRT from the alpha1 chain carboxyl-terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1-nidogen can support the branching morphogenesis of submandibular epithelium even if LN1-nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.     

Polycystin‐1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.     

Establishment of endometrial glandular epithelial cell subculture in a serum-free,hormonally defined medium,on a basement membrane matrix

Summary— Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serumfree Ham's F12 containing defined chemicals including 17β-estradiol. while epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix-failed to spread, they formed on poly-d -lysine plus serum-coated dishes, a subconfluent monolayer (5–7 days) enriched in cytokeratin-immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly-d -lysine plus serum-coated dishes in serum-free hormonally defined medium, were passaged on Matrigel-coated dishes in serum-free hormonally defined medium. These subcultures contained, at confluence (4–5 days), a high percentage (> 95%) of cytokeratin-immunostained cells. These monolayers consisted of well-differentiated cells which exhibited ultrastructural features characteristics of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undectable after longterm treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well-defined model for the study of protein synthesis and secretion by endometrial glandular cells under hormonal control.     

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Vα14Jα18 T cell receptor α-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver (∼0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-γ (IFN-γ) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-γ in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.     

Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system

Basement membranes (BMs) are specialized extracellular matrices that have important roles in cell attachment, migration, growth and differentiation. The murine teratocarcinoma cell line, M1536-B3, has been shown to produce a model BM composed of laminin, entactin and heparan sulfate proteoglycans but lacking collagen. Therefore, M1536-B3 cells are an excellent model system in which to study the role of non-collagenous components in BM assembly. We have used these cells to test for a requirement of mature heparan sulfate (HS) chains in BM assembly. Growth of M1536-B3 cells in the presence of chlorate, an inhibitor of activated sulfate synthesis, resulted in a dose-dependent decrease in the sulfation of glycosaminoglycans and reduction in the charge density of the isolated HS. The undersulfated HS from chlorate-treated cells had a decreased binding capacity for laminin when compared with control HS. Concurrent with these changes in sulfation, chlorate treatment of M1536-B3 cells resulted in the failure of BM assembly, which was restored upon removal of the chlorate from the growth medium. These results were not due to major alterations in cell attachment, spreading, growth, protein synthesis, or to an inability of the cells to synthesize and secrete laminin. These data suggest that the sulfation of HS and its subsequent ability to interact with other BM components play major roles in the assembly and structure of BMs.