Biomedical subjects
Publications and source records attributed to K Tryggvason.
MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules. The structure and function of the molecule is described. Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection. The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.
BACKGROUND: Congenital nephrotic syndrome (NPHS1) is a rare disease inherited as an autosomally recessive trait. The NPHS1 gene mutated in NPHS1 children has recently been identified. The gene codes for nephrin, a cell-surface protein of podocytes. Two mutations, named Fin-major and Fin-minor, have been found in over 90% of the Finnish patients. In this study, we correlated the NPHS1 gene mutations to the clinical features and renal findings in 46 Finnish NPHS1 children. METHODS: Clinical data were collected from patient files, and kidney histology and electron microscopy samples were re-evaluated. The expression of nephrin was studied using immunohistochemistry, Western blotting, and in situ hybridization. RESULTS: Nephrotic syndrome was detected in most patients within days after birth regardless of the genotype detected. No difference could be found in neonatal, renal, cardiac, or neurological features in patients with different mutations. Nephrin was not expressed in kidneys with Fin-major or Fin-minor mutations, while another slit diaphragm-associated protein, ZO-1, stained normally. In electron microscopy, podocyte fusion and podocyte filtration slits of various sizes were detected. The slit diaphragms, however, were missing. In contrast to this, a nephrotic infant with Fin-major/R743C genotype expressed nephrin in kidney had normal slit diaphragms and responded to therapy with an angiotensin-converting enzyme inhibitor and indomethacin. CONCLUSIONS: The most common NPHS1 gene mutations, Fin-major and Fin-minor, both lead to an absence of nephrin and podocyte slit diaphragms, as well as a clinically severe form of NPHS1, the Finnish type of congenital nephrotic syndrome.
CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. We have examined the patterns of expression of both CD2AP and nephrin in developing mouse and human kidney. Both proteins were first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP was also observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap -/- mice initially exhibited normal nephrin localization, but as the mice aged and foot processes became effaced, nephrin disappeared. In laminin-beta(2) mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli was aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP was observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues.
Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.
BACKGROUND: Treatment decisions for cervical cancer, a common disease worldwide, depend on demonstrating whether or not tumor invasion of the surrounding tissue has occurred. Invasion can be difficult to assess by standard histopathologic methods, especially when limited amounts of tissue are available. Several studies of a variety of cancers have reported increased expression of laminin-5-an important attachment protein for epithelial cells-in invasive carcinomas. This study was designed to investigate whether the presence of laminin-5 is related to the invasive capacity of cervical lesions. METHODS: We used immunohistochemical methods to stain archival, paraffin-embedded sections of cervical lesions with a polyclonal antibody specifically targeting the gamma2 chain of human laminin-5 protein. The study sample included 23 lesions of mild and moderate dysplasia (cervical intraepithelial neoplasia [CIN] 1 and 2, respectively), 32 lesions of severe dysplasia or carcinoma in situ (CIN 3), 15 lesions of microinvasive cancer, and 20 lesions of frankly invasive cancer. Cellular proliferative activity was also investigated by the use of monoclonal MIB-1 (directed against the antigen Ki-67) and anticyclin A antibodies. RESULTS: Invasiveness of cervical lesions was positively associated with immunohistochemical staining of the gamma2 chain of laminin-5 (two-sided P =.001). All CIN 1 and CIN 2 lesions-except one CIN 2 lesion later shown to be invasive cancer-and 21 CIN 3 lesions tested negative for the gamma2 chain of laminin-5. Eleven CIN 3 lesions and all invasive cancers tested positive for this protein. One lymph node metastasis and a pleural metastasis from one of the patients with invasive cancer showed strong immunohistochemical positivity. Proliferative activity increased with advancement of the lesion but was not confined to cells positive for the gamma2 chain of laminin-5. CONCLUSIONS: These data suggest that antibodies directed against the gamma2 chain of laminin-5 can identify cervical lesions with invasive capacity and thus may be useful as a sensitive marker of early invasion.
We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.
Matrix metalloproteinases (MMPs) catalyze extracellular matrix degradation. Control of their activity is a promising target for therapy of diseases characterized by abnormal connective tissue turnover. MMPs are expressed as latent proenzymes that are activated by proteolytic cleavage that triggers a conformational change in the propeptide (cysteine switch). The structure of proMMP-2 reveals how the propeptide shields the catalytic cleft and that the cysteine switch may operate through cleavage of loops essential for propeptide stability.
The structures of the human and mouse genes for the macrophage receptor with collagenous structure were determined. Both genes have 17 exons, of which exons 4-15 encode the collagenous domain. The transcription initiation sites in the mouse gene were identified using primer extension, SI nuclease mapping, and 5' capturing rapid amplification of cDNA ends assays. All three methods revealed two major initiation sites, one starting 27 bp downstream of a TATA box and another at positions -63 and -66 downstream of an AT-rich region. Several potential binding sites for transcription factors were identified in the promoter region, neither gene has a CAAT box or GC boxes. The human and mouse genes were localized to syntenic regions on chromosomes 2 and 1, respectively, using fluorescence in situ hybridization.
A novel laminin gamma3 chain was identified from the expressed sequence tag data base at the National Center for Biotechnology Information. A complete cDNAderived peptide sequence reveals a 1592-amino acid-long primary translation product, including a tentative 33-amino acid-long signal peptide. Comparison with the laminin gamma1 chain predicts that the two polypeptides have equal spatial dimensions. In addition, the well conserved domains VI and III(LE4) predict that gamma3 containing laminins are able to integrate to the laminin network and also via nidogen connect to other protein networks in the basement membranes. Combination of Northern analysis and in situ hybridization experiments indicate that expression of the gamma3 chain is highly tissue- and cell-specific, being significantly strong in capillaries and arterioles of kidney as well as in interstitial Leydig cells of testis.
Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR-/-) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 +/- 5, 77 +/- 2, and 85 +/- 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of approximately 70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1-reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 +/- 5 and 47 +/- 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected approximately 70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 approximately 40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.
MARCO is a novel macrophage-specific receptor structurally related to macrophage class A scavenger receptors. It is constitutively expressed in macrophages of the marginal zone of the spleen and in lymph nodes and is up-regulated in other tissues during systemic bacterial infections. In this study, we show that ectopic expression of MARCO in cell lines such as Chinese hamster ovary, HeLa, NIH3T3, and 293 induces dramatic cell shape changes. Typically these changes include formation of large lamellipodia-like structures and of long dendritic processes. The morphological changes are accompanied by disassembly of actin stress fibers and often also by complete loss of focal adhesions. The MARCO-induced changes are dependent on cell adhesion and are inhibited, but not completely abolished, when the cells are plated on fibronectin-coated surfaces. Similarly, a dominant-negative mutant of the Rho family GTPase Rac1 partially inhibited the morphogenic effects of MARCO in Chinese hamster ovary cells, whereas a dominant-negative form of a related protein, Cdc42, did not. Expression studies with a variety of truncated MARCO forms indicated that the proximal segment of the cysteine-rich domain V is important for the morphoregulatory activity. The results indicate that expression of MARCO has a direct effect in generating the phenotype of activated macrophages necessary for the trapping and removal of pathogens and other foreign substances.
Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between -2722 and -7745 allowed for expression in osteoclasts and migrating keratinocytes, i. e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the -2722 to -7745 base pair region.
The scavenger receptors expressed by macrophages are thought to play an important role in the immune response against bacteria by mediating binding and phagocytosis. A novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (MARCO), has recently been identified. In this study we have generated a panel of mAbs with specificities for different domains of this receptor. Two of those reacting with the C-terminal cysteine-rich domain block ligand binding of MARCO. The in vivo expression of this murine receptor is normally restricted to distinct populations of macrophages in the spleen and lymph nodes. During bacillus Calmette-Guérin (BCG) infection, during bacterial sepsis, or after the injection of purified LPS, however, the expression of MARCO is rapidly induced on macrophages in other tissues, including Kupffer cells in the liver. Using the mouse macrophage cell line J774.2, it was shown that LPS stimulation up-regulates surface expression of MARCO in a dose- and time-dependent fashion. The proinflammatory cytokines IL-1, IL-6, TNF-alpha, and IFN-gamma had little or no effect. Using inhibitory mAbs, the relevance of MARCO for the clearance of circulating bacteria in vivo was determined. Although the overall elimination of live Escherichia coli and Staphylococcus aureus from the blood did not appear to be affected by treatment with these Abs, the capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen was clearly inhibited. This study suggests a role for MARCO in the host antibacterial defense.
Sixty-four malignant lung tumours and 12 of their regional lymph node metastases were analysed for expression of the laminin gamma2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin gamma2 chain was strongest in squamous cell carcinomas, followed by adenocarcinomas and large cell carcinomas. Positive cells, except for large cell carcinomas, were located at the epithelial-stromal interface of tumour clusters. An important exception was small cell lung carcinoma, with only a low level of laminin gamma2 chain expression. Apart from tumour type, this may reflect the relatively scanty fibrous stroma in these tumours and supports previous observations that small cell lung carcinoma cells, contrary to other types, lack surface expression of alpha(6)beta(4) integrin, the specific laminin-5 binding receptor. In frozen sections, immunohistochemistry showed linear basement membranes around tumour clusters in squamous cell carcinomas and adenocarcinomas. This shows that carcinoma cells are capable of heavy deposition of the laminin gamma2 chain around tumour clusters and suggests that a laminin gamma2 chain-containing substrate may be of significance for the spread and growth of malignant tumours.
In an attempt to develop gene therapy for Alport syndrome, we have evaluated surgical methods for gene transfer into pig kidneys. For gene transfer we used an adenovirus expressing the Escherichia coli beta-galactosidase gene as a reporter gene. The viral preparation was first infused in vivo into the porcine renal artery. Then explanted kidneys were perfused ex vivo at body temperature for 12 hours with the viral solution and, finally the kidney perfusions were carried out in vivo via laparotomy for 60 and 120 minutes. Gene transfer was determined visually on histological cryosections after 5-bromo-4-chloro-3-indoyl-beta-galactopyranoside (X-gal) and periodic acid-Schiff (PAS) staining. Perfusion of whole porcine kidneys ex vivo resulted in strong expression in about 80% of glomeruli. The in vivo kidney perfusion via laparotomy for 120 minutes resulted in reporter gene expression of about 75% of the glomeruli examined after 4 days. Expression was observed almost exclusively in glomeruli, while little if any expression was found in other renal structures. The present results suggest that operatively performed kidney perfusion may be used for gene transfer in treatment of glomerular disease. This surgical approach may also prove useful for somatic gene therapy of other organs.
The effects of laminin-5 and its subunit gamma2 chain on cell adhesion and migration were studied, and a migration-related cis-acting element was identified in the gamma2 chain gene (LAMC2) using promoter-reporter gene constructs in transgenic mice. Intact laminin-5 molecules, but not recombinant gamma2 chain promoted cell adhesion of human keratinocytes and mouse squamous carcinoma cells, indicating that the gamma2 chain does not contain a cellular binding site. However, the gamma2 chain as such is probably involved in the process of cell locomotion, as antibodies against the short arm of the chain inhibited migration of carcinoma cells in an in vitro assay. Further evidence for the involvement of the gamma2 chain in cell migration was obtained by the identification of a cis-acting element in a promoter-lacZ reporter gene construct that was active in migratory epithelial cells of healing wounds in mice made transgenic by microinjection of the construct into fertilized oozytes. The migration active element was located in the sequence between -613 and +55. The same construct, and another one containing 5900 base pairs of the 5' flanking region, yielded very limited expression in cells of normal tissues. The limited expression was, however, only observed in epithelial cells of different tissues, i.e. cell types that normally express laminin-5 in vivo. The results show that the sequence between -613 and +55 contains elements that can drive expression during epithelial cell migration and that also partially confers more general epithelium expression. However, elements outside -5900 and +55 are needed for normal epithelium expression of the LAMC2 gene.
Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and in regulation of cell-matrix interactions during organ development. The activity of MMPs is regulated by members of the TIMP (tissue inhibitors of metalloproteinase) family. We analyzed by in situ hybridization the expression of gelatinase A (MMP-2) and gelatinase B (MMP-9) as well as Timp-1, -2 and -3 during different stages of mouse tooth development. Gene expression was generally found in mesenchymal tissues except for Timp-3, which also was found in dental epithelial cells. During early tooth development, gelatinase A and Timp-2 were widely expressed in the branchial arch, while gelatinase B and Timp-1 and Timp-3 expression showed clear association with epithelial morphogenesis and was restricted to the mesenchyme at the tip of the growing tooth bud. Gelatinase A and Timp-1 showed transient expression in secretory odontoblasts at the time of basement membrane degradation, while Timp-2 expression continued throughout the dental papilla. At the time of tooth eruption, Timp-3 was expressed in most dental epithelial cells except secretory ameloblasts, and gelatinase B was intensely expressed in osteoclasts in the jaw bone. The exact co-localization of gelatinase A and Timp-1 in secretory odontoblasts, and the correlation between gelatinase B and Timp-3 during bone resorption may indicate interaction of the proteins during degradation of the basement membrane and in the control of ECM turnover in connection with tooth eruption.
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin.