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Riko Kitazawa

Publications and source records attributed to Riko Kitazawa.

13 recordsLinked to original sources
PubMed2003

Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells.

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.

Animals↗
PubMed2003

Vitamin D3 supports osteoclastogenesis via functional vitamin D response element of human RANKL gene promoter.

Receptor activator of NF-kappaB ligand (RANKL) has been identified as requisite for osteoclastogenesis. To elucidate the molecular mechanism that conducts its catabolic action on bone, the effect of 1alpha,25 dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on osteoclastogenesis and RANKL mRNA expression was examined by coculture, RT-PCR and nuclear run-on studies. By accelerating the transcription rate of the RANKL gene in SaOS2 osteoblastic cells, 1alpha,25(OH)(2)D(3) enhanced in vitro osteoclast formation from peripheral monocytes. Cloning and characterization of the 5'-flanking region of the human RANKL gene revealed that the basic promoter comprises inverted TATA- and CAAT-boxes flanked by RUNX2 binding sites. Both electrophoresis mobility shift assay (EMSA) and transfection studies demonstrated that 1alpha,25(OH)(2)D(3) activated human RANKL promoter through vitamin D responsive elements (VDRE) located at -1584/-1570 by binding VDR and RXRalpha heterodimers in a ligand-dependent manner. The results provide direct evidence that 1alpha,25(OH)(2)D(3) augments osteoclastogenesis by transactivating the human RANKL gene in osteoblastic cells through VDRE.

Base Sequence↗
PubMed2003

MeCP2 and promoter methylation cooperatively regulate E-cadherin gene expression in colorectal carcinoma.

Reduced expression of E-cadherin (E-cad) owing to aberrant 5'CpG island hypermethylation has been regarded as one of the main molecular events involved in the dysfunction of the cell-cell adhesion system. The molecular mechanisms providing diversity and heterogeneity of E-cad expression in colorectal carcinoma were explored. In 29 cases of colorectal carcinoma in Indonesia, the expression of E-cad was analyzed by immunohistochemical staining, the methylation status of the E-cad promoter was determined by methylation-specific PCR, and the expression of methyl-CpG-binding protein (MeCP) 2 was studied by in situ hybridization. E-cad expression was strong, and no methylation was observed in normal colon mucosa and most of the well-differentiated adenocarcinoma. In contrast, both signet-ring cell carcinoma and mucinous adenocarcinoma showed fully methylated patterns and strong MeCP2 expression. In moderately- and poorly-differentiated adenocarcinomas, however, E-cad expression was rather heterogeneous, especially at the front of invasion and in the dissociated areas, where loss of MeCP2 expression correlated with E-cad reexpression even in the presence of E-cad promoter methylation. We conclude that both CpG methylation of the E-cad promoter and significant MeCP2 expression cooperatively and epigenetically regulate E-cad expression in colorectal cancer.

Adenocarcinoma↗
PubMed2003

Functional giant parathyroid cyst with high concentration of CA19-9 in cystic fluid.

A 63-year-old man was admitted to our hospital for the evaluation of hypercalcemia and anterior neck mass. Laboratory findings revealed hypercalcemia, hypophosphatemia, and hypercalciuria, as well as elevated serum levels of parathyroid hormone (PTH) and alkaline phosphatase. Computerized tomography and magnetic resonance images showed that the mass contained a cystic area. Parathyroid scintigraphy using either 99mTc-sestamibi alone or 201Tl-chloride in conjunction with 99mTc-pertechnetate for thyroid image subtraction showed uptake of the radioactivity into the cyst wall, suggesting that the mass originated from the parathyroid. Fine needle aspiration biopsy revealed that the cyst fluid was serous and bloody with extremely high concentrations of both PTH and CA19-9. The patient was diagnosed as primary hyperparathyroidism caused by parathyroid cyst and cervical exploration was performed. The cyst was dissected away along with the right lobe of the thyroid gland. After tumor removal, serum calcium and PTH levels were normalized. Histological study showed that the tumor possessed malignant potential with capsular invasion as well as moderate cellular atypia with trabecular pattern in arrangement. Parathyroid cells in the wall of the cystic tumor were immunostained positively for CA19-9, suggesting that CA19-9 in the cyst fluid was produced from the cells.

CA-19-9 Antigen↗
PubMed2003

Decrease in vitamin D receptor and calcium-sensing receptor in highly proliferative parathyroid adenomas.

OBJECTIVE: A significant decrease in vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) protein expression has been demonstrated recently in parathyroid (PT) adenomas. In this study, we investigated the relationships between the proliferative activity of parathyroid glands (PTGs) and the expression of VDR as well as CaSR, and compared it with the clinical severity in patients with primary hyperparathyroidism (1 degrees HPT). DESIGN: Seven patients with 1 degrees HPT were included in this study. Four patients with thyroid carcinoma served as controls. METHODS: Immunohistochemical staining was performed on serial sections of PTGs with specific antibodies against CaSR, VDR, and Ki67. Areas examined in each section were selected at random in relation to the gland size. The number of Ki67-positive cells was expressed as a labeling index (LI; positive cells per 1000 PT cells). The expression of CaSR and VDR was semi-quantitatively analyzed based on the intensity of staining. After averages of the scores from all areas were calculated, CaSR and VDR scores, and Ki67 LI were assigned to each gland for use in statistical analyses. RESULTS: In PT adenomas, scores of VDR and CaSR were markedly lower than in normal PTGs (P<0.01), while the proportion of Ki67-positive cells in PT adenomas was significantly higher than in normal PTGs (P<0.01). Single regression analyses revealed that Ki67 LI was positively correlated with serum levels of intact parathyroid hormone and Ca, and PTG weight (R=0.70, P<0.05, R=0.78, P<0.01 and R=0.84, P<0.05 respectively). Ki67 LI was negatively correlated with CaSR and VDR scores (R=-0.78, P<0.01 and R=-0.72, P<0.05 respectively). Moreover, there was a strong positive relationship between CaSR and VDR expression (R=0.95, P<0.001). CONCLUSIONS: Marked decreases in VDR and CaSR expression could, at least in part, be responsible for the high proliferation of PT cells and the pathological progression of 1 degree HPT.

Adenoma↗
PubMed2002

Transforming growth factor-beta induces expression of receptor activator of NF-kappa B ligand in vascular endothelial cells derived from bone.

Vascular endothelial cells in bone are thought to have significant roles on pathological bone resorption such as bone metastasis and hypercalcemia because this resorption is often seen where blood vessels are abundant. However, the detailed mechanisms have not yet been elucidated. Here, we focused on transforming growth factor-beta (TGF-beta) and studied its effects on vascular endothelial cells because TGF-beta is abundantly stored in bone matrix and is released and activated during bone resorption. We found that TGF-beta up-regulated the expression of receptor activator of NF-kappa B ligand (RANKL) mRNA and protein in bone marrow-derived endothelial cells and in primary vascular endothelial cells but not in osteoblasts. Further analysis revealed that TGF-beta promoted phosphorylation of cAMP response element-binding protein and p38. Protein kinase A inhibitor KT5720 and p38 inhibitor SB203580 significantly reduced the TGF-beta-induced RANKL expression. Moreover, we found two CRE-like domains in murine RANKL promoter region that were critical for TGF-beta-dependent RANKL expression. Therefore, protein kinase A and p38 signaling pathways are involved in TGF-beta-induced RANKL expression by stimulating transcription factors that bind to the CRE-like domains. Our findings indicate that TGF-beta stimulates osteoclastogenesis by promoting RANKL expression in endothelial cells under pathological conditions.

Animals↗
PubMed2002

Epigenetic control of mouse receptor activator of NF-kappa B ligand gene expression.

Receptor activator of NF-kappa B ligand (RANKL) is a membrane-bound signal transducer requisite for differentiation and maintenance of osteoclasts. RANKL expression on stromal/osteoblastic cells is tightly regulated to maintain physiological serum calcium levels and bone mass. These stromal/osteoblastic cells, however, comprise a rather heterogeneous population ranging from immature mesenchymal cells to mature osteoblasts and also respond differently to bone resorptive stimuli. In the mouse coculture system, we also have demonstrated the passage-dependent difference of cultured mouse stromal cells in supporting osteoclastogenesis due to altered RANKL gene expression. To address the issue of what molecular mechanism gives the diversity of RANKL gene expression to stromal/osteoblastic cells, we characterized the mouse RANKL gene promoter that contains two CpG clustering regions; one around the transcription start site, and the other downstream of the vitamin D response element (VDRE). Using earlier- and later-passage mouse ST2 cells, we analyzed the CpG methylation status by sodium bisulfite mapping and found that CpG loci around the transcription start site (-66/+246) were predominantly methylated in later-passage ST2 cells. Moreover, earlier- and later-passage ST2 cells transfected with a RANKL promoter construct showed the same steady-state level of luciferase activity and of the inducible effect of 1,25(OH)(2)D(3). Furthermore, the introduction of methylation to the promoter construct silenced promoter activity. The results suggest that CpG methylation around the transcription start site of the mouse RANKL gene is an important epigenetic event, and that its heterogeneity might cause the diversity of the stromal/osteoblastic cells in RANKL gene expression.

Animals↗
PubMed2002

RANK ligand, RANK, and OPG expression in type II collagen-induced arthritis mouse.

Rheumatoid arthritis (RA) is a systemic disorder characterized by synovial inflammation and subsequent destruction and deformity of synovial joints. The articular lesions start with synovitis, focal erosion of unmineralized cartilage, and then culminate in the destruction of subarticular bone by pannus tissue. Periarticular osteopenia and systemic osteoporosis follow as late complications of RA. Osteoclasts, specialized cells that resorb bone, play a central role in developing these osteolytic lesions. To elucidate the mechanism of osteoclastogenesis and bone destruction in autoimmune arthritis, we investigated the expression of RANK ligand (RANKL), RANK, and osteoprotegerin (OPG) mRNA in a mouse type II collagen-induced arthritis (CIA) model by in situ hybridization. The results indicated that most of the TRAP-positive mono- and multinucleated cells in the inflamed and proliferating synovium and in the pannus were RANK-positive authentic osteoclasts and their precursors. In the inflamed synovium and pannus of the mouse CIA model, synovial fibroblastic cells around these RANK-positive cells were strongly positive for RANKL. Moreover, RANKL-positive osteoblasts on the endosteal bone surface, at a distance from the affected synovial joints, increased significantly in the mouse CIA model prior to periarticular osteopenia and systemic osteoporosis. These data indicated that the RANKL-RANK system plays an important role for osteoclastogenesis in both local and systemic osteolytic lesions in autoimmune arthritis, and can therefore be a good target for therapeutic intervention.

Animals↗
PubMed2002

Vitamin D(3) augments osteoclastogenesis via vitamin D-responsive element of mouse RANKL gene promoter.

Receptor activator of NF-kappaB ligand (RANKL) is a membrane-bound signal transducer necessary for the induction and maintenance of osteoclasts. To clarify the molecular mechanism by which 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) augments osteoclasts, we characterized the promoter region of the mouse RANKL gene. Mirroring in vitro osteoclastogenesis demonstrated by a coculture of bone marrow macrophages with ST2 stromal cells, Northern blot, and nuclear run-on analyses showed that 1,25-(OH)(2)D(3) upregulate RANKL gene expression at the transcriptional level. Using a series of deletion mutants of mouse RANKL promoter-luciferase reporter gene constructs, transient transfection studies revealed that the inductive effect of 1,25-(OH)(2)D(3) was abolished when the region up to -723 was deleted. An electrophoretic motility shift assay demonstrated that the VDR-RXRbeta heterodimer bound to AGGTCAGCCTGGTTCA (-937/-922), and VDRE/nuclear protein supershift complexes that bound to anti-VDR and -RXRbeta antibodies were detected in the nuclear extract of 1,25-(OH)(2)D(3)-treated ST2 cells. Furthermore, induction of mutation to the putative VDRE also diminished the inductive effect of 1,25-(OH)(2)D(3). We therefore concluded that mouse RANKL gene is one of the target genes of 1,25-(OH)(2)D(3) containing a functional VDRE in the promoter region.

Animals↗
PubMed2002

RANK ligand is a prerequisite for cancer-associated osteolytic lesions.

Breast cancer is frequently associated with osteolytic bone metastasis, where osteoclasts play a major role in bone destruction. Recently, osteoclast differentiation factor (RANKL) has been identified as a prerequisite for the formation and maintenance of osteoclasts from haematopoietic precursors. To elucidate the mechanism of osteoclastogenesis and bone destruction in bone-residing breast cancer, PTHrP-producing (MCF-7) and -non-producing (MCF-7UP) human breast cancer cells were subcutaneously injected into the forehead of nude mice maintained without oestrogen supplement. One, two, and three weeks thereafter, the expression of RANKL and PTHrP mRNA, and osteoclastogenesis were analysed by in situ hybridization and TRAP staining. In MCF-7 cells, at early stages, spindle-shaped stromal cells and osteoblasts on the bone surface expressed RANKL, then numerous osteoclasts were induced on the periosteal bone surface. Three weeks after the transplantation, MCF-7 cancer cells migrated onto the eroded bone surface, where they survived apoptosis. At all stages, RANKL expression was confined to the stromal/osteoblastic cells, whereas PTHrP was confined to the MCF-7 breast cancer cells. On the other hand, PTHrP was negative in MCF-7UP cells at all stages, and neither induction of osteoclasts nor infiltrative growth of cancer cells was observed. Moreover, in vitro treatment with PTHrP resulted in increased RANKL mRNA expression and transcription activity in the MC3T3-E1 mouse osteoblastic cell line. Thus PTHrP induces osteoclastic bone resorption through the transactivation of the RANKL gene on stromal/osteoblastic cells, affording a bone microenvironment conducive to the survival of PTHrP-producing cancer cells.

Animals↗
PubMed2002

Expression of parathyroid hormone-related protein (PTHrP) in multiple myeloma.

Multiple myeloma is a plasma cell neoplasia often associated with multiple skeletal lesions and hypercalcemia. Several cytokines, including interleukin (IL)-1, IL-6 and tumor necrosis factor-beta (TNF-beta), derived from myeloma cells are thought to accelerate osteoclastic bone resorption and cause hypercalcemia through a paracrine mechanism. We report on a case of a 69-year-old man with multiple myeloma associated with hypercalcemia and advanced osteolytic lesions. After bisphosphonate treatment and MP (melphalan and prednisolone) therapy, the patient's serum calcium level was successfully but transiently recovered to the normal range. Biochemical analysis showed a remarkable increase in serum parathyroid hormone-related protein (PTHrP; 3.7 pmol/L) and IL-6 (22.0 pg/mL). On the other hand, parathyroid hormone and 1alpha,25(OH)2 vitamin D3 were suppressed. By immunohistochemistry and in situ hybridization on aspiration-biopsied bone marrow clot sections, PTHrP mRNA and protein were detected in the cytoplasm of myeloma cells. The rate of PTHrP-positive myeloma cells was estimated to be at least one-third. Since PTHrP can, as an endocrine factor, systemically act on bone and kidney, hypercalcemia in this case might have been caused through both local osteolytic hypercalcemia and humoral hypercalcemia of malignancy mechanisms.

Aged↗