Riko Kitazawa

Promoter structure of mouse RANKL/TRANCE/OPGL/ODF gene

Receptor activator of NF-kappa B ligand (RANKL)/tumor necrosis factor-related activation induced cytokine (TRANCE)/osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) is a membrane-bound signal transducer responsible for differentiation and maintenance of osteoclasts. To elucidate the mechanism regulating RANKL/TRANCE/OPGL/ODF gene expression, we cloned the 5-flanking basic promoter region of the mouse RANKL/TRANCE/OPGL/ODF gene and characterized it by transient transfection studies and genomic Southern blot analysis. Inverted TATA- and CAAT-boxes and a putative Cbfa1/Osf2/AML3 binding domain constituted the basic promoter structure. The repeated half-sites for the vitamin D3 (VitD3) and glucocorticoid receptors were located at -935 and -640, respectively. Transient transfection studies revealed that short-term treatment with 1alpha,25(OH)2 VitD3 or dexamethasone increased luciferase activity up to 204% and 178%, respectively; on the other hand, treatment with dibutyryl cyclic AMP did not affect the promoter activity. Since the expression of Cbfa1/Osf2/AML3 is also regulated by VitD3, 1alpha,25(OH)2 VitD3 might affect RANKL/TRANCE/OPGL/ODF gene expression both directly and indirectly. CpG methylation was observed dominantly in mouse stromal cells, ST2, of a later passage which ceased to support in vitro osteoclastogenesis, suggesting that the methylation status of the CpG loci in the RANKL/TRANCE/OPGL/ODF gene promoter may be one of the influential cis-regulating factors.

Transcriptional Regulation of Rat Cyclin D1 Gene by CpG Methylation Status in Promoter Region

Cyclin D1, a G1/S cell cycle-regulating oncogene, is known to be transcriptionally regulated by numerous growth factors. We cloned and characterized the rat cyclin D1 gene 5′-flanking region and, by species- and subspecies-matched transient transfection studies, found that a basic promoter structure with a cAMP response element and two continuous Sp1-binding sites was crucial for the steady-state expression of the cyclin D1 gene. Furthermore, the methylation status especially around two continuous Sp1-binding sites was found to be an important epigenetical mechanism determining the steady-state expression level in rat leukemic cell lines K4D, K4DT, and K4D16. Whether or not epigenetic control of the cyclin D1 gene existed among normal rat tissues was further examined by high sensitivity mapping of the methylated cytosine. In normal rat tissues, the methylated cytosines at non-CpG loci within two continuous Sp1-binding sites were observed in uterine stromal cells of the basal layer and found to be demethylated in the functioning layer, possibly by a passive demethylation mechanism through cell division. Since in the passive demethylation process Sp1-binding sites remain methylated in a part of the cell population, methylated cytosines at Sp1-binding sites may be essential for keeping a number of the stromal cells in the basal layer live against estrogen-induced proliferation that leads to either apoptosis or compaction.

Estrogen via the Estrogen Receptor Blocks cAMP-Mediated Parathyroid Hormone (PTH)-Stimulated Osteoclast Formation†

Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)‐induced bone resorption in vivo and in vitro. However, its precise mechanism remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH‐related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH‐induced osteoclast‐like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) as well as in osteoblastic MC3T3‐E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM‐CSF as well as in MC3T3‐E1 cells. The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17β‐estradiol (17β‐E2) but not 17α‐E2 dose dependently antagonized Ocl formation stimulated by human (h) PTH(1–34) at a minimal effective concentration of 10−10 M in the hemopoietic blast cell culture. 17β‐E2 also significantly inhibited Ocl formation stimulated by 10−8 M hPTHrP(1–34), while it did not affect 1,25‐dihydroxyvitamin D3–induced Ocl formation. However, 10−8 M 17β‐E2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10−4 M) and Sp‐cAMPS (10−4 M), an activator of cAMP‐dependent protein kinase (PKA) as well as forskolin (10−5 M). In contrast, 17β‐E2 did not affect Ocl formation by either phorbol myristate acetate (10−7 M), an activator of protein kinase C (PKC), or A23187 (10−7 M), a calcium ionophore. The pretreatment with 17β‐E2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with PTH and Rp‐cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits PTH‐stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PKA pathway but not a calcium/PKC pathway.

Identification of methylated cytosine from archival formalin-fixed paraffin-embedded specimens.

T he use of archival formalin-fixed, paraffinembedded pathological specimens for molecular biological analysis has become increasingly important. Also, DNA methylation is one of the essential epigenetical mechanisms controlling gene expression. We show that formalin fixation, for up 72 hours, does not impair the accessibility of methylated cytosine, and that DNA degradation, as an adverse effect of formalin fixation, is an advantage in the PCR-mediated detection of methylated cytosine. With this technique, retrospective and morphology-oriented epigenetical studies as well as clonal analysis are possible using microdissected samples from paraffin-embedded pathological specimens. As an epigenetic event, DNA methylation, especially cytosine methylation, plays a major role in the regulation of sequential and tissue-specific expression of the gene (Lewis et al, 1992). In this study, we first analyzed the effect of the formalin-fixation on the accessibility of the methylated cytosine, then applied the agarosebead-mediated bisulfite modification and the PCR technique to investigate the methylation status of the 59-flanking region of the rat cyclin D1 gene from microdissected fragments of paraffin sections. Two established cell lines, K4D and K4D16 (Kitazawa et al, 1999), were used as a model for assessing the effects of the fixative condition on the detection of methylated-cytosine residues. Freshly prepared 30 ml of 10 mM hydroquinone and 520 ml of 3 M sodium bisulfite at pH 5 were added to the extracted DNA. Each sample was incubated under mineral oil at 50° C for 16 hours. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 minutes at room temperature, then by ethanol precipitation. Bisulfite-modified DNA (100 ng) was amplified with PCR using the following primers: 59-GTGTTGATGAAATTGAAAGAAGTTG-39: sense 59-ACTTTACAACTTCAACAAAACTCCCCTAT-39: anti-sense The PCR condition was as follows: 30 cycles of 94° C for 30 seconds, 60° C for 30 seconds, 72° C for 30 seconds, and the final elongation step for 5 minutes at 72° C. When intact DNA was fragmented, a clear PCR product of 560 bp was formed (Fig. 1, Lane 2, EcoRI 1 0). When cells were fixed with 10% formalin for 24, 48, and 72 hours, a clear PCR product was formed without predigestion with EcoRI restriction endonuclease (Fig. 1, upper panel, Lanes 3 to 5). No PCR products were, however, formed when fixation with 10% formalin reached 120 hours (Fig. 1, upper panel, Lane 6). The PCR products yielded from samples after 72 hours of fixation with 10% formalin were eluted, purified, and analyzed by sequencing. The methylated cytosine in the 19th CpG locus in the K4D cell line was detectable from the DNA extracted from K4D fixed with 10% formalin for 72 hours (Fig. 1, lower panel, arrows). When K4D and K4D16 cells were mixed and injected intravenously into Long-Evans rats, the cells developed numerous metastatic nodules resembling myeloblastoma in most of the organs. The lung tissues were removed and fixed with 10% formaldehyde for 24 hours and embedded in paraffin. Sections were cut, and selected areas of the interest were microdissected and collected in 1.5 ml microcentrifuge tubes. The samples were deparaffinized for a total of 30 minutes in 1 ml xylene before dehydration with 100% ethanol in the microcentrifuge tubes. The samples were further dehydrated in a vacuum for 10 minutes, redissolved, and shredded into minute pieces by pipetting in 10 ml nuclease-free water, then immediately mixed with an equal amount of preheated (80° C) and melted 3.2% low-temperature melting agarose (SeaPlaque, Takara, Kyoto, Japan) by pipetting. The agarose beads were made by dropping 5 ml of the mixed samples into a 1.5 ml microcentrifuge tube containing 250 ml mineral oil kept at room temperature (Olek et al, 1996). One milliliter of 0.2 mg/ml proteinase K in 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 20 mM EDTA was added to each tube kept at 50° C until the shredded samples became translucent (usually for 24–48 hours) in a water bath. After cooling the tubes at room temperature, the agarose beads were treated twice with 1.5 ml of 0.3 N NaOH for 15 minutes each, and once with 1.5 ml of 0.1 N NaOH. To inactivate proteinase K, samples were heated for 15 minutes at 80° C and cooled on ice for 5 minutes. One milliliter of freshly prepared bisulfite solution (3.5 M NaHSO3, 1 mM Hydroquinone, pH 5.0) was added to the microcentrifuge tubes, and the samples were incubated for 24 hours at 50° C under a lightprotected condition. Beads were then washed twice with 1 ml of 10 mM Tris-HCl (pH 8.0), 10 mM EDTA for 15 minutes each, and desulfonated by three washes with 1 Received November 16, 1999. Address reprint requests to: Dr. S. Kitazawa, Second Department of Pathology, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe S650–0017, Japan. Fax: 81 78 362 0297; E-mail: kitazawa@med.kobe-u.ac.jp 0023-6837/00/8002-275

Promoter structure of human sonic hedgehog gene.

03.00/0 LABORATORY INVESTIGATION Vol. 80, No. 2, p. 275, 2000 Copyright

Molecular cloning and analysis of the 5′-flanking region of the human bone morphogenetic protein-6 (BMP-6)

Sonic hedgehog (Shh) is a secreted signal transducer responsible not only for patterning of the anterior-posterior axis during early limb and neuronal development, but also for generating cell-type diversity at later developmental stages. To elucidate the mechanism regulating human Shh gene expression, we cloned the 5-flanking region of the human Shh gene and characterized it by transient transfection studies. Two transcription start sites were identified by primer extension analysis. Two TATA-boxes, a CCAAT-box and a palindrome-like structure constituted the basic promoter structure. Furthermore, two continuous E-boxes and a putative homeodomain containing an ATTA-box were located around 350-450 bp upstream of the upper TATA-box. Consensus binding sites of the RA, estrogen, D3 and glucocorticoid/progesterone receptors were not found within the cloned sequence. Short-term treatment with TPA increased luciferase activity up to 2.1-fold; on the other hand, treatment with dibutyryl-cyclic AMP decreased it to 0.8-fold. Retinoic acid (RA), vitamin D3, dexamethazone (DEX) and estradiol (E2) had no effect on the luciferase activity. Since the zebrafish Shh promoter contains two closely spaced axial (HNF3beta) binding sequences on its basic promoter, the palindrome-like structure located in the corresponding site of the human Shh promoter may be a crucial binding domain regulating human Shh gene expression.

TYPE IIA EARLY GASTRIC CANCER WITH PROLIFERATION OF XANTHOMA CELLS

We cloned a genomic fragment of the 5-flanking region of the gene encoding bone morphogenetic protein-6 (BMP-6) and assessed its promoter activity. Primer extension revealed the presence of one major transcription start site 178 bp upstream of the translation start site. The promoter region lacked a canonical TATA box but did contain a GC-rich region. A putative tramtrack responsive element, a Drosophila transcriptional factor regulating the segment polarity, was found in the promoter region. Known steroid hormonal responsive elements, however, were not found. Although BMP-6 is classified as a member of the vgr-1 family, the structure of the promoter was similar to that of BMP-2 and 4.

Analysis of 5'-flanking region of human Smad4 (DPC4) gene.

Abstract: We report a type IIa early gastric cancer associated with xanthoma cell proliferation in a 61-year-old man. The patient was admitted to our hospital because of a gastric polyp detected at a medical checkup. An irregular protruding lesion with xanthoma cell proliferation was detected endoscopically. Histological examination showed a well differentiated tubular adenocarcinoma in the mucosa associated with xanthoma cell proliferation. The distribution of the xanthoma cells in the stroma corresponded closely with that of the cancer cells. Neither atypism nor mitotic figures were recognized in the xanthoma cells. In an immunohistochemical study, almost all the xanthoma cells were stained positive for α1-antitrypsin, while relatively few exhibited positive S-100 protein staining. Specific monocyte chemotactic and activating factor immunoreactivity was present only in the xanthoma cells, and not in the cancer cells. On the basis of these findings, it was speculated that the gastric cancer cells may have caused the xanthoma cell proliferation via an autocrine mechanism i.e., by a chemical mediator acting in a paracrine or juxtacrine manner.

Expression of Bone Morphogenetic Proteins (BMPs) in Fractured Mouse Bone Tissue: In Situ Hybridization with Polymerase Chain Reaction (PCR) -derived Antisense DNA Probe

Among the transducers of the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) receptor signaling proteins, Smad4 and Smad1 act together in BMP 2/4 signaling pathways, and Smad4 and Smad2 act in TGF-beta/activin signaling. To investigate how the Smad4 gene is regulated at the transcriptional level, we cloned and characterized its 5-flanking region. The major transcription start site mapped by primer extension analysis was 132 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes; it did, however, contain a TATA-like structure (TAAAAT) 32 bp upstream of the transcription start site. A consensus sequence for homeoprotein HoxA-5 (TTTAAAAATTA) was identified at 171 bp upstream of the transcription start site. Within 600 bp upstream of the transcription start site, two Pit-1 and four F2F binding sites were found. One putative AP-1 site was located at -1129. These findings suggest that these homeoproteins could conduct their signals specifically by controlling both inter- and intracellular signal transduction pathways.