Alkaline Phosphatase Phenotypes Research Articles

Disease-Associated Extracellular Matrix Suppresses Osteoblastic Differentiation of Human Periodontal Ligament Cells Via MMP-1

Fibronectin (FN) fragments found in chronic inflammatory diseases, including periodontal disease and arthritis, may contribute to tissue destruction in part via induction of matrix metalloproteinases (MMPs). We previously showed that the 120-kDa FN fragment containing the central cell binding domain (120FN) dose dependently induces MMP-1 (collagenase-1) in human periodontal ligament (PDL) cells, whereas intact FN did not elicit this response. Recently, we found that an increase in MMP-1 expression is accompanied by a decreased osteoblastic phenotype in PDL cells. We hypothesized that 120FN inhibits osteoblastic differentiation of PDL cells by inducing MMP-1. Effects of increasing concentrations of 120FN on MMP-1 expression and on osteoblastic markers were assessed in cultured PDL cells using Western blotting, qRT-PCR, and collagen degradation and alkaline phosphatase (AP) activity assays. The 120FN dose dependently increased MMP-1 expression and activity, concomitant with a decrease in AP activity. The increase in collagenase activity was largely attributed to increased MMP-1 expression. Concurrent with the decrease in AP activity, the 120FN reduced baseline and dexamethasone-induced gene expression of specific osteoblastic markers, Runx2 and osteonectin, and diminished mineralized nodule formation. Finally, siRNA inhibition of 120FN-induced MMP-1 reduced collagenase expression and rescued the AP phenotype to baseline levels. These findings suggest that disease-associated 120FN, in addition to having direct effects on tissue destruction by upregulating MMPs, could contribute to disease progression by impeding osteoblastic differentiation of osteogenic PDL cells and, consequently, diminish bone regeneration.

Isoforms of Bone Alkaline Phosphatase, Stem Cells, and Osteoblast Phenotypes

Isoforms of Bone Alkaline Phosphatase, Stem Cells, and Osteoblast Phenotypes

Novel Mouse Model of Autosomal Semidominant Adult Hypophosphatasia Has a Splice Site Mutation in the Tissue Nonspecific Alkaline Phosphatase Gene Akp2

Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.

Dexamethasone's enhancement of osteoblastic markers in human periodontal ligament cells is associated with inhibition of collagenase expression

Although dexamethasone (Dex) substantially enhances the osteoblastic phenotype in osteogenic cells, including human periodontal ligament (PDL) cells, the basis for this response remains poorly understood. Since the accretion of a collagenous matrix is important for an osteoblastic response and dexamethasone is known to decrease collagenase expression, we examined whether osteoblastic differentiation mediated by Dex is linked to a decrease in collagenase expression in PDL cells. Early passage human PDL cells were exposed to Dex, or ascorbic acid (AA) or β-glycerophosphate (βGP) alone, or in various combinations in serum-free media for 3 or 5 days. Cells exposed to Dex alone or any combinations of treatments that included Dex demonstrated increased core binding factor alpha 1 (Cbfa1), alkaline phosphatase (AP), osteonectin (ON), osteopontin (OP), bone sialoprotein (BSP) and collagen I (α1) expression when compared to control cells or those exposed to AA or βGP. The induction of these osteoblastic markers was accompanied by a decrease in collagenase-1 expression. Collagenase activity showed a statistically significant strong negative relationship to Cbfa1 (Pearson's r = − 0.97), AP ( r = − 0.87), OP ( r = − 0.95) and BSP ( r = − 0.82) in 5-day cultures, and moderately strong relationship to ON ( r = − 0.74) from 3 days culture. Dex also produced a dose-dependent increase in AP that was paralleled by a decrease in collagenase activity ( r = − 0.98). Addition of collagenase inhibitors increased AP expression while concomitantly suppressing collagenase activity. Conversely, addition of exogenous collagenase decreased the AP phenotype of the cells, which was more marked in the absence then in the presence of Dex. The findings indicate that Dex enhances specific markers of osteoblastic differentiation in PDL cells by decreasing collagenase expression, and suggest that endogenous collagenase may regulate osteoblastic differentiation of these cells.

Ascorbic acid induces collagenase-1 in human periodontal ligament cells but not in MC3T3-E1 osteoblast-like cells: potential association between collagenase expression and changes in alkaline phosphatase phenotype.

Ascorbic acid (AA) enhances osteoblastic differentiation by increasing collagen accumulation, which in turn, results in increased alkaline phosphatase (AP) expression in some osteogenic cells. However, in other cells, including human periodontal ligament (PDL) cells, additional osteoinductive agents are required for this response. To understand the potential basis for the maintenance of the AP phenotype of PDL cells exposed to AA, we examined the modulation of the tissue-degrading matrix metalloproteinases (MMPs) and their inhibitors by AA in short-term cell cultures. Early passage PDL cells in serum-free medium were exposed to AA for 5 days. The samples were analyzed for MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), AP, collagen I(alpha1), and osteocalcin. We found that AA dose-dependently increased the expression of collagenase-1, and minimally TIMP-1, but not stromelysin-1 or TIMP-2. Additionally, AA caused substantial increases in levels of type I collagen. AA was unable to increase AP activity or osteocalcin messenger RNA in PDL cells. However, the cells retained the ability to show a significantly greater AP expression in high- versus low-density cultures, and increased osteocalcin as well as AP levels when cultured in the presence of dexamethasone. Moreover, in cells exposed to dexamethasone, increases in AP and osteocalcin were accompanied by a repression of collagenase-1 expression. In contrast to PDL cells, AA did not induce collagenase but produced a significant increase in AP expression in MC3T3-E1 cells. These findings provide the first evidence that AA, by modulating both collagen and collagenase-1 expression in PDL cells, most likely contributes to a net matrix remodeling response in these cells. Furthermore, the relationship between changes in collagenase expression and alterations in AP activity in PDL and MC3T3-E1 cells suggests a potential role for collagenase in modulating the AP phenotype of cells with osteoblastic potential.

Effects of androgens on subpopulations of the human osteosarcoma cell line SaOS2

Previously, we showed that androgens stimulate murine and human osteoblast-like cell proliferation and differentiation by mechanisms involving increased responses to mitogenic growth factors (GF) and increased GF production. To explain this dual action of androgens on primary osteoblastic cell populations we advanced the hypothesis that androgens exert differential effects on osteoblastic subpopulations. We subcloned a human osteosarcoma cell line (SaOS2) into subpopulations expressing high (HAS) and low (LAS) levels of alkaline phosphatase (ALP). The obtained subclones differed significantly in their ALP production and expressed a high and low ALP phenotype, respectively, for the entire experimental period. Dihydrotestosterone (DHT) increased specific ALP activity and type-I procollagen peptide secretion in both HAS and LAS. DHT pretreatment enhanced the mitogenic action of basic fibroblast growth factor (bFGF) and insulinlike growth factor 2 (IGF2) only in HAS. The enhanced mitogenic effect of IGF2 in HAS after DHT pretreatment was associated with increased IGF2-receptor mRNA levels. Therefore, we conclude that androgens exert their osteoanabolic action (1) by stimulating differentiated functions of osteoblastic cells with a high and a low ALP phenotype, and (2) via increased growth factor receptor expression and thereby enhancing mitogenic growth factor responses only in HAS.

The human placental immunoglobulin G receptor and immunoglobulin G transport

The objective of this study was to quantitatively determine an immunoglobulin G receptor, placental alkaline phosphatase, and its ligand immunoglobulin G in maternal and fetal blood and to study the transport capacity of the receptor. Venous blood samples from 66 term pregnant women and cord samples from their fetuses were obtained, together with the corresponding placentas. Mean placental alkaline phosphatase levels were determined to be 23.7 ng/ml and 1.2 ng/ml in maternal and fetal blood, respectively. Mean immunoglobulin G level of the fetal samples was significantly higher than that of the maternal samples (12.6 vs 9.5 gm/L, p < 0.0001). The placental alkaline phosphatase phenotype S had a larger dissociation constant to immunoglobulin G than did type F and was found to have mean fetal immunoglobulin G levels higher than those of the F type (13.3 vs 9.7 gm/L). The placental immunoglobulin G receptor placental alkaline phosphatase is found in the fetal circulation. The placental alkaline phosphatase phenotype was found to be related to the levels of its ligand immunoglobulin G in fetal blood, although the mechanism for this remains to be established. Immunoglobulin G is actively transported to fetal blood to reach higher levels than in the maternal circulation.

Allelic Amino Acid Substitutions Affect the Conformation and Immunoreactivity of Germ-Cell Alkaline Phosphatase Phenotypes

The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.

Molecular cloning of the wild-type phoM operon in Escherichia coli K-12.

A metastable bacterial alkaline phosphatase (Bap) phenotype is seen in phoR mutants, which alternately express a Bap-constitutive or -negative phenotype. The alteration is affected by mutations in the phoM region near 0 min. By molecular cloning of the wild-type phoM operon onto a multicopy plasmid and recombining onto the plasmid the pho-510 mutation that abolishes variation, the phoM operon, rather than some nearby gene, was shown to control variation. Complementation tests indicated that the wild-type phoM allele is dominant to the pho-510 mutation when both are in single copy, but whichever allele is present in higher copy appears as dominant when multicopy plasmids are examined. The alternating phenotypic variation of BAP synthesis was not seen in phoR+ cells with multicopy wild-type phoM plasmids, thus showing that the variation is associated with phoM-dependent Bap expression. The alternation acted at the level of phoA transcription; it was also recA independent. BAP clonal variation is phenotypically similar to Salmonella phase variation, which is controlled by a DNA rearrangement. No evidence was found for a DNA change near the phoM operon that might be responsible for the variable Bap phenotype.

1,25 Dihydroxyvitamin D3 is required for growth-independent expression of alkaline phosphatase in cultured rat osteoblasts.

Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D3 (10(-9) M) or 24,25-dihydroxyvitamin D3 (10(-9) M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)2D3 led to a significant (P less than 0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)2D3 (10(-9) M) but not 24,25(OH)2D3 maintained the basal percentage of Alk Pase positive cell units in non-proliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)2D3 was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.

Eutopic expression of placental-like alkaline phosphatase in testicular tumors.

Very high levels of placental-like alkaline phosphatases (PLAP-like enzymes) were observed in tissues from 13 typical seminomas. Four tumors with seminoma components contained these enzymes to varying degrees, and other testicular tumors had them in smaller or non-detectable amounts. Analysis using monoclonal antibodies produced against the common placental alkaline phosphatase (PLAP) phenotypes and enzyme inhibition studies with amino acids and peptides showed the PLAP-like enzymes present in seminoma to be similar to those PLAP-like enzymes which are expressed in lower amounts in two embryonal carcinomas and in trace amounts in normal testicular tissue. These similarities suggest that the increased expression of PLAP-like enzymes in seminomas results from enhanced eutopic expression of enzymes found in normal testis.

Determination of placental alkaline phosphatase phenotypes by starch-gel and acrylamide gel electrophoresis

The polymorphism of placental alkaline phosphatase (ALP) is usually studied by butanol extraction followed by starch gel electrophoretic separation at two different pH's, 8.6 and 6.0. Acrylamide gel electrophoresis (7.5%) in the presence of Triton X-100 is shown to be preferable in speed, reliability and reproducibility of results. All the placental ALP phenotypes detected had the same molecular mass by gradient acrylamide electrophoresis. The enzyme butanol extract is not sufficiently pure to enable the different placental ALP's to be distinguished by isoelectric focusing on polyacrylamide gels.

Frequency Distribution of Genetic Markers in Guernsey Cattle1,2

Ten polymorphic systems of blood antigens and blood proteins were analyzed in 6,802 Guernsey cows from 77 herds and 11 states. The numbers of alleles identified with frequencies >1% in the A, B, C, F, J, L, M, S, and Z blood antigen systems and the alkaline phosphatase system were 3, 17, 11, 2, 2, 2, 1, 3, 2, and 2. Gene frequencies were determined by maximum likelihood, allocation, and square root methods for the dominant systems and by the gene counting method for the codominant system. Distributions varied greatly in the different systems. Comparisons among these and other blood and milk genetic marker systems revealed lack of independence of J and alkaline phosphatase phenotypes, J and milk protein β-lactoglobulin phenotypes, and of combined phenotypes of the three systems. More cows than expected were J-negative when alkaline phosphatase A was present or when β-lactoglobulin B was present. Phenotypes of blood antigen system A and hemoglobin were also different from independent assortment. More hemoglobin B cows had blood antigen system A phenotype A than the other phenotypes. The blood antigen locus F had more heterozygous cows than were expected for Hardy-Weinberg equilibrium.

Serum alkaline phosphatase phenotypes and secretor status in several Jewish populations in Israel.

The frequency of serum alkaline phospatase phenotypes and secretor trait is determined for Israeli Jews originating from three distinct geographical regions: Eastern and Central Europe (group I), North Africa (group II) and the Middle East (group III). The frequency of the Pp 2 serum alkaline phosphatase phenotype in each of the three groups were 33.56% (group I), 29.79% (group II) and 24.07% (group III). The average frequency for the three groups combined was 30.58%. The corresponding frequencies of the se gene in the three groups were 0.4765, 0.5149 and 0.5468, respectively, and the average frequency for the total number examined was 0.4978.

Sex ratio in man: an analysis of the relationship with ABO blood groups and placental alkaline phosphatase phenotype.

Secondary sex ratio (SR) in man is influenced by various genetic and environmental factors. It has been observed that SR in subjects of blood group B compatible with their mothers is higher than in other subjects. The analysis of 676 newborns of the Rome population and 1,684 newborns of the New Haven (Connecticut) population have confirmed a higher SR in B group subjects compatible with their mothers. The data also indicate that placental alkaline phosphatase is another genetic factor influencing SR in man and that there is a strong interaction among ABO phenotype, fetomaternal ABO compatible status and PAP phenotype concerning their effects on SR.

Placental-like alkaline phosphatase in malignant and benign ovarian tumors

Alkaline phosphatase (ALP) components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalariine inhibition and reactivity against antiplacental ALP antiserum. Seven of the eighteen tumors had ALP components which most closely resembled the ALP isozyme normally found in placenta and were clearly distinguished from all other tissue ALPs. The proportion of tumors with the placental-like ALP in the malignant group (five out of seven) was significantly greater than the proportion in the benign group (two out of eleven). The fraction (78%) of the malignant tumors with the isozyme represents a larger percentage than has previously been found by examination of cancer patients' sera. The electrophoretic mobilities of the placental-like ALPs in the tumors were in no case identical to the mobilities of any of the six common placental ALP phenotypes. The tumor ALPs may thus be determined by rare variant alleles at the ALP locus, or alternatively, the enzyme molecules may have been subject to structural modification. At least two of these tumors contained an electrophoretically slow. heat-stable, leucine-sensitive ALP, which may correspond to what has been termed the D-variant of placental ALP found in some other tumors.

Heterogeneity of alkaline phosphatases in different HeLa lines.

Alkaline phosphatase (ALP) components in 8 cell lines of HeLa were examined. Line to line heterogeneity in ALP expression was observed using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylanine inhibition, and reactivity against antiplacental ALP antiserum. Six lines contained a placentallike ALP isozyme and varying amounts of a liverlike ALP isozyme. One line contained a liverlike ALP isozyme only. One line contained a new ALP form which was clearly distinguished from the placental, liver, bone, and intestinal ALPs. Thus, derepression of the placental ALP structural locus appeared to have occurred in 6 of the 8 lines. However, where expressed, the placentallike ALP varied electrophoretically from line to line, and in only one case was the mobility identical to that of a common placental ALP phenotype. This phenotypic heterogeneity of the ""derepressed'' placentallike ALP contrasts markedly with the phenotypic stability of other enzymes expressed in HeLa cells.

Placental enzyme polymorphisms in Canadian populations. III. Alkaline phosphatase.

Placental alkaline phosphatase phenotypes have been determined for a large Canadian population. The 'common' alleles, PL1, PL2, and PL3 have frequencies similar to those of European populations. Five new phenotypes are described and the incidence of rare phenotypes is compared for several populations.

Alkaline phosphatase phenotypes in tumour and non-tumour cell lines: Not an invariable marker for neoplastic transformation

The cytochemical localisation and presumed isoenzyme type (based on selective inhibition experiments) of alkaline phosphatase in 5 cell lines derived from normal human, rat, mouse and hamster tissues, 6 human lymphoblastoid lines and 6 human and mouse tumour-derived cell lines are described. Enzyme activity varied between the cell lines. An isoenzyme inhibited by L-phenylalanine was present in 3 normal lines, 3 lymphoblastoid lines and 2 tumour lines. The presence of this isoenzyme cannot be used as a marker of neoplastic transformation.

L-leucine, a specific inhibitor of a rare human placental alkaline phosphatase phenotype.

FISHMAN et al. reported that human placental alkaline phosphatase (Regan isoenzyme) was found in the serum and tumour tissue of a patient with lung cancer1. Two electrophoretically different variant forms of Regan alkaline phosphatase with a greater sensitivity to inhibition by L-leucine have since been described: the ‘Nagao isoenzyme’2, and a hepatoma-specific alkaline phosphatase3–5. Inglis et al.6 reported that the L-leucine-sensitive Nagao isoenzyme found in some cancer patients closely resembled the ‘D variant’ of human placental alkaline phosphatase, a rare phenotype defined electrophoretically on starch gel which is found in less than 1% of the general population of pregnancies7. An example was reported of a pregnancy serum D-phenotype enzyme presumably of placental origin, confirmed by electrophoresis established as L-leucine-sensitive6. Most interesting was the occurrence of the variant at high frequency in the ovarian cancer patients surveyed6,8. To extend the observations of Inglis et aL, screening for the phosphatases in placentae was carried out, both to establish the frequency of the L-leucine-sensitive placental phenotypes (which should match the frequency of the electrophoretically defined D variant phenotypes) and to provide enough enzyme to enable purification and characterisation. Supply of cancer tissue is generally restrictive for this purpose.