Correlation between RsaI restriction fragment length polymorphism and electrophoretic types of human placental alkaline phosphatase

Restriction fragment length polymorphism (RFLP) of human alkaline phosphatases was studied in a population sample from northern Sweden using a placental alkaline phosphatase (PLAP) cDNA probe. After digestion of human genomic DNA with RsaI the Southern blots showed DNA fragments most probably derived from three genes: PLAP, germ cell alkaline phosphatase (PLAP-like) and intestinal alkaline phosphatase. In agreement with a previous study, a two-allele polymorphism was found in PLAP with bands at 1.6 kilobases (A1) and 1.8 kilobases (A2). The gene frequencies of A1 and A2 were 0.46 and 0.54, respectively. There was a significant correlation between the RsaI RFLPs and electrophoretic types of PLAP; RSAI A2 showed an association with the ALP2p allele of PLAP.     

Phorbol ester-induced shedding of the prostate cancer marker transmembrane protein with epidermal growth factor and two follistatin motifs 2 is mediated by the disintegrin and metalloproteinase-17

The transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in prostate and brain and shed from the cell surface in a metalloproteinase-dependent fashion. Neither the sheddase(s) responsible for TMEFF2 shedding nor the physiological significance or activity of the soluble TMEFF2 ectodomain (TMEFF2-ECD) has been identified. In the present study we present new evidence that a disintegrin and metalloproteinase-17 (ADAM17) is responsible for phorbol 12-myristate 13-acetate-induced release of TMEFF2-ECD using small interfering RNA to ablate ADAM17 expression or by inhibiting enzymatic activity. A single well shedding assay monitoring the release of alkaline phosphatase-tagged TMEFF2-ECD into medium and the generation of 22- and 14-kDa C-terminal fragments in lysates were dependent on ADAM17 activity. A gamma-secretase inhibitor prevented the formation of a 10-kDa fragment in cell lysates, thus establishing TMEFF2 as a novel substrate for regulated intramembrane proteolysis. We assigned proliferation-inducing activity to TMEFF2. Inhibition of TMEFF2 shedding using synthetic metalloproteinase inhibitors or small interfering RNA targeting TMEFF2 expression yielded a statistically significant reduction of cell proliferation in the lymph node-derived prostate cancer cells (LNCaPs) and a human embryonic kidney (HEK293) cell line overexpressing TMEFF2. The TMEFF2-ECD was able to induce ERK1/2 phosphorylation in an epidermal growth factor receptor (or ErbB1)-dependent manner in HEK293 cells. Our data suggest that TMEFF2 contributes to cell proliferation in an ADAM17-dependent autocrine fashion in cells expressing this protein.     

The TMEFF2 tumor suppressor modulates integrin expression,RhoA activation and migration of prostate cancer cells

Cell adhesion and migration play important roles in physiological and pathological states, including embryonic development and cancer invasion and metastasis. The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate and its expression is deregulated in prostate cancer. We have previously shown that TMEFF2 can function as a tumor suppressor by inhibiting cell migration and invasion of prostate cells. However, the molecular mechanisms involved in this inhibition are not clear. In this study we demonstrate that TMEFF2 affects cell adhesion and migration of prostate cancer cells and that this effect correlates with changes in integrin expression and RhoA activation. Deletion of a 13 basic-rich amino acid region in the cytoplasmic domain of TMEFF2 prevented these effects. Overexpression of TMEFF2 reduced cell attachment and migration on vitronectin and caused a concomitant decrease in RhoA activation, stress fiber formation and expression of αv, β1 and β3 integrin subunits. Conversely, TMEFF2 interference in 22Rv1 prostate cancer cells resulted in an increased integrin expression. Results obtained with a double TRAMP/TMEFF2 transgenic mouse also indicated that TMEFF2 expression reduced integrin expression in the mouse prostate. In summary, the data presented here indicate an important role of TMEFF2 in regulating cell adhesion and migration that involves integrin signaling and is mediated by its cytoplasmic domain.     

TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer

TMEFF2 is a type I transmembrane protein with two follistatin (FS) and one EGF‐like domain over‐expressed in prostate cancer; however its biological role in prostate cancer development and progression remains unclear, which may, at least in part, be explained by its proteolytic processing. The extracellular part of TMEFF2 (TMEFF2‐ECD) is cleaved by ADAM17 and the membrane‐retained fragment is further processed by the gamma‐secretase complex. TMEFF2 shedding is increased with cell crowding, a condition associated with the tumour microenvironment, which was mediated by oxidative stress signalling, requiring jun‐kinase (JNK) activation. Moreover, we have identified that TMEFF2 is also a novel substrate for other proteases implicated in prostate cancer, including two ADAMs (ADAM9 and ADAM12) and the type II transmembrane serine proteinases (TTSPs) matriptase‐1 and hepsin. Whereas cleavage by ADAM9 and ADAM12 generates previously identified TMEFF2‐ECD, proteolytic processing by matriptase‐1 and hepsin produced TMEFF2 fragments, composed of TMEFF2‐ECD or FS and/or EGF‐like domains as well as novel membrane retained fragments. Differential TMEFF2 processing from a single transmembrane protein may be a general mechanism to modulate transmembrane protein levels and domains, dependent on the repertoire of ADAMs or TTSPs expressed by the target cell.     

The tumor suppressor activity of the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) correlates with its ability to modulate sarcosine levels

The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in brain and prostate and overexpressed in prostate cancer, but its role in this disease is unclear. Several studies have suggested that TMEFF2 plays a role in suppressing the growth and invasive potential of human cancer cells, whereas others suggest that the shed portion of TMEFF2, which lacks the cytoplasmic region, has a growth-promoting activity. Here we show that TMEFF2 has a dual mode of action. Ectopic expression of wild-type full-length TMEFF2 inhibits soft agar colony formation, cellular invasion, and migration and increases cellular sensitivity to apoptosis. However, expression of the ectodomain portion of TMEFF2 increases cell proliferation. Using affinity chromatography and mass spectrometry, we identify sarcosine dehydrogenase (SARDH), the enzyme that converts sarcosine to glycine, as a TMEFF2-interacting protein. Co-immunoprecipitation and immunofluorescence analysis confirms the interaction of SARDH with full-length TMEFF2. The ectodomain does not bind to SARDH. Moreover, expression of the full-length TMEFF2 but not the ectodomain results in a decreased level of sarcosine in the cells. These results suggest that the tumor suppressor activity of TMEFF2 requires the cytoplasmic/transmembrane portion of the protein and correlates with its ability to bind to SARDH and to modulate the level of sarcosine.     

TMEFF2 modulates the AKT and ERK signaling pathways

The transmembrane protein with epidermal growth factor (EGF) and two follistatin (FS) motifs 2 (TMEFF2) has a limited tissue distribution with strong expression only in brain and prostate. While TMEFF2 is overexpressed in prostate cancer indicating an oncogenic role, several studies indicate a tumor suppressor role for this protein. This dual mode of action is, at least in part, the result of metalloproteinase-dependent shedding that generates a soluble TMEFF2 ectodomain with a growth promoting function. While recent studies have shed some light on the biology of different forms of TMEFF2, little is known about the molecular mechanisms that influence its oncogenic/tumor suppressive function. In several non-prostate cell lines, it has been shown that a recombinant form of the TMEFF2 ectodomain can interact with platelet derived growth factor (PDGF)-AA to suppress PDGF receptor signaling and can promote ErbB4 and ERK1/2 phosphorylation. However, the role of the full length TMEFF2 in these pathways has not been examined. Using prostate cell lines, here we examine the role of TMEFF2 in ERK and Akt activation, two pathways implicated in prostate cancer progression and that have been shown to cross talk in several cancers. Our results show that different forms of TMEFF2 distinctly affect Akt and ERK activation and this may contribute to a different cellular response of either proliferation or tumor suppression.     

A difference in the regulation of mRNA expression between the phenotypic and the embryonic alkaline phosphatase genes in human cancer cells

The steady-state levels of mRNAs encoding alkaline phosphatase isoenzymes were examined in two human breast carcinoma cell lines. MDA-MB-157 cells expressed the phenotypic breast alkaline phosphatase and BT20 cells expressed the nonphenotypic placental alkaline phosphatase isoenzyme, frequently reexpressed in neoplasms. Dexamethasone (DEX), which elicits a general effect on phosphatase expression, and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), a promoter of cell differentiation that correspondingly effects embryonic phosphatase expression, were chosen as perturbing agents for these experiments. RNA blot analysis showed a single RNA species of approximately 2.6 kb under all treatment conditions in BT20 cells and a single RNA species of 2.6 kb under each condition in MDA-MB-157 cells. The results showed that the expression of both the AP isoenzyme mRNA phenotypic of breast produced by MDA-MB-157 cells and the embryonic alkaline phosphatase isoenzyme (PLAP) mRNA produced by BT20 cells was increased by treatment with DEX. By comparison 1,25(OH)2D3 caused an increase in the tissue-unspecific AP mRNA in the MDA-MB-157 cells, but caused a decrease in PLAP mRNA levels in BT20 cells. The level of each isoenzyme mRNA species is altered by either hormone in a dose- and time-dependent manner in both cell lines. In BT20 cells, treatment with cycloheximide showed that ongoing protein synthesis is not required to potentiate the PLAP mRNA response to DEX, but is required for the action of 1,25(OH)2D3. However, protein synthesis is required for the action of both hormones in the MDA-MB-157 cells which make the breast phenotypic AP. These data demonstrate that the DEX- and 1,25(OH)2D3-regulated expression of both of these alkaline phosphatase isoenzymes occurs via a complex mechanism involving control of mRNA abundance, not translational control of constant message levels.     

Effect of sodium butyrate on the expression of genes transduced by retroviral vectors

We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial β-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2–8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the β-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR. J. Cell. Biochem. 69:201–210, 1998. © 1998 Wiley-Liss, Inc.     

Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

A tryptophan hydroxlyase 1 reporter that directs Cre recombinase extinguishable placental alkaline phosphatase expression in serotonergic (5-HT) neurons and peripheral tissues

The serotonergic (5-HT) system modulates many behaviors and has been implicated in psychiatric disorders, but the density of 5-HT processes has complicated analyses. We have used regulatory regions from the Tryptophan hydroxylase 1 (Tph1) gene to drive expression of LoxP-flanked placental alkaline phosphatase (PLAP) to generate the Tph1-Lox-PLAP reporter mouse line. In these mice, PLAP is expressed in the hindbrain raphe nuclei and in peripheral tissues known to express Tph1. Tph1 is expressed at low levels in neurons. While, in Tph1-Lox-PLAP mice, most PLAP-expressing neurons are monoaminergic, PLAP was expressed in only 5-10% of neurons expressing the predominant neuronal 5-HT biosynthetic enzyme Tph2, serotonin transporter (SERT) or aromatic amino acid decarboxylase (AADC). To test this reporter further, we examined the brains of mice carrying the anorexia (anx) mutation, in which increased overall density of 5-HT immunoreactivity had been previously observed at P21. PLAP-labeling of processes in anx/anx and anx/+ mice was reduced at P0. By P10, distribution of PLAP-labeled processes in anx/+ and +/+ cortices was indistinguishable, but differed markedly from that seen in the cortical layers of anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter revealed a dosage sensitive role of the anx mutation in the early 5-HT system and later cortical layer-specific differences in 5-HT process distribution in anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter provides a sensitive indicator for analyses of serotonergic cells in the brain and periphery.     

Expression pattern of the orphan receptor LGR4/GPR48 gene in the mouse

Leucine-rich G-protein-coupled Receptors (LGR) constitute a subfamily of receptors related to glycoprotein hormone receptors. Amongst them, LGR4, LGR5 and LGR6 form a cluster for which natural agonists are still unknown. By an extensive gene trapping approach, Leighton et al. (2001) obtained a mouse line in which the LGR4 gene is disrupted by a trap vector carrying two biological markers, beta-geo (a fusion between bacterial beta-galactosidase and neomycin phosphotransferase) and a placental alkaline phosphatase (PLAP). Due to perinatal lethality, characterization of adult mice homozygous for this insertion has been impaired. In the present study we have investigated LacZ and PLAP activity patterns in heterozygous mice as a marker for LGR4 natural expression at both macroscopic and histological levels. We present a detailed atlas of LGR4 expression, which displays very wide expression with particularly strong activity in cartilages, kidneys, reproductive tracts and nervous system cells.     

Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells. Electron microscopic observations using gold-labeled antibodies

The cell-surface distribution of human placental alkaline phosphatase (PLAP) on cultured cancer cells, A431 and HeLa TCRC-1, and on normal syncytial cells of placental tissue was examined in immunoelectron transmission microscopy using the gold-labeling technique. Chemically fixed cells were reacted with affinity-purified rabbit polyclonal antibodies to PLAP, and the antibodies were visualized using gold particles tagged with goat antirabbit IgG. On all cells PLAP was observed in clusters distributed throughout the membrane surface, including microvilli, but it was not expressed in desmosomes or along other dense regions on the membrane. Previous histochemical and immunochemical techniques failed to demonstrate clusters. The results show that (1) the gold-labeling technique allows a more precise localization of PLAP on the cell surface than previously employed methods, and (2) the distribution of the enzyme is the same on cultured cancer cells and on normal placental syncytial cells. The clustered distribution of PLAP is thus a general phenomenon and is probably influenced by the physiological function of the enzyme, which has yet to be defined.     

DNA polymorphism of alkaline phosphatase isozyme genes: linkage disequilibria between placental and germ-cell alkaline phosphatase alleles.

The use of human placental alkaline phosphatase (PLAP) cDNA as a probe allows the detection and identification of restriction DNA fragments derived from three homologous genes, i.e., intestinal alkaline phosphatase (AP), germ-cell AP (GCAP), and PLAP. In previous RFLP studies we have reported linkage disequilibria between an RsaI and two PstI (a and b) polymorphic restriction sites and electrophoretic types of PLAP. In this report we present evidence that, in spite of the strong correlation with PLAP types, PstI(b) is an RFLP of GCAP. The data indicate close linkage between the PLAP and GCAP loci.     

The mechanism of placental alkaline phosphatase induction in vitro

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG-IIIa was investigated in vitro by enzyme-cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme-cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat-stable ALP) in prednisolone-treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene. These findings suggest that the induction of PLAP mRNA in SKG-IIIa cells by prednisolone in turn increased the levels of PLAP.     

Interethnic variability in birth weight and genetic background: A study of placental alkaline phosphatase

The relationship between human placental alkaline phosphatase (PLAP) genotype and birth weight is investigated in a sample of white, black and Puerto-Rican new-born infants from New Haven, Connecticut (total 710 subjects). Black and Puerto-Rican infants show a higher incidence of growth retardation and a higher frequency of ALPp^*1/^*1 genotype as compared to whites. The proportion of newborns with a low birth weight (below the 10th percentile) is lower in infants with ALPp^*1/^*1 genotype than in those with other PLAP genotypes, especially among non-whites. It is argued that the higher frequency of ALPp^*1 allele among non-whites might be, at least in part, a consequence of their adaptation in the past to environmental conditions adverse to optimal intrauterine development. © 1996 Wiley-Liss, Inc.     

Expression of Plxdc2/TEM7R in the developing nervous system of the mouse

Plexin-domain containing 2 (Plxdc2) is a relatively uncharacterised transmembrane protein with an area of nidogen homology and a plexin repeat (PSI domain) in its extracellular region. Here, we describe Plxdc2 expression in the embryonic mouse, with particular emphasis on the developing central nervous system. Using light microscopy and optical projection tomography (OPT), we analyse RNA in situ hybridization patterns and expression of two reporter genes, beta-geo (a fusion of beta-galactosidase to neomycin phosphotransferase) and placental alkaline phosphatase (PLAP) in a Plxdc2 gene trap mouse line (KST37; [Leighton, P.A., Mitchell, K.J., Goodrich, L.V., Lu, X., Pinson, K., Scherz, P., Skarnes, W.C., Tessier-Lavigne, M., 2001. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174-179]). At mid-embryonic stages (E9.5-E11.5) Plxdc2-betageo expression is prominent in a number of patterning centres of the brain, including the cortical hem, midbrain-hindbrain boundary and the midbrain floorplate. Plxdc2 is expressed in other tissues, most notably the limbs, lung buds and developing heart, as well as the spinal cord and dorsal root ganglia. At E15.5, expression is apparent in a large number of discrete nuclei and structures throughout the brain, including the glial wedge and derivatives of the cortical hem. Plxdc2-betageo expression is particularly strong in the developing Purkinje cell layer, especially in the posterior half of the cerebellum. The PLAP marker is expressed in a number of axonal tracts, including the posterior commissure, mammillotegmental tract and cerebellar peduncle. We compare Plxdc2-betageo expression in the embryonic brain with the much more restricted expression of the related gene Plxdc1 and with members of the Wnt family (Wnt3a, Wnt5a and Wnt8b) that show a striking overlap with Plxdc2 expression in certain areas.     

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.