The short arm of laminin gamma2 chain of laminin-5 (laminin-332) binds syndecan-1 and regulates cellular adhesion and migration by suppressing phosphorylation of integrin beta4 chain

The proteolytic processing of laminin-5 at the short arm of the gamma2 chain (gamma2sa) is known to convert this laminin from a cell adhesion type to a motility type. Here, we studied this mechanism by analyzing the functions of gamma2sa. In some immortalized or tumorigenic human cell lines, a recombinant gamma2sa, in either soluble or insoluble (coated) form, promoted the adhesion of these cells to the processed laminin-5 (Pr-LN5), and it suppressed their migration stimulated by serum or epidermal growth factor (EGF). Gamma2sa also suppressed EGF-induced tyrosine phosphorylation of integrin beta4 and resultant disruption of hemidesmosome-like structures in keratinocytes. Gamma2sa bound to syndecan-1, and this binding, as well as its cell adhesion activity, was blocked by heparin. By analyzing the activities of three different gamma2sa fragments, the active site of gamma2sa was localized to the NH(2)-terminal EGF-like sequence (domain V or LEa). Suppression of syndecan-1 expression by the RNA interference effectively blocked the activities of domain V capable of promoting cell adhesion and inhibiting the integrin beta4 phosphorylation. These results demonstrate that domain V of the gamma2 chain negatively regulates the integrin beta4 phosphorylation, probably through a syndecan-1-mediated signaling, leading to enhanced cell adhesion and suppressed cell motility.     

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.     

Effects of acidic phospholipids, nucleotides, and heparin on the activity of lipase from rat liver lysosomes

Purification and characterization of endogenous lipid factors that stimulate rat liver lysosomal lipase has led to the identification of cardiolipin, phosphatidylserine, and phosphatidic acid as stimulators of this activity. Bovine heart cardiolipin (half-maximal stimulation at 1.5 x 10(-4) m) and bovine brain phosphatidylserine (half-maximal stimulation at 9.5 x 10(-4) m) were the most potent of the phospholipids from other sources tested. The major rate-enhancing effect of phosphatidylserine is expressed as a 35-fold increase in the apparent V(max) of the enzyme. The effect is produced by acid phospholipids specifically, since in no case was there greater than a twofold stimulation by synthetic detergents, zwitterionic phospholipids, taurocholic acid, or gum acacia. The observed degree of stimulation depends upon the detergent used to disperse tripalmitin substrate and the relative concentrations of factor and detergent in reaction mixtures. The concentration of phosphatidylserine to produce half-maximal stimulation is directly dependent upon the Triton X-100 concentration, but the effects of this detergent on cardiolipin stimulation are more complex. Enzyme activity is inhibited 50% by 1 mm nucleoside triphosphate and 2.5 mm ADP, 80% by 1 mm PP(i), 100% by 20 U/ml heparin and 0.25 mg/ml chondroitin sulfate, and 80% by 10 mm sulfate ion. Inhibition is partially prevented by phosphatidylserine.     

Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration

Laminin-332 (α3?3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ?1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3?1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6?4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6?4 and α3?1, whereas unassembled soluble Lm332 supports cell migration.     

A parallel comparison of age-related peripheral nerve changes in three different strains of mice.

Strain-specific differences contributing to spontaneous age-related peripheral nerve changes were examined in three different strains of 100-week-old female mice housed under the same conditions over the same period: inbred C57BL and C3H strains, and the hybrid B6C3F1 strain. A lower incidence of obesity and significantly lower body weight, grasping power of fore- and hind-limbs, blood lipid level, tail-flick latency and motor nerve conduction velocity were observed in C57BL mice; significantly lower body temperature, blood glucose and HbA1c levels were observed in C3H mice. Histological examination conducted on isolated sciatic nerves and brachial plexuses revealed peripheral nerve lesions, characterized by axonal degeneration and remyelination, in all strains. Although the extent of histopathologic change in nerve fibers was similar in quality to those observed in all three mouse strains, the incidence and severity of nerve lesions in B6C3F1 and C3H mice were significantly greater than those observed in C57BL mice.     

ESR studies on the oxidation of N,N-dimethyl-p-anisidine and its analogues catalyzed by myeloperoxidase

N,N-Dimethyl-p-anisidine (DMA) was used as a substrate to differentiate between the direct, or chloride-independent, and the indirect, or chloride-dependent, pathways characteristic of myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7). The chemical oxidation by sodium hypochlorite and the horseradish peroxidase-catalyzed oxidation by H2O2 were also investigated for a comparison. The chemical oxidation of DMA by NaOCl (DMA/NaOCl = 1) gave the p-N,N-dimethylaminophenoxy radical at pH 5 and 7. p-Benzoquinone and formaldehyde were determined as stable end-products. On the other hand, the cation radical of DMA was detected and p-benzoquinone was not obtained in the horseradish peroxidase-H2O2-Cl- system. In the presence of Cl- the myeloperoxidase-catalyzed oxidation at pH 5 gave nearly the same result as did the oxidation by NaOCl, whereas in the absence of Cl- the result of the oxidation was similar to that of the horseradish peroxidase-catalyzed oxidation, except for a low yield of formaldehyde formation, which was ascribed to the decomposition of H2O2 by the catalase activity of myeloperoxidase. Although the myeloperoxidase-catalyzed oxidation of DMA at pH 7 in the presence of Cl- gave only the cation radical of DMA, a fairly large amount of p-benzoquinone was obtained as a product. This result indicates that the indirect chloride-dependent oxidation is also operating at pH 7. The reaction mechanism for the myeloperoxidase-catalyzed oxidation of DMA is proposed.     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.