Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase.

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.

Genomic structure and chromosomal mapping of the mouse hic-5 gene that encodes a focal adhesion protein.

The hic-5 gene encodes a focal adhesion protein that has striking similarity to paxillin. Genomic clones of the mouse hic-5 gene were isolated, and included 10 exons that covered the whole mouse mRNA sequence. Comparison of the sequence with those in the expressed sequence tag database suggested that the hic-5 gene contained an extra exon (named exon 1') located about 1kb upstream of exon 1, and mouse cells seemed to express two alternatively spliced forms of mRNA. All the exon-intron boundaries followed the GT/AG rule. Physical mapping and fluorescent in situ hybridization analysis indicated that the hic-5 gene is located on mouse chromosome 7, 60. 0cM from the centromere.

Decrease in the expression of a novel TGF beta1-inducible and ras-recision gene, TSC-36, in human cancer cells.

TSC-36, a TGF beta1-inducible gene, encodes a polypeptide that shows significant similarity to SPARC (secreted protein rich in cysteine), and follistatin, an activin-binding protein. The expression of the TSC-36 gene was reported to be extinguished in v-ras-transformed mouse fibroblastic cells, and also was found also to be abrogated in cells transformed with v-myc. The level of expression was, however, not affected in cells transformed with v-src, v-abl, or v-raf. The TSC-36 cDNA was first isolated from mouse cells, and recently its rat and human homologues have been reported which show striking similarity with each other. In various human tumor cells, TSC-36 mRNA was almost undetectable. TSC-36 mRNA was detected in various mouse organs, but its level was the highest in the lung. TSC-36 mRNA level was the highest in the lung among mouse organs, and on in situ hybridization, the TSC-36 transcript was detected in the alveolar epithelium but not in the bronchial epithelium. These features suggest possible usage of TSC-36 as one of the markers in human tumors.

Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis.

In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.

In vivo production of various cytokines in splenocytes of sheep erythrocyte-immunized mice after intravenous administration of bacterial lipid A.

The effects of an intravenous administration of lipid A from Salmonella minnesota R595 lipopolysaccharide on the in vivo production of interleukin-2 (IL-2), IL-4, IL-5 and IL-6 in the spleens of mice intravenously immunized with sheep red blood cell (SRBC) antigen were investigated. The increased number of antigen-specific IgM antibody-producing cells and the titer of the IgM serum antibody were measured using the plaque-forming cell (PFC) assay and an enzyme-linked immunosorbent assay (ELISA). Simultaneous injections of SRBC antigen and lipid A adjuvant enhanced IgM-PFC number on days 3 and 4 and the serum IgM titer on days 4 and 5 after the immunization. We found that the enhanced IL-4 and IL-5 levels correlated with the PFC number and IgM titer. When lipid A was injected intravenously 2 days after immunization with SRBC, the PFC number in lipid A-treated groups were similar to those in controls 3 and 4 days after the immunization. However, it was found that a twofold increase in the IgM titer in serum was induced by lipid A 5 days after immunization. In relation to this increase, lipid A stimulated the production of only IL-5 among the cytokines tested.

Neutralization of Shwartzman-inducing activity by antibodies recognizing the Re core or lipid A structures of lipopolysaccharide from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477.

Antibodies recognizing the Re core or lipid A structures of lipopolysaccharide (LPS) derived from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477 were tested for the ability to neutralize the preparatory activity of endotoxin using the local Shwartzman reaction. Shwartzman-inducing activity of R595 LPS (Re-form) was strongly suppressed when the LPS was incubated with the rabbit anti-R595 antiserum or the purified IgG antibody which recognizes core region of the LPS. The antiserum also suppressed the preparatory activity of LPS from S. typhimurium SL1102 (Re) and Escherichia coli F515 (Re), but not that of either S. typhimurium LT-2 (S) LPS or R595 lipid A. Moreover, it was found that the murine monoclonal antibody (MAb), SmRe100G (IgG2a) which recognizes the core region of R595 LPS, significantly suppressed the preparatory activity of R595 LPS. Both conventional antibodies specific to R595 lipid A, which contains a 1,4'-bisphosphorylated beta-D-glucosaminyl-alpha-D-glucosamine disaccharide structure, and JCM1477 lipid A, which contains a monophosphorylated 3-amino-D-glucosamine disaccharide structure, neutralized the preparatory activity of homologous and a closely related lipid A, but not that of LPS. In addition, it was observed that MAb Sm5G (IgG2b) specific to enterobacterial lipid A preparations (especially R595 lipid A) neutralized the preparatory activity of R595 lipid A, although the effect was somewhat weak as compared with that of rabbit antiserum. These results suggest that anti-Re LPS antibody binding to the core of Re LPS is involved in suppressing the endotoxic activity of Re LPS, and that the direct binding of anti-lipid A antibody to some specific epitopes of lipid A is important in neutralizing the endotoxic activity.

Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence.

Transforming growth factor (TGF) beta 1 is a potent cytokine that inhibits the growth of several types of cells. Our earlier study suggested that the mouse osteoblastic cell line, MC3T3-E1, was sensitive to growth inhibition by TGF beta 1 and that this effect was partly mediated by H2O2. To identify the molecules that participate in the negative regulation of growth by these stimuli, we carried out differential screening of cDNA libraries and isolated a set of genes induced by TGF beta 1. Among the clones isolated, one originally named tsc-5 was found to be induced by H2O2 as well as TGF beta 1. Analysis of this cDNA renamed hic (hydrogen peroxide-inducible clone)-5 suggested that Hic-5 protein has four LIM motifs, each of which contained two (or one) putative zinc fingers. The expression of hic-5 mRNA was repressed in Ki-ras-transformed mouse fibroblasts and in several cell lines established from human tumor. On the other hand, its expression was augmented in the in vitro senescent process of human diploid fibroblasts. Among the mouse organs examined, hic-5 was highly expressed in the lung and spleen. Finally, a colony formation assay using an hic-5 expression vector driven by the cytomegalovirus promoter suggested that hic-5 overexpression had a cytostatic effect on cellular growth, depending upon the cell type. Although the relationship between hic-5 function and the signal transduction pathway of TGF beta 1 remains unresolved, these results implied that hic-5 has some role in the growth-inhibitory pathway associated with in vitro senescence, and that down-regulation of hic-5 contributes to tumorigenesis.

Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes.

The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.

Pseudomonas diminuta LPS with a new endotoxic lipid A structure.

Lipid A that contains mainly 2,3-diamino-2,3-dideoxy-D-glucose, phosphate and fatty acids in the molar ratio 2:1:5-6 was found in Pseudomonas diminuta lipopolysaccharide. The lipid A was considered to have a diamino-sugar disaccharide structure that carries a nonglycosidic phosphomonoester group and amide-bound acyloxyacyl and 3-hydroxy fatty acyl groups. The lipopolysaccharide exhibited endotoxic activities including lethal toxicity, pyrogenicity, local Shwartzman activity, body weight-decreasing toxicity and Limulus activity. The free lipid A was also endotoxic.

Tumor necrosis factor-inducing activities of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis.

Tumor necrosis factor (TNF)-inducing activities of lipid A preparations from P. diminuta and P. vesicularis, which contain mainly 2 mol of 2,3-diamino-2,3-dideoxy-D-glucose and 1 mol of nonglycosidic phosphate as the backbone component and have partly different fatty acid compositions, were examined. TNF was induced by injecting various lipid A fractions into mice that had previously been sensitized with Mycobacterium bovis BCG vaccine. A major component of lipid A of both strains, referred to as A3 fraction, exhibited stronger TNF-inducing activity than A2 fraction having incomplete acyl residues. The removal of ester-linked fatty acyl groups by mild hydrazinolysis of the P. diminuta lipid A results in a marked decrease of the activity. These results suggest that the structure of the hydrophobic part, including the amide-linked acyloxyacyl group(s), of the lipid A molecule play an important role in inducing TNF in the sera of mice.

Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide.

Transforming growth factor(TGF)beta 1 is a potent inhibitor of growth in mouse osteoblastic MC3T3-E1 cells. To isolate genes that are induced by TGF beta 1, the differential screening method was adopted using a cDNA library constructed from cells treated with TGF beta 1 for 4 h. Six independent cDNA clones were isolated (TGF beta-stimulated clone, TSC-5, TSC-36, TSC-115, TSC-128, TSC-160 and TSC-161), the expression of which was increased by TGF beta 1-treatment with maximal expression at 6-10 h. The steady-state levels of TSC-36, TSC-128 and TSC-160 increased almost tenfold, whereas those of TSC-5, TSC-115 and TSC-161 were elevated at most threefold. From partial nucleotide sequences, TSC-160 was found to be identical to rrg (ras-recision gene, lysyl oxidase), and TSC-115 had 80% similarity with tropomyosin cDNA, whereas other genes seemed novel. Expression of TSC-36 and TSC-160 was dramatically decreased in v-Ki-ras-transformed MC3T3 cells or in transformed NIH 3T3 cells (DT), and was recovered to normal levels in a flat revertant (C11). A nearly full-length copy of TSC-36 cDNA was isolated, and an open reading frame indicated that it encodes a protein of 35 kDa. An antiserum was raised against the C-terminal peptide predicted from the nucleotide sequence, and a polypeptide with an approximate molecular mass of 38 kDa was detected in cultured medium of MC3T3-E1 cells. The amino acid sequence of TSC-36 protein was found to have some similarity with follistatin, an activin-binding protein, and a limited similarity with the secreted protein rich in cysteine (SPARC).

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria.

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.

In vitro antigenic reactivity of synthetic lipid A analogues as determined by monoclonal and conventional antibodies.

Cross-reactivities of synthetic lipid A analogues with monoclonal and conventional antibodies against Salmonella lipid A were studied. It was shown that the in vitro antigenicity of a synthetic compound 506, beta-(1----6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, was practically indistinguishable from that of the natural E. coli lipid A preparation, and that both phosphates in positions 1 and 4' as well as ester- and amide-linked fatty acyl residues, particularly 3-acyloxyacyl group, of the glucosamine disaccharide are involved in the cross-reactivity of lipid A as important antigenic determinants.

Structural heterogeneity regarding local Shwartzman activity of lipid A.

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.

Synthetic Salmonella-type lipid A antigen with high serological specificity.

A synthetic compound (compound 516), beta(1-6)-linked D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated by (R)-3-hexadecanoyloxytetradecanoyl, (R)-3-hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3-tetradecanoyloxytetradecanoyl groups at positions 2,3,2', and 3', respectively, exhibited in vitro antigenic reactivity of high specificity comparable to that of free lipid A from Salmonella minnesota R595. This was confirmed by an enzyme-linked immunosorbent assay and an enzyme-linked immunosorbent assay inhibition test with monoclonal and conventional antibodies. The results of comparative analysis performed with several synthetic lipid A analogs as well as three monosaccharide derivatives suggested that the complete structure involving both phosphate groups at the C-1 and C-4' positions and the 3-acyloxyacyl groups at the C-2, C-2', and C-3' positions of the glucosamine disaccharide are required for the expression of the serological specificity of Salmonella-type lipid A. This was deduced from the observations that compound 506, a synthetic Escherichia coli-type lipid A which has the same structure as that of compound 516, except that 3-hydroxytetradecanoyl group is substituted for an acyloxyacyl residue at the C-2 position, exhibited significantly reduced antigenic reactivity as compared with compound 516 and that the replacement by the hydrogen atom of the phosphoryl group at the C-1 position or by 3-hydroxytetradecanoyl or tetradecanoyl groups of acyl residues at the 2, 3, 2', and 3' positions of compound 516 results in a marked reduction of reactivity with monoclonal antibodies 5G and 36G. Similar results were obtained by assays with conventional rabbit antibodies, but the structural difference between compounds 516 and 506 could not be distinguished by these polyclonal antibodies. The results of cross-reactions among synthetic analogs with monoclonal antibodies 161M and 1-9M, which have been confirmed to exhibit different serological specificities from the 5G or 36G antibody, also suggested that the nature and linkage of fatty acyl residues as well as the backbone structure of lipid A play an important role in determining serological specificity of the lipid A molecule.

Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant.

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type lipid A.