Expression and functional analysis of fibulin-1 (Fbln1) during normal and abnormal placental development of the mouse.

The extracellular matrix protein fibulin-1 (FBLN1) is an important component of blood vessel walls, as shown by the lethality of mice with homozygous targeted deletion of the Fbln1 gene. Here, we show that a murine placental overgrowth phenotype is associated with elevated Fbln1 transcript levels, suggesting that the gene and its product have a functional role in placentation. Fbln1 exhibits a specific expression pattern in the mouse placenta. Transcripts could not be detected prior to day 12. In subsequent stages, Fbln1 was expressed strongly in the spongiotrophoblast. Other sites of expression were endothelia of large fetal blood vessels, a tissue type reported to not express this gene. In addition, a subset of giant cells expressed the gene. This giant cell specific expression was strongly increased in hyperplastic placentas. Analysis of the placentation in fibulin null mice did not show any abnormality. Attempts to rescue the placental phenotypes of a congenic model of interspecies hybrid placental dysplasia (IHPD) by normalizing expression of Fbln1 proved that Fbln1 alone is not the key cause of phenotypes in these models of placental hyperplasia.

Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis.

BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions.

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.

Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice.

The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

Studies of early hepatocellular proliferation and peroxisomal proliferation in Wistar rats treated with herbicide diclofop.

A study was performed to determine whether diclofop (2-(4-(2,4-dichlorophenoxy) phenoxy)propionic acid), introduced as a herbicide, exhibits the properties of peroxisome proliferators (PPs). Diclofop was administered orally at 7-56 mg/kg body weight per day to male Wistar rats for 2, 4, 7 or 14 consecutive days and some effects regarded as early hepatic markers of PPs were studied. The early changes in rat liver, produced by short-term treatment with diclofop consisted of mitogenesis and, time- and dose-related increase in liver weight. Hepatomegaly was typically associated with proliferation of smooth endoplasmic reticulum (SER) and peroxisomes. The parallel biochemical measurements showed that there was a dose-dependent increase in peroxisomal palmitoyl-CoA oxidation and catalase activity in treated rats. Markers of hepatocellular proliferation (S- and M-phase) indicated that mitogenesis was transient and declined despite continuation of diclofop treatment. The threshold exposure level for the palmitoyl-CoA oxidation (one of the peroxisome proliferation markers) was approximately the same (14 mg/kg body weightxper day) as for the stimulation of mitogenesis in Wistar rats. However, for hepatomegaly and catalase activity the threshold exposure level was 7 mg/kg body weightxper day. The results presented here demonstrate clearly that diclofop belongs to a class of rodent PPs.

Early hepatic changes in rats induced by permethrin in comparison with DDT.

In this study permethrin [(3-phenoxyphenyl)-methyl-3-(2,2-dichloroethenyl)-2,2-dim ethylcyclopropanecarboxylate] and DDT [1,1-(2,2,2 trichloroethylidene)-bis-(4-chlorobenzene)] were compared in rats for their effects on early hepatic changes, proposed in the literature to be useful endpoints in screening for non-genotoxic hepatocarcinogenesis and/or liver tumour promotion. We compared the effects of both insecticides on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and liver pathology. Male Wistar rats received permethrin (PERM) or DDT in one, three, five and 14 daily oral doses (at 24-h intervals) equivalent to 1/10 LD50. Distinct differences in early liver response between PERM and DDT were observed. DDT stimulated the early effect consisting of hepatomegaly accompanied by an increase in hepatocellular proliferation with signs of cell necrosis. Thus, it might be concluded, that the mitogenic effect of DDT was at least partly related to a regenerative liver response. Although PERM significantly affected DNA synthesis and increased binuclear hepatocytes, this compound did not increase the number of mitotic figures. These results suggest that PERM may inhibit of phase G2 in the cell cycle and consequently it may suppress the cell entering into the stage of mitosis (M-phase). In addition, the present findings provide evidence for the occurrence of abnormal mitoses in the hepatocytes of rats treated with DDT.

Sequence, recombinant expression and tissue localization of two novel extracellular matrix proteins, fibulin-3 and fibulin-4.

Fibulin-1 and fibulin-2 have previously been identified as basement membrane and microfibrillar proteins with a broad binding repertoire for other extracellular ligands. Here we report on the cloning and sequence analysis of human fibulin-3 (487 residues), also known as protein S1-5, and fibulin-4 (443 residues). These novel members of this protein family are most closely related to fibulin-1C. They consist of a C-terminal globular domain III, also shared by the fibrillins, a central rod-like element composed of five calcium-binding epidermal growth factor-like (EG) modules (domain II) and an N-terminal interrupted EG module (domain I) which replaces the anaphylatoxin-like modules of the other fibulins. This predicted domain structure was supported by electron microscopy of fibulin-4, which demonstrated short rods. Northern blots showed that both novel fibulins are expressed in several human tissues to a variable extent and that they are up-regulated in quiescent fibroblasts. Specific antibodies which were raised against each of the novel fibulins did not cross-react with fibulin-1. Immunohistology of adult mouse tissues showed that fibulin-3, fibulin-4 and fibulin-1 have overlapping but distinct extracellular tissue localizations. A particularly prominent feature was the staining of variable sets of large and small blood vessels.

Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican.

The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca2+-dependent, with KD values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.

Complete exon-intron organization of the mouse fibulin-1 gene and its comparison with the human fibulin-1 gene.

Fibulin-1 is a 90 kDa calcium-binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C-termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin-1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3' end. By searching the database we identified most of the exons encoding the human fibulin-1 gene and showed that its exon-intron organization is similar to that of the mouse gene.

[Molecular mechanisms of chemically induced carcinogenesis]. / Molekularne mechanizmy chemicznej kancerogenezy.

In this review recent point of view concerning the molecular mechanisms of chemically induced carcinogenesis is presented. The new and promising trends of neoplasia investigations are based on discovery of protooncogenes and tumor suppressor genes, which maintain tissue homeostasis by controlling cellular proliferation and differentiation. It is generally recognised, that mutations induced by genotoxic carcinogens, particularly those resulting in activation of protooncogenes and inactivation of suppressor genes, play a crucial role in the initiation step of multistage process of tumorigenesis. Tumor promotion is recognized as a process whereby initiated cells are stimulated to selective growth and then, to develop into the cancer during progression step. Tumor promotion can be affected by many nongenotoxic carcinogens. In this review the attention is given to the mutational activation of the c-ras oncogenes and inactivation of p53 suppressor gene in rodent and human cancers by genotoxic carcinogens. Moreover, the significance of nongenotoxic carcinogens and the mechanisms by which these compounds may accelerate tumorigenesis are discussed.

[The role of cell proliferation in carcinogenesis]. / Rola proliferacji komórkowej w kancerogenezie.

Cell proliferation is regulated by the cell cycle which is controlled by a number of cyclin-dependent kinases (CDKs). The functions of CDKs are critical for cell cycle and are required to traverse checkpoints. A network of inhibitors (CKI) of the cyclin-dependent kinases provide the important function of regulating the activity of the cyclin complexes. Deregulation of these results in either uncontrolled proliferation or cell death (apoptosis). Cell proliferation is an important factor in the development of carcinogenesis induced by genotoxic as well as nongenotoxic carcinogens. It is an integral part of the process of converting DNA adducts to mutations, it also decreases the time that is available for DNA repair and is required to clonal expansion of initiated cell populations. Moreover, cell proliferation increases the number of initiated cells by blocking cell death (apoptosis) and pertrubing checkpoints in the cell cycle. Two major mechanisms of induction of cell proliferation (regenerative and mitogens-stimulated) were discussed in relation to their potential roles in the carcinogenicity.

[The role of apoptosis in cell physiology and pathology]. / Rola apoptozy w fizjologii i patologii komórki.

The cell death is a natural physiological process that occurs in every living organism. It is clear that programmed cell death--apoptosis--is an important mechanism maintaining cell number in a multicellular organism. This review summarises recent progress in the field of apoptosis. Extracellular signals, such as various growth factors and antigen Fas ligand can trigger apoptosis via cell surface receptors. Within the cell the tumor suppressor gene p53 and oncogenes c-myc, c-fos and c-jun tend to activate apoptosis, while other genes such as most members of the bcl-2 family, tend to suppress it. Many of these signals regulate a family of cysteine proteases--related to interleukin 1 beta converting enzyme (ICE)--caspases--which play a crucial role in apoptosis. Many factors that affect apoptosis also affect the cell cycle. For example, p53 appears to be an important mediator of both apoptosis and cycle arrest. If DNA damage is repaired during cycle arrest, the cell survives. Special attention is paid to the abnormalities in the regulation of apoptosis that may contribute to different pathogenic processes.

Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules.

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.

Sequence and expression of a novel member (LTBP-4) of the family of latent transforming growth factor-beta binding proteins.

Overlapping cDNA clones from human heart and melanoma libraries were used to establish the 1587-residue sequence of a novel protein (LTBP-4) belonging to the family of extracellular microfibrillar proteins which also bind transforming growth factor-beta. LTBP-4 consists of 20 EG modules, 17 of them with a consensus sequence for calcium binding, 4 TB modules with 8 cysteines and several proline-rich regions. Northern blots demonstrated a single 5 kb mRNA which is highly expressed in heart but also present in skeletal muscle, pancreas, placenta and lung. The modular structure predicts that LTBP-4 should be a microfibrillar protein which probably also binds TGF-beta.

[The effect of permethrin and DDT on the activity of cytochrome P-450 1A and 2B molecular forms in rat liver]. / Wplyw permetryny i DDT na aktywnosc form molekularnych cytochromu P-4501A i 2B w watrobie szczura.

The effect of permethrin on relative liver weight (RLW) and the activity of hepatic monooxygenase system related to cytochrome P-4502B and 2A was studies. The effect of permethrin was compared with DDT used as phenobarbital-type of monooxygenase inducer (induces cyt. P-4502B). Male Wistar rats received permethrin and DDT for 4 days at 24 h intervals in daily oral doses of 1/10, 1/50 and 1/100 LD50. 3-methylocholantrene and phenobarbital which served as inducers of cytochrome P-4501A and 2B, respectively and were used as positive controls. The activities of cytochrome(s) P-450 were measured by 7-pentoxy- and 7-etoxyresofurin O-dealkylation by S-9 fraction of rat liver; these two compounds have been shown to be the substrates for reactions mediated by cytochrome P-4502B and 2A. Thus this biochemical procedure permits to determine whether tested compound belongs to one of two main types of inducers of the cytochrome P-450 monoxygenase system. Treatment of rats with both pesticides resulted in significant increase in RLW, to 30 and 15% of control, respectively. In animals treated with permethrin the metabolism of 7-pentoxyresofurin increased in a dose dependent manner. Phenobarbital and the highest dose of permethrin (620 mg/kg b.w. x day-1) induced similar (about 30-fold) increase in O-dealkylation of 7-pentoxyresofurin. DDT stimulated metabolism of 7-pentoxyresofurin to much higher degree as compared with phenobarbital. It should be noted that both pesticides induced only slight increase in O-dealkylation of 7-etoxyresofurin (cyt. P-4501A-mediated reaction). The present results indicate that permethrin as well as DDT shows the ability to induce the phenobarbital-type of cytochrome P-4502B.

Early hepatic changes induced in rats by two hepatocarcinogenic organohalogen pesticides: bromopropylate and DDT.

The aim of the present studies was to describe the effect of two organohalogen pesticides: DDT and bromopropylate, on early changes in rat liver, proposed in the literature to be useful endpoints in screening of non-genotoxic hepatocarcinogens and/or liver tumor promoters. We investigated the effects on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and cytochrome CYP2B1-dependent monooxygenase induction. The histological and cytochemical changes in the liver were also recorded. Male Wistar rats received bromopropylate in one, three or five daily oral doses of 125, 250, and 500 mg/kg body wt. day-1. DDT was applied as one, three, and five daily oral doses of 24 mg/kg body wt. day-1 (this dose is close to the mean hepatocarcinogenic dose in male Wistar rats: 34.1 mg/kg body wt. day-1). In the case of both pesticides the early effects observed consisted of hepatomegaly accompanied by an increase in the p-nitroanisole O-demethylase activity and hepatocyte proliferation. Hepatocyte proliferation was elevated during the total experimental period. Vacuolated cytoplasm and evident focal necrosis may suggest that the maximal increase in hepatocyte proliferation, preceding hepatomegaly, is at least partly related to a regenerative liver response to pesticides. In addition to the above-mentioned early changes, the present findings provide new evidence for the occurrence of dose-dependent abnormal mitoses (and c-mitoses) in the hepatocytes of the bromopropylate and DDT treated rats.

[The effect of selected polychlorinated hydrocarbon pesticides on proliferation of cells in rat liver (14 day experiment)]. / Wplyw wybranych pestycydów z grupy polichlorowanych weglowodorów na proliferacje komórkowa w watrobie szczurów (doswiadczenie 14 dniowe).

The aim of present studies was to describe the effect of two organochlorine pesticides: nuarimol and DDT on the changes in rat liver, proposed in the literature to be useful endpoints in screening of non-genotoxic hepatocarcinogens and/or liver tumor promoters. The effects on the following endpoints: mitogenesis (DNA synthesis and mitotic activity), hepatomegaly as well as histological changes in rat liver have been investigated. Male Wistar rats received nuarimol or DDT in one, five and fourteen daily oral administration of the doses of 125 and 12 mg/kg b.w. day-1 respectively. In the case of both pesticides the effects observed consisted of hepatomegaly and hepatocyte proliferation (DNA synthesis and mitotic activity), although our studies indicated several distinct differences in the hepatic response between nuarimol, on the one hand and DDT on the other. The differences were reflected in the character and the basal rate of hepatocyte proliferation. Nuarimol elicited rapid but transient wave of hepatocyte proliferation during the first day of exposure. As opposite to nuarimol, DDT induced sustained hepatocyte proliferation during experimental period (14 days). Moreover, DDT induced evident focal necrosis and abnormal mitoses whereas nuarimol caused only slight vacuolated cytoplasm. Thus it can be concluded that nuarimol induced in rat liver direct mitogenic effect. On the other hand, DDT which is well known hepatocarcinogen, was found to produce mitogenic effect appearing to be related to regenerative response, since histological signs of necrosis were apparent.

Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

Early hepatic changes induced by nuarimol in rats.

It is commonly believed that in short-term tests hepatic cytochrome P-450b inducers stimulate liver enlargement and mitogenesis in the absence of overt hepatotoxic effects. In this investigation male Wistar rats received naurimol (an organochlorine pesticide) in one, three and five oral doses of 31.5, 62.5 and 125 mg kg-body wt. day-, whereupon the effects on liver were determined. The early effects were dose-dependent increases in p-nitroanisole metabolism, hepatocyte proliferation (DNA synthesis and mitotic activity) and liver weight. Five administrations of the lowest does (31.5 mg kg-1 body wt. day-1) did not change liver weight, despite increased p-nitroanisole metabolism and hepatocyte proliferation. In contrast to p-nitroanisole metabolism and hepatomegaly, proliferation was only transient and disappeared even when treatment continued. The increase in binuclear hepatocytes and signs of necrosis suggested that the hepatomitogenic effect of nuarimol reflected a regenerative response, which may simulate the proliferation caused by partial hepatectomy.

Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.

Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.