A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein.

Heat shock proteins of chick embryo fibroblasts were analyzed on SDS polyacrylamide gradient gels and were found to include not only three previously well-characterized proteins of 25,000, 73,000, and 89,000 D, but also a 47,000-D protein. Two-dimensional gel electrophoresis revealed that this protein was unusually basic (pI = 9.0) and corresponded to a recently characterized, major gelatin- and collagen-binding protein. The induction of synthesis of this 47,000-D membrane glycoprotein after heat stress of fibroblasts was particularly apparent in preparations isolated by gelatin-affinity chromatography. Regulation of this 47,000-D phosphoprotein was more sensitive than that of three major heat shock proteins in that a substantial stimulation of synthesis occurred at even 42 degrees C, as well as at higher temperature. Phosphorylation of the 47,000-D protein was not altered after heat shock. These studies establish this phosphorylated membrane glycoprotein as a member of the heat shock/stress protein family, and they add collagen binding to the unexpectedly diverse spectrum of biochemical activities induced by exposure of cells to stress.

pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein.

A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Double-staining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation- and heat shock-regulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.

Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection.

The molecules of the collapsin/semaphorin gene family have been thought to play an essential role in axon guidance during development. Semaphorin III/D is a member of this family, has been shown to repel dorsal root ganglion (DRG) axons in vitro, and has been implicated in the patterning of sensory afferents in the spinal cord. Although semaphorin III/D mRNA is expressed in a wide variety of neural and nonneural tissues in vivo, the role played by semaphorin III/D in regions other than the spinal cord is not known. Here, we show that mice homozygous for a targeted mutation in semaphorin III/D show severe abnormality in peripheral nerve projection. This abnormality is seen in the trigeminal, facial, vagus, accessory, and glossopharyngeal nerves but not in the oculomotor nerve. These results suggest that semaphorin III/D functions as a selective repellent in vivo.

Enhanced fibronectin receptor expression in Rous sarcoma virus-induced tumors.

The mechanisms by which cells acquire the capacity to invade interstitial connective tissues during malignancy are as yet uncertain. Since the fibronectin receptor complex has been implicated in transient, developmentally regulated steps of migration and morphogenesis in embryogenesis, we examined whether this receptor might be reexpressed at elevated levels in tumors. Immunofluorescence revealed increased expression of the receptor throughout frozen sections of Rous sarcoma virus-induced tumors in chickens, and expression was enhanced 4.7-fold after such malignant transformation of fibrocytes in vivo. Frozen thin sections showed that the increased antigen was localized diffusely in the plasma membrane. Western immunoblotting with monoclonal antibodies and immunoprecipitation analyses indicated that the tumor cell receptor contained all three known avian receptor subunits, i.e., Bands 1, 2, and 3. This type of induction of a key extracellular matrix receptor involved in cell migration may be a prerequisite for tumor cell invasion.

Involvement of transforming growth factor beta1 in autocrine enhancement of gelatinase B secretion by murine metastatic colon carcinoma cells.

We have reported previously that highly metastatic LuM1 cells derived from colon carcinoma colon 26 secrete larger amounts of gelatinase B than NM11 cells with poor metastatic potential, and that an increase in this gelatinase B secretion can be induced by autocrine factors (Hyup et A, Cancer Res., 54: 3611-3616, 1994). In the present study, a partial characterization was achieved by comparison of the autocrine factor preparation (fraction G) from serum-free medium conditioned with metastatic LuM1 cells with soluble factors known to stimulate gelatinase B secretion. Secretion of gelatinase B by LuM1 cells was augmented by tumor necrosis factor alpha, transforming growth factor beta1 (TGF-beta1), interleukin 1beta, or epidermal growth factor, and specific neutralizing antibodies abolished the induced increases. Platelet-derived growth factor and insulin-like growth factor 1 had no effect on gelatinase B secretion by LuM1 cells. The enhancement of gelatinase B secretion by fraction G was partially inhibited by the antibody to TGF-beta1. TGF-beta1 was detected in both active and latent forms in serum-free medium conditioned with LuM1 or NM11 cells, with the amount of TGF-beta1 higher in the former case. Gelatinase B secretion by LuM1 cells was enhanced by the addition of TGF-beta1 to the culture medium, but that by NM11 cells was not seriously affected, although the latter bound more of the factor. These results indicate the involvement of this growth factor in the autocrine stimulation of gelatinase B secretion by LuM1 cells. However, the autocrine factor effect was not fully explained by TGF-beta1 in the medium, and the involvement of some other unknown factor(s) was thus indicated.

Autocrine factor enhancing the secretion of M(r) 95,000 gelatinase (matrix metalloproteinase 9) in serum-free medium conditioned with murine metastatic colon carcinoma cells.

We previously reported that murine tumor cells with a high spontaneous metastatic potential to the lung secrete higher amounts of M(r) 95,000 gelatinase (matrix metalloproteinase 9, MMP9) than do poorly metastatic cells. The present study, conducted to clarify the mechanisms underlying the increase in MMP9, revealed an autocrine factor that enhances the secretion of M(r) 95,000 gelatinase (MMP9). The secretion of MMP9 by highly metastatic colon carcinoma LuM1 cells, detected by zymography, was augmented 10-fold when cultured in medium supplemented with serum-free medium conditioned with LuM1 cells. Because the secretion of M(r) 60,000 gelatinase (MMP2), as well as total protein, by the same cells was not affected under these conditions, the augmentation appears specific for MMP9. The steady-state level of MMP9 mRNA was elevated in LuM1 cells cultured in the presence of the supernatant. The amount of the factor in the culture medium increased with time in culture, indicating that it was produced by the LuM1 cells. It was found to be heat stable but sensitive to trypsin digestion. Conditioned medium from poorly metastatic NM11 cells did not stimulate the secretion of gelatinases by NM11 cells, suggesting that autocrine stimulation of MMP9 secretion is a characteristic of metastatic cells. This factor could account for the augmented secretion of MMP9 by murine tumor cells with spontaneous metastatic potential to the lung.

Ultrastructural characterization of the retrovirus particles (Sm-MTV) liberated from the mammary tumor cell line (Sm-MT) of a house musk shrew, Suncus murinus (Insectivora).

Detailed ultrastructure of a new type of retrovirus (Sm-MTV) released by cultured cells (Sm-MT) of a spontaneous mammary tumor from a house musk shrew Suncus murinus, Insectivora, is described. The virus particles were revealed as three forms: intracellular; budding; and extracellular. The intracellular type A particles were similar in profile to those associated with mouse mammary tumor cells and tended to form a small cluster of several particles in the cytoplasm. In addition, horseshoe-shaped particles as well as smaller particles in clusters, with doughnut-shaped morphology similar in structure to type A particles, were identified near the clusters of type A particles, although in smaller numbers. The budding particles contained a doughnut-shaped nucleoid, although the nucleoids decreased in size as compared with intracytoplasmic type A particles. The extracellular virions consisted of an envelope and a centrally located nucleoid. In routinely fixed specimens, the former was covered with irregularly distributed fuzzy materials in its surface, and the latter was further composed of a small electron dense core surrounded by an intermediate layer. Tannic acid treatment of cells resulted in the visualization of surface projections on the envelope of virions. Similar projections were also detected exclusively on the plasma membrane where virus budding took place, and not on the normal plasma membrane. The presence of surface projections on the viral envelope was further confirmed by the whole-cell-mounting technique. Together with our previous results of biochemical and immunological investigations, we concluded that Sm-MTV seemed to have closer phylogenetic relatedness with type D viruses of primates than with murine mammary tumor virus.

Establishment and characterization of an immature human megakaryoblastic cell line, MEG-A2.

We have established a novel human megakaryoblastic cell line, designated as MEG-A2, from a patient with megakaryoblastic crisis of Philadelphia (Ph) chromosome positive chronic myelogenous leukemia. MEG-A2 cells showed positive phenotypes for periodic acid Schiff and alpha-naphthylbutyrate esterase reactions, but were negative for myeloperoxidase and naphthol ASD chloroacetate esterase reactions. Flow cytometric analyses of cell surface markers revealed that MEG-A2 cells had a low level of GP IIb/IIIa expression as well as apparent expressions of CD4, CD7, CD13, CD33 and CD34 antigens, but no expression of GP Ib nor glycophorin A. Stimulation with phorbol 12-myristate 13-acetate (PMA) dramatically increased the expression of megakaryocyte-related markers such as HPL-3, J15, Pit-1, Y2/51 and AN51 in MEG-A2 cells. The PMA-stimulation also induced expression of platelet peroxidase (PPO) in MEG-A2 cells on electromicroscopic observation. Proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or erythropoietin were observed, and the expression of GP IIb/IIIa was increased by stimulation with GM-CSF, IL-3, erythropoietin and interleukin-6 (IL-6). Protein S mRNA expression was seen in cultured cells on Northern blot analysis. Expression of platelet factor 4 mRNA was induced in PMA-stimulated cells, and a marked accumulation of protein was observed in the culture medium. In conclusion, a new cell line, MEG-A2, belongs to the relatively immature megakaryocytic lineage and has markedly increased megakaryocytic characteristics with PMA stimulation.

AlphaB-crystallin phosphorylated at Ser-59 is localized in centrosomes and midbodies during mitosis.

We have reported that the three serine residues in alphaB-crystallin are phosphorylated under various stress conditions. We prepared affinity-purified antibodies recognizing each of the phosphorylated serine residues (Ser-19, Ser-45, and Ser-59, respectively) in alphaB-crystallin with peptides (p19S, p45S, or p59S) that contained the corresponding phosphorylated serine residue. Immunocytochemically anti-p45S antibodies stained the cytoplasm of mitotic cells (J. Biol. Chem. 273, 28,346-28,354). We have now found that the anti-p59S antibodies recognize centrosomes and midbodies of dividing cells. alphaB-Crystallin was the only protein recognized by the anti-p59S antibodies in Western blot analyses of isolated centrosome fractions. alphaB-Crystallin phosphorylated at Ser-59 was localized at the microtubule organizing centers by means of double staining with anti-beta-tubulin antibody in aster formation analysis and was co-localized with gamma-tubulin in centrosomes. Gamma-Tubulin was co-immunoprecipitated with alphaB-crystallin in U373 glioma cell extracts. On the other hand, the location of the phosphorylated alphaB-crystallin deviated from that of alpha-tubulin or gamma-tubulin in the midbody region. Taken together with the evidences that several chaperones are distributed to centrosomes, these results suggest that alphaB-crystallin as a chaperone might be also involved in the quality control of proteins.

Conformational changes in mutant lysozymes detected with monoclonal antibody.

A monoclonal antibody (mAb) against hen egg white lysozyme (HEWL) with the exquisitely sensitive specificity to native conformation was prepared to detect the conformational changes in mutant lysozymes constructed by genetic modification in a yeast expression system. The binding of mAb with lysozyme was decreased both by denaturation with heat and guanidine-HCl, corresponding to the denaturation curves of lysozyme. These results demonstrate that mAb is a powerful probe to monitor the conformational changes in the lysozyme molecule. By using this probe, the conformational change of various mutant lysozymes was detected. A good correlation was observed between the binding with mAb and the delta G (Gibbs free energy change), reflecting the conformational stability of wild-type and seven mutant lysozymes. This result suggests that a monoclonal antibody with the specificity for native conformation can be used as a powerful probe of protein conformation.

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts.

Changes in the expression of alpha and beta subunits of proline 4-hydroxylase (PH alpha and PH beta) and HSP47, implicated as a molecular chaperone specific for procollagen processing, were examined in human embryonal fibroblasts in relation to in vitro aging. For this purpose a model with treatments causing the decreased hydroxylation of proline residues in procollagens was used. In cells at a low population doubling level (PDL) induction of PH alpha, PH beta, and HSP47 by depletion of ascorbate or addition of alpha-alpha' dipyridyl could be clearly demonstrated by immunoprecipitation and Northern blotting. In contrast, the induction of PH alpha and HSP47 expression was markedly attenuated in high PDL cells, indicating an age-related decrease in response to procollagen retention in the ER caused by hypohydroxylation of proline residues of procollagens.

Age-related attenuation of HSP47 heat response in fibroblasts.

The collagen-binding heat shock protein of molecular weight 47,000 (HSP47), resident in the endoplasmic reticulum (ER), is assumed to play a specific role as a molecular chaperon in the processing of procollagen molecules. The present investigation of age-related alteration in the HSP47 heat response in cultured murine and human fibroblasts revealed expression in cells with a low population doubling level (PDL) derived from young mice and people more inducible by heat treatment than those from older mice and people. On the other hand, cells with a high PDL showed a very low heat response in terms of HSP47 expression regardless of the donor age. Northern blot analysis of HSP47 m-RNA indicated that the age related attenuation of HSP47 expression was regulated by transcriptional mechanisms. Furthermore, immunofluorescent analysis using a monoclonal antibody against the carboxylterminal propeptide of type I procollagen revealed far greater retention of procollagen molecules in the ER lumen of cells from old persons than in those from young persons. This was particularly prominent in heat-treated cells from old persons, indicating the possibility that the observed decrease in HSP47 heat response might cause blockage of procollagen transport to the Golgi and therefore secretion.

Inhibition of complement-mediated immune hemolysis by peptides derived from the constant domain of immunoglobulin.

High-dose administration of intravenous immunoglobulin is reported to be useful for inhibiting complement-dependent immune cytolysis. We have found that, among the proposed C1q-binding sites of the Fc portion of human IgG1, only residues 282-292 inhibited pig red blood cell lysis by human serum. Moreover, a hexadecemeric multiple antigen peptide of residues 282-292 from IgG showed significantly greater activity in suppressing complement-mediated immune cytolysis and can be used in place of high-dose intravenous immunoglobulin, which is extracted from donors and thus is expensive.

Distribution of the collagen binding heat-shock protein in chicken tissues.

We examined the tissue distribution of heat-shock protein MW 47,000 D, hsp47, which binds to native and denatured collagen including Types I, III, and IV, in various chicken tissues by Western blotting and immunohistochemical methods. hsp47 was located on fibrocytes or fibroblasts in the connective tissue in various organs, chondrocytes in the cartilage, smooth muscle cells in the gastrointestinal tract and blood vessels, vitamin A storage cells in sinusoidal area of liver, endothelial cells in blood vessels, and epithelial cells of renal glomeruli, tubules, and basal layer of epidermis. These cells also co-expressed a certain type of collagen molecule. Furthermore, in developing embryos, fibroblasts and chondrocytes expressed hsp47 before the deposition of collagen Type I or Type II in the surrounding tissue. These results indicate that the binding of hsp47 to collagen molecules has important biological significance.

Distinct distribution of protein disulfide isomerase family proteins in rat tissues.

We investigated the distribution of protein disulfide isomerase (PDI) family proteins PDI, ERp72, and ERp61 in rat tissues and compared their localization by immunohistochemical double staining. The extent of their expression was diverse in different cell types, although they were ubiquitously distributed in a wide variety of cell types. Prominent staining for all three proteins was detected in thyroid follicular epithelia, tracheal mucous gland cells and chondrocytes, and chief cells and mucous neck cells of the stomach. Hepatocytes, neuronal cells in brain and spinal cord, and pancreatic acinar cells were also consistently and uniformly stained, but with low intensity. On the other hand, distinct differences were observed for the expression of the three proteins in plasma cells, pancreatic islet cells, goblet cells, and Paneth's cells of intestines, seminiferous epithelia, and salivary gland ductal epithelia. The similarities and differences in the distribution of the three proteins provide helpful clues to their biological functions.

Simple sandwich enzyme immunoassay for quantification of mouse mammary tumor virus in mouse milk.

We have developed a sandwich enzyme immunoassay in order to measure quantitatively mouse mammary tumor virus (MMTV) in mouse milk. In this assay, the antibody-beta-D-galactosidase complex and antibody-bound silicon rubber pieces pieces as solid phase are used. The assay is able to detect 10 ng/ml of MMTV in the milk sample.

Genetic resistance to mammary tumorigenesis in a mouse strain with high murine mammary tumor virus expression.

Although II-TES mice release large amounts of murine mammary tumor virus (MMTV) in milk, they are resistant to mammary tumorigenesis. High mammary tumor incidence was observed in (BALB/ca X II-TES)F1 and (C57BL/6N X II-TES)F1, whereas no mammary tumors developed in BALB/ca X OZ-F)F1. Mammary tumors developed in 68% of (OZ-F X (OZ-F X II-TES)F1 and 45% of (II-TES X (OZ-F X II-TES)F1). These results suggest that the II-TES mouse carries a recessive gene for mammary tumor resistance which does not inhibit MMTV release, and two independent dominant mammary tumor promoting genes which are inhibited by the resistant gene.

Matrix metalloproteinase 2 and 9 in esophageal cancer.

To search for the biochemical properties of esophageal carcinoma relevant to its aggressive behavior, we studied metalloproteinases released from surgical specimens of the carcinoma. In an assay with [H-3]-labeled gelatin, esophageal carcinoma tissues showed gelatinolytic activities clearly higher than those of paired normal mucosae. EDTA and TIMP-1 could strongly suppress these activities, suggesting that the activities belong to metalloproteinases. In addition, levels of TIMP-1 expression did not show good correlation with these activities, suggesting that tumor-specific elevation of gelatinolytic activity depended on metalloproteinase per se rather than the suppression of TIMP-1-secretion. By zymographic analysis, two gelatinase bands of 82- and 62-kDa were found specifically in carcinoma tissues, in addition to the other 6 bands detected both in normal and carcinoma tissues. Immunoprecipitation and immunoblotting of gelatinases with anti-MMP-9 or anti-MMP-2 monoclonal antibody, and treatment of the enzymes with APMA showed that these 82- and 62-kDa gelatinases were cleaved products of MMP-9 and MMP-2, respectively. These results imply that enhanced secretion and proteolytic activation of MMP-2 and MMP-9 take place specifically in the esophageal carcinoma tissues. Moreover, the levels of gelatinolytic activity expressed good correlation with the organ metastasis rate of the carcinoma, suggesting that MMPs play an important role in tumor metastasis.

Oral adsorbent ameliorates renal TGF-beta 1 expression in hypercholesterolemic rats.

BACKGROUND: A spontaneously hypercholesterolemic Imai rat has recently been reported as a model of focal glomerulosclerosis that causes nephrotic syndrome followed by renal failure. This study was designed to determine if an oral adsorbent, AST-120, ameliorates renal lesions and TGF-beta 1 expression in the rats. METHODS: AST-120 was given orally to the Imai rats for 32 weeks, and renal function and pathology were compared between the AST-120-administered and control Imai rats. RESULTS: AST-120-administered rats showed significantly lower level of blood urea nitrogen, serum creatinine, urinary protein, serum total-cholesterol, serum triglyceride, and serum and urinary indoxyl sulfate, and significantly higher levels of serum albumin and creatinine clearance than control rats. AST-120 reduced the glomerular sclerosis index, interstitial fibrosis area, and the extent of glomerular lipid deposition. Immunohistochemistry demonstrated that AST-120 reduced the expression of transforming growth factor (TGF)-beta 1 and tissue inhibitor of metalloproteinase (TIMP)-1 as well as interstitial infiltration of macrophages in the renal cortex of the Imai rats. CONCLUSIONS: AST-120 prevented the progression of nephrotic syndrome and renal failure in the Imai rats by ameliorating glomerular sclerosis and interstitial fibrosis, accompanied with reduced expression of TGF-beta 1 and TIMP-1, and reduced infiltration of macrophages in the kidneys.

A synthetic peptide of human apoprotein E with antibacterial activity.

In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicalpha-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133-162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133-162 revealed close similarities to CAP18.