Fat feeding stimulates only one of the two mRNAs encoding rat intestinal membranous and secreted alkaline phosphatase

We have identified two mRNAs in rat intestinal mucosa by Northern blot analysis, using cloned cDNAs encoding human placental alkaline phosphatase (PLAP). Probes from both the NH2- and COOH-terminal ends of the human PLAP coding region identified, in rat intestine (especially duodenum), an mRNA of nearly identical size (3 kb) to that found in human placenta. A smaller mRNA (2.7 kb), detected only with the COOH-terminal probe, was more prevalent in jejunum. Following feeding of triacylglycerols, the prevalence of the 2.7 kb mRNA increased over 2-fold. The tissue distribution and response of the 2.7 kb mRNA to fat feeding corresponds exactly with the known behavior of the secreted alkaline phosphatase.     

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.     

Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.

The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.     

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.     

中胚叶叉头-1在骨成形蛋白-2诱导肌胚细胞C2C12转化成骨细胞过程中的作用

为了研究中胚叶叉头-1(MFH-1)基因在骨骼形成和细胞分化中的作用,利用基因重组、杂交瘤技术制作MFH-1单克隆抗体, 利用蛋白质印迹和RNA印迹分析观察了骨成形蛋白-2 (BMP-2)诱导小鼠肌胚细胞C2C12表达MFH-1、产生碱性磷酸酶和骨钙蛋白.小鼠肌胚细胞C2C12低水平地表达内源性MFH-1蛋白以及导入小鼠MFH-1 cDNA的人膀胱癌细胞HTB9也表达小鼠MFH-1蛋白,这种蛋白质定位于细胞核中.用BMP-2处理后, MFH-1蛋白和mRNA在C2C12细胞中的表达显著地增加.用反义MFH-1序列转染小鼠肌胚细胞C2C12可降低内源性MFH-1水平, BMP-2不能诱导导入反义MFH-1序列的肌胚细胞C2C12产生MFH-1蛋白,也不能诱导碱性磷酸酶(ALP)活性和骨钙蛋白量的增加.结果表明, BMP-2诱导的MFH-1蛋白在调节肌胚细胞C2C12向成骨细胞分化方面起关键作用.     

Human bone marrow osteoprogenitors express estrogen receptor-alpha and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation.

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.     

Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia.

Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene.     

Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)     

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.