Stabilization of peroxisome proliferator-activated receptor alpha by the ligand.

Peroxisome proliferator-activated receptor (PPAR) constitutes a subfamily among a large group of ligand-activated transcription factors, the nuclear receptor superfamily. We studied the effects of ligand on the intracellular behaviors of PPARalpha. Although nuclear localization of PPARalpha was not affected by a selective ligand, Wy14643, we observed that exogenously expressed PPARalpha was rapidly degraded in HeLa cells, and the ligand significantly stabilized the protein. The stability of PPARalpha was also improved by coexpression of the heterodimer partner retinoid X receptor (RXR) alpha, and further stabilization was not observed with the ligand. These results indicate that PPARalpha is stabilized through heterodimerization with RXR, and the excess protein unpaired with RXR is rapidly turned over, if not bound by an appropriate ligand. These observations on PPARalpha are in sharp contrast to the ligand-stimulated degradation reported on PPARgamma. The ligand-dependent stabilization would have physiological significance when the synthesis of PPARalpha is elevated exceeding the available level of RXR.

Nuclear receptor binding factor-2 (NRBF-2), a possible gene activator protein interacting with nuclear hormone receptors.

A protein named nuclear receptor binding factor-2 (NRBF-2) was identified by yeast two-hybrid screening, as an interaction partner of peroxisome proliferator-activated receptor alpha as well as several other nuclear receptors. NRBF-2 exhibited a gene activation function, when tethered to a heterologous DNA binding domain, in both mammalian cells and yeast.

NXP-1, a human protein related to Rad21/Scc1/Mcd1, is a component of the nuclear matrix.

Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions. In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library. This clone was partially represented a known cDNA clone HA1237. HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1. NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library. We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST. Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells. Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1. Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16-128 amino acid) of NXP-1 has an important role in nuclear matrix distribution.

Nuclear receptor binding factor-1 (NRBF-1), a protein interacting with a wide spectrum of nuclear hormone receptors.

To identify the proteins which may modulate the functions of peroxisome proliferator-activated receptor (PPAR), a rat liver cDNA library was screened by a yeast two-hybrid system, using the mouse PPARalpha as a bait. A protein named nuclear receptor binding factor-1 (NRBF-1) was identified, which interacts not only with PPARalpha, but also with various nuclear hormone receptors in the presence of the respective ligands. Both the hinge and ligand-binding domains of PPARalpha are required for the interaction. NRBF-1 seems to be translocated to the nucleus by a piggyback mechanism, together with PPARalpha. NRBF-1 has a significant homology to the yeast protein MRF1, a putative transcription factor regulating the expression of mitochondrial respiratory proteins. NRBF-1 might be another type of nuclear receptor co-operator.

An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors.

The yeast two-hybrid screening was applied to cloning cDNAs of proteins that interact with peroxisome proliferator-activated receptor alpha (PPAR alpha). We obtained from a rat liver cDNA library a clone encoding a protein related to the ligand-binding domain of the members of nuclear hormone receptor superfamily, whereas apparently lacking the zinc-finger DNA-binding domain. This protein interacted with the activated forms of several nuclear receptors, and thus is a novel type of heterodimer-forming nuclear receptor.

Intracellular localization and biochemical function of variant beta-actin, which inhibits metastasis of B16 melanoma.

We analyzed the biochemical nature of beta m-actin protein found in mouse B16 melanoma. When we carried out immunostaining with the antibody specific to beta m-actin, filamentous immunofluorescence was observed in B16-F1, a low-metastatic cell line expressing beta m-actin, but not in highly metastatic B16-F10 that did not express beta m-actin. When a purified actin fraction containing beta m-actin was polymerized and immunoprecipitated with anti-beta m-actin antibody, the immunoprecipitate contained beta m-, beta- and gamma-actin. This indicated that the beta m-actin was incorporated into an actin filament together with beta- and gamma-actin in vitro, and this phenomenon was consistently suggested by cellular double immunostaining with anti-beta m-actin and common anti-actin antibody. When the actin fraction containing beta m-actin under a regular depolymerizing condition was subjected to immuno-adsorption assay using anti-beta m antibody and protein-A Sepharose, the immunoadsorbed aggregates contained beta m-, beta- and gamma-actin. This indicates that the actin fraction was not completely depolymerized and contained beta m-actin-containing oligomers, which were too small to be precipitated with anti-beta m-actin antibody alone. The incomplete depolymerization of the beta m-actin-containing fraction was also suggested by the much lower DNase 1 inhibition activity of the beta m-actin-containing fraction than that of beta- and gamma-actin fraction. Furthermore, a DNase 1 binding assay showed that cytoplasmic supernatant prepared from B16-F1 under a low-ionic condition contained less monomeric actin than the cytoplasmic preparation from B16-F10. These results suggested that beta m-actin protein in B16 melanoma probably inhibits the dynamic conversion between the monomeric and polymerized forms of actin, leading to a decrease in cell motility and consequently the suppression of invasiveness and metastasis.

A variant actin (beta m) reduces metastasis of mouse B16 melanoma.

We recently reported an acidic actin co-expressed with beta and gamma actin in mouse B16 melanoma, whose expression was inversely correlated with the metastatic potential. The cDNA for this actin is slightly different from the hitherto recognized mouse beta actin cDNA, and we designated it beta m actin. In order to directly investigate the effects of beta m actin on metastasis, we transfected the beta m actin cDNA into a re-cloned B16-BL6 cell line which is more invasive than the highly metastatic cell line, B16-F10; we have already reported the suppressive effect of beta m actin on the invasiveness of B16-F10. Here we report on the decline in the metastatic ability of beta m-transfected cells. In the beta m-transfected B16-BL6 cell line, we observed an increase in the organization of actin stress fibers, accompanied by a decrease in metastasis to the lung, in the invasion of collagen gels, in in vivo invasiveness, and in cell migration on a glass plate covered with colloidal gold particles. We observed no correlation of beta m actin expression either with cell attachment to Matrigel, or with type-IV collagenase expression. These results suggest that beta m actin can play a role in reducing the invasiveness of mouse B16 melanoma, most probably through decreasing cell motility, which may thus result in suppression of the metastatic ability of cells.

Effect of (-)-epigallocatechin gallate, the main constituent of green tea, on lung metastasis with mouse B16 melanoma cell lines.

(-)-Epigallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, inhibits tumor promotion and chemical carcinogenesis in animal experimental systems. Here we report that the peroral administration of EGCG inhibited metastasis of B16 melanoma cell lines, such as B16-F10 and BL6, in both experimental and spontaneous systems.

Differential expression of vinculin between weakly and highly metastatic B16-melanoma cell lines.

We previously reported on the altered expression of a third actin in mouse-B16 melanoma associated with malignant progression. While further investigating the relationship of cytoskeletal proteins to malignancy, we found that the expression of vinculin was higher in weakly metastatic B16-F1 cells than in highly metastatic B16-F10 cells. By Northern blot analysis, the mRNA expression of vinculin in B16-F1 was also shown to be higher than in B16-F10. Immunofluorescence staining showed a clear dotted distribution of vinculin in B16-F1, but only a weak and diffuse distribution in B16-F10. The dotted distribution tended to be larger in B16-F1 and when cultured on Matrigel and fibronectin than on laminin and type IV collagen. An alteration in the expression of vinculin was also observed in other cell systems. Vinculin was detected in both normal 3Y1 and in relatively weakly malignant transformed 3Y1 cell lines, while vinculin was either scarcely detected or not detected at all in more malignant cell lines. These results suggest that the suppression of vinculin is closely related to malignant progression in both the B16-melanoma and 3Y1 cell systems.

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450.

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.

Newly identified type of beta actin reduces invasiveness of mouse B16-melanoma.

Low metastatic parent B16 melanoma and isolated B16-F1 cell lines have a third actin designated as beta m(Ax:previously). beta m actin is scantily or not at all detected in highly metastatic cell lines, such as B16-F10 and BL6. To directly assess the physiological role of beta m in phenotypic changes of B16 melanoma, we transfected expression plasmids of beta m into B16-F10 cells. The actin expressed in the transfectants is located largely in cytoskeletal fractions. The transfectants exhibited a larger number of stress fibers and a lower invasiveness than did the recipient cells. Thus, beta m actin plays an important role in the organization of actin stress fibers, the result being a decrease in invasiveness of B16 melanoma.

Alteration in expression of smooth muscle alpha-actin associated with transformation of rat 3Y1 cells.

Expression of actin was examined in a cultured rat embryonic cell line 3Y1 and transformed cell lines that originated from 3Y1. An alpha-actin in addition to cytoplasmic beta- and gamma-actins was detected in 3Y1 by two-dimensional gel electrophoresis. This alpha-actin was hardly detected at all in the transformants induced by Rous sarcoma virus, v-H-ras oncogene or adenovirus type 12, while the alpha-actin was retained in the transformed cell lines induced by N-methyl-N'-nitro-N-nitrosoguanidine or in SV40, which are cell lines of relatively low malignancy. Western and Northern blot analyses established that this alpha-actin was a smooth muscle alpha-isoform. An immunofluorescence study revealed that smooth muscle alpha-actin in 3Y1 cells is present in stress fibers. Thus, smooth muscle alpha-actin is also a component of actin stress fibers, as beta- and gamma-actins are in 3Y1 cells. An alteration in the expression of this actin isoform may be related to phenotypical changes accompanying transformation.

High invasiveness associated with augmentation of motility in a fos-transferred highly metastatic rat 3Y1 cell line.

We previously reported that v-fos transfer to a src-transformed rat 3Y1 cell line enhanced lung metastasis. To clarify the mechanism of this enhancement, we compared various biological factors related to metastatic potential between a fos-transferred highly metastatic cell line (fos-SR-3Y1-202) and the control cell line transferred with genetic marker (pSV2-neo) plus pBR322, neo-SR-3Y1-200. Lung arrest, the effect of lung extract on cell growth, or sensitivity to natural killer cells could not explain the higher metastasis of fos-SR-3Y1-202, compared to findings with neo-SR-3Y1-200. The invasiveness, assessed by penetration through a Matrigel-coated filter was about 5 times higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200; high invasiveness in vitro was also observed in a fos-transferred mixed-population cell line (fos-SR-3Y1-200) and fos-transferred highly metastatic clones. Histopathological evidence of an in vivo tumor also showed the high invasiveness of fos-SR-3Y1-202 cells. To elucidate the causes of the increased invasiveness of fos-SR-3Y1-202, attachment of the cells to Matrigel and its components, such as laminin and type IV collagen, type IV collagenase activity, and motility were examined. Attachment of the cells to the substrate coated on Petri dishes or the activity of type IV collagenase did not differ significantly. On the other hand, cell motility, determined by a new method to directly quantitate alteration of cell shape continuously, using video image analysis and computer techniques, was higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200. Thus, the fos-transferred cell line, fos-SR-3Y1-202 has a high invasiveness, in association with augmentation of motility, hence the enhancement of metastatic potential.

Differential expression of smooth muscle alpha-like actin between benign and malignant human pigment tissues.

When examining proteins in human pigment tissues, we found that a third actin-like protein, in addition to beta- and gamma-actin was more frequent and in a larger amount in benign tissues such as blue nevus and nevus pigmentosus, than in malignant melanoma. This third actin-like protein was immunologically stained with monoclonal antibodies reacting with several actin species and specific for smooth muscle alpha-actin. We propose that this third actin-like protein is probably smooth muscle alpha-actin and that different expressions of this third actin may possibly serve as a sensitive biochemical marker for the diagnosis of human malignant melanoma.

Altered expression of a third actin accompanying malignant progression in mouse B16 melanoma cells.

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two-dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found AX actin (Mr = 43,000, pI = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high-metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony-forming ability following iv administration, increased with increase in the F number. The half life of AX actin was much the same as that of beta- and gamma-actin and the different expressions of AX actin between the low- (F = 1) and high-metastatic (F = 10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of AX actin. The AX actin was incorporated into the cytoskeletal fraction with the same efficiency as beta- and gamma-actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in AX expression. The actin stress fibers, observed staining with rhodamine-conjugated phalloidin, were organized better in a low-metastatic cell line (F = 1) than in a high-metastatic one (F = 10). These results suggest to us that depression of AX actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.

cDNA cloning and sequence of a new type of actin in mouse B16 melanoma.

Parent B16 melanoma and B16-F1 cell lines express a third actin (Ax) in addition to beta- and gamma-actin. It has the same molecular mass (43,000 daltons) and a more acidic isoelectric point (pI = 5.2) than the latter two actins (pI = 5.3) (Taniguchi, S., Kawano, T., Kakunaga, T., and Baba, T. (1986) J. Biol. Chem. 261, 6100-6106). We constructed a cDNA library from poly(A)+ RNA of B16-F1 and then isolated Ax actin candidate clones. According to the nucleotide sequencing analysis for one of the candidate clones, pMA 30, the predicted amino acid sequence was composed of 375 amino acids and was similar to that of beta-actin, but differed at the 28th amino acid in that leucine replaced the arginine of beta-actin. When RNA synthesized from the clone pMA 30 with the SP6 transcription system was translated in vitro using reticulocyte lysate, we identified a polypeptide which had the same isoelectric point and molecular weight as Ax actin; the polypeptide had binding activity to DNase I, a common characteristic of native actin. These observations provide evidence that the clone pMA 30 encodes the mRNA for Ax actin. In the nucleotide sequence of the Ax cDNA, there are: 1) one base change in the coding region which causes a loss of the SmaI site and an amino acid exchange, as mentioned above; 2) four deletion sites in the 3'-noncoding region; 3) one insertion site in the 3'-noncoding region; and 4) one base change in the 5'-noncoding region, as compared with hitherto known mouse beta-actin cDNA. These differences between Ax and beta-actin cDNA indicate that the Ax actin is encoded by an unique gene set, independent of beta-actin.

Malignant progression of a transformed rat cell line by transfer of the v-fos oncogene.

Transfer of the v-fos oncogene into a rat cell line transformed by Rous sarcoma virus increased both the spontaneous and experimental lung-metastasis. Metastatic ability of each v-fos transferred cell line was dependent on both the manner of integration and transcriptional amount of the v-fos oncogene, but did not correlate with the growth rate in vivo. Expression of the src, myc or ras genes were not altered by transfer of the v-fos gene, except that the myc expression was enhanced in the cell line, which acquired augmentation of growth rate in vivo but not metastatic potential to the lung. Cells of the metastatic lung nodules of each cell line also possessed exogenous fos DNA and the transcripts. These results suggest that v-fos oncogene functions in the transfected cells and causes malignant progression.

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis.

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.