Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners.

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.

Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action.

The present study reports the developmental patterns of expression of adrenomedullin (AM) in rat and mouse embryos. AM is a novel multifunctional peptide recently isolated from a human pheochromocytoma, which has been shown to promote growth in a variety of mammalian cell lines. We have applied several techniques to investigate the localization of both the AM peptide and its receptor throughout development. Immunocytochemical detection has been performed using different specific antibodies against AM and its gene-related peptide pro-AM N-terminal 20 peptide. In situ hybridization showed the localization of the messenger RNAs for AM and its receptor. Western blot analysis together with reverse transcription-PCR gave further support to the localization of AM and its receptor in a variety of embryonic tissues. The localization of the receptor paralleled that of AM itself, suggesting an autocrine or paracrine mode of action. The spatio-temporal pattern of expression of AM in cardiovascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs is described. The primitive placenta, especially the giant trophoblastic cells, shows high levels of AM and AM receptor. The heart is the first organ that expresses AM during development. The kidney, lung, and developing tooth, in which epithelial-mesenchymal interactions are taking place, show specific patterns of AM expression. In several regions of the embryo, the patterns of AM expression correspond to the degree of differentiation. The possible involvement of AM in the control of embryonic invasion, proliferation, and differentiation is discussed.

Adrenomedullin expression in human tumor cell lines. Its potential role as an autocrine growth factor.

Although adrenomedullin (AM) previously has been identified in human tumors, its role has remained elusive. Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed AM mRNA in 18 of 20 human normal tissues representing major organs, and 55 of 58 (95%) malignant cell lines. Western blot and high performance liquid chromatography analysis showed immunoreactive AM species of 18, 14, and 6 kDa that are consistent with the precursor, intermediate product, and active peptide, respectively. Immunohistochemistry and in situ RT-PCR performed on paraffin-embedded tumor cell lines of various tissue origins exhibited AM cytoplasmic staining. Neutralizing monoclonal antibody to AM inhibits tumor cell growth in a concentration-dependent manner, an effect that was reversed with the addition of exogenous AM. Responding tumor cells were shown to have approximately 50,000 AM receptors per cell by Scatchard analysis with 125I-AM and expressed AM receptor mRNA by RT-PCR. Our data showed 36 of 48 (75%) tumor cell lines expressed AM receptor mRNA by RT-PCR assessment, all of them also expressed AM. In the presence of AM, cAMP levels were shown to increase in tumor cells. Our collective data demonstrate that AM and AM receptor are expressed in numerous human cancer cell lines of diverse origin and constitute a potential autocrine growth mechanism that could drive neoplastic proliferation.

Regulation of insulin secretion and blood glucose metabolism by adrenomedullin.

Adrenomedullin (AM), a recently discovered hypotensive peptide, is expressed in the endocrine pancreas of different species, as demonstrated by immunocytochemistry. Electron microscopic studies with double immunogold showed colocalization of AM and pancreatic polypeptide. A homogeneous expression of AM receptor was found throughout the islet using in situ hybridization. Six different insulin- producing cell lines have been analyzed by reverse transcription-PCR and showed expression of both AM and its receptor. Two experimental models have been used to study the effects of AM in pancreatic physiology. 1) Analysis of isolated rat islets shows that AM inhibits insulin secretion in a dose-dependent manner. The monoclonal antibody MoAb-G6, which neutralizes AM bioactivity, was able to increase insulin release 5-fold; this effect was reversed by the addition of synthetic AM. 2) Oral glucose tolerance tests showed that iv injection of AM reduces the levels of insulin in the bloodstream with a concomitant increase in circulating glucose. These studies implicate AM as a newly defined factor of the insulin regulatory system that could be involved in disorders such as diabetes and obesity.

Insulin-like growth factor expression in human cancer cell lines.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis.

Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4.

We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.

Adrenomedullin-like immunoreactivity in the nervous system of the starfish Marthasterias glacialis.

The nervous system of the starfish Marthasterias glacialis was investigated immunocytochemically using an antiserum specific for adrenomedullin (AM), a new regulatory peptide. Immunoreactivity was only found in nerves of the basiepithelial plexus of cardiac and pyloric stomachs and pyloric caeca, while the radial nerve cords and the other digestive organs were negative. The strongest AM-like immunoreactivity was located in the current-producing areas of the cardiac stomach. The distribution of this peptide suggests different functions in echinoderms involving regulation of muscle movement and neurotransmission. The presence of an AM-like substance in echinoderms points to an early phylogenetic origin for this regulatory system.

Expression of adrenomedullin in normal human lung and in pulmonary tumors.

Adrenomedullin (AM) is a potent hypotensive peptide recently discovered in extracts of human pheochromocytoma. In this report we present evidence, using reverse transcriptase-polymerase chain reaction, immunocytochemistry, and in situ reverse transcriptase-polymerase chain reaction, that AM is synthesized by several cell populations of the normal lung, tumor cell lines of pulmonary origin, and tumor specimens. Among the normal cell populations of the lung, we found AM expression in the columnar epithelium, some glands, neurons of the pulmonary parasympathetic nervous system, endothelial cells, chondrocytes, alveolar macrophages, and smooth muscle cells. In tumors, AM expression was located in most of the nonsmall cell lung carcinomas and in half of the small cell lung carcinomas studied. These findings suggest that AM may play a broad role in respiratory homeostasis and lung carcinogenesis.

Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections.

Technological developments have made possible extension of polymerase chain reaction (PCR) analysis to individual cells to localize DNA/RNA with non-radioactive labels at the light microscopic level. This approach, in situ PCR, is particularly useful in resolving low-frequency message expression in mixed populations of cells and tissues. We have established a working protocol for direct in situ PCR and have utilized several controls to validate our results. In this report we outline the procedures for detecting either DNA or RNA in a rapid and reproducible manner. We evaluate the sequential steps required for this analysis, such as protease hydrolysis, DNAse digestion, "hot start" capabilities, and detection methods. We have applied these methods in several applications, including detection of the p53 gene in human tumor samples, localization of insulin-like growth factor-IA mRNA in cell lines with low levels of expression, and distribution of transferrin mRNA in lung cancer cell lines and tumors. We demonstrate from this study that the in situ PCR technique is an investigative approach capable of detecting specific DNA/RNA sequences at the cellular level and of identifying cells with low levels of mRNA expression.

Analysis of small cell lung cancer cell growth inhibition by 13-cis-retinoic acid: importance of bioavailability.

13-cis-Retinoic acid can mediate differentiation of transformed cells and slow the proliferation of malignant cells, suggesting its use as a potential intervention tool. Specific cDNA probes for retinoic acid receptors demonstrated the expression of mRNAs for the different retinoic acid receptor isoforms in small cell lung cancer cell lines. Addition of 13-cis-retinoic acid to small cell lung cancer cells cultured using serum-free, hormonally defined medium resulted in a 5-8-fold increase in the level of the retinoic acid receptor-beta mRNAs; in medium containing serum, the increase in expression of the retinoic acid receptor-beta mRNAs was less pronounced, usually no more than 2-fold. Using an in vitro proliferation assay, addition of 13-cis-retinoic acid resulted in a significant dose-dependent, growth-inhibitory effect on the small cell lung cancer cell lines tested using serum-free conditions. These inhibitory effects decreased when cells were cultured in medium containing serum or serum components. Molecular size exclusion chromatography and native gel electrophoresis showed that the causative serum component eluted and migrated with serum albumin. Preincubating serum with triglycerides restored the inhibitory effects of 13-cis-retinoic acid demonstrated in serum-free systems. These data suggest that 13-cis-retinoic acid preferentially binds to serum albumin, restricting its inhibitory effects on epithelial cell receptors. Blocking retinoic acid-albumin interactions with a fatty acid source may improve the bioavailability of 13-cis-retinoic acid and significantly enhance the inhibitory effect in vivo.

Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation.

Tissue inhibitor of metalloproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of 3H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 +/- 12 nM (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture plastic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo.

Cellular activation of the 72 kDa type IV procollagenase/TIMP-2 complex.

Members of the collagenase family of enzymes have been implicated as central mediators of a number of both physiologic and pathologic processes. The 72-kDa type IV collagenase is secreted as a latent proenzyme, complexed with tissue inhibitor of metalloproteinase-2 (TIMP-2). Like other members of the collagenase family, this enzyme complex must be converted to a catalytically active form for proteolytic remodeling of extracellular matrix to occur. In the current study we demonstrate an inducible cell-mediated activation of the 72-kDa type IV procollagenase/TIMP-2 complex. Isolation of the 62 kDa activated enzyme/TIMP-2 complex from conditioned media of concanavalin A treated WI-38 fibroblasts demonstrated that the cell activated species was proteolytically active and amino terminal sequencing gave the sequence YNFF. This is identical to that of the 62 kDa species generated following organomercurial activation of purified 72-kDa type IV procollagenase/TIMP-2 complex. We have also isolated biosynthetically 35S-labeled 72-kDa type IV procollagenase/TIMP-2 complex and used this to further study the cellular activation process. In cell lines tested the activator was retained in the residual cell fraction following lysis in the presence of 0.2% (wt/vol) Brij-35. Inhibitor studies demonstrated that processing and activation of 72-kDa type IV procollagenase/TIMP-2 complex by the residual fraction was inhibited by 5 mM ethylenediaminetetraacetic acid and 0.5 mM 1,10-phenanthroline demonstrating a metal atom dependence. The species responsible for activation could be partially recovered in soluble form with 0.5% (vol/vol) Triton X-100 and 0.25% (wt/vol) CHAPS but was not salt extractable.(ABSTRACT TRUNCATED AT 250 WORDS)

Higher-order complex formation between the 72-kilodalton type IV collagenase and tissue inhibitor of metalloproteinases-2.

The collagenases are a class of matrix degradative enzymes whose actions are important in physiological and pathological processes. The human 72-kDa type IV collagenase (matrix metalloproteinase-2) and its proteinase inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2), are produced as a proenzyme-inhibitor complex by numerous cell lines. We analyzed the quaternary structure of and enzyme-inhibitor interactions in the native enzyme-inhibitor complex by studying the pattern of complexes demonstrated by molecular weight determination in nondenaturing polyacrylamide gels and evaluating the products formed by reaction of the native complexes with cross-linking agents. Electrophoresis in native polyacrylamide gels demonstrates that approximately 79% of the latent enzyme is present in a 1:1 bimolecular complex with the inhibitor TIMP-2, with 21% present as a complete tetrameric complex of two molecules of collagenase combined with two molecules of TIMP-2. The enzyme complex activated with organomercurials displays a shift to a higher proportion of the bimolecular complex with only 5% present as higher molecular weight complexes. Cross-linking of the latent and active forms of the complex with bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) tartarate demonstrates both the 1:1 and 2:2 complexes as well as an intermediate form that appears to be a complex composed of two molecules of collagenase and one of TIMP-2. The distribution of cross-linked products is unchanged with the addition of excess TIMP-2 to the reaction mix, implying that the binding sites for TIMP-2 to the initial enzyme-inhibitor complex are all occupied when the stoichiometry is 1 to 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.