241 research outputs found
High Extracellular Calcium Increased Expression of Ank, PC-1 andOsteopontin in Mouse Calvarial Cells
In the process of bone remodeling, mineral phase of bone
is dissolved by osteoclasts, resulting in elevation of calcium
concentration in micro-environment. This study was performed
to explore the effect of high extracellular calcium
(Ca
2+
e) on mineralized nodule formation and on the expression
of progressive ankylosis (Ank), plasma cell membrane
glycoprotein-1 (PC-1) and osteopontin by primary cultured
mouse calvarial cells. Osteoblastic differentiation and mineralized
nodule formation was induced by culture of mouse
calvarial cells in osteoblast differentiation medium containing
ascorbic acid and β-glycerophosphate. Although Ank, PC-1
and osteopontin are well known inhibitors of mineralization,
expression of these genes were induced at the later stage of
osteoblast differentiation during when expression of osteocalcin,
a late marker gene of osteoblast differentiation, was
induced and mineralization was actively progressing. High
Ca
2+
e (10 mM) treatment highly enhanced mRNA expression
of Ank, PC-1 and osteopontin in the late stage of osteoblast
differentiation but not in the early stage. Inhibition of p44/42
MAPK activation but not that of protein kinase C suppressed
high Ca
2+
e-induced expression of Ank, PC-1 and
osteopontin. When high Ca
2+
e (5 mM or 10 mM) was present
in culture medium during when mineral deposition was
actively progressing, matrix calcifiation was significantly
increased by high Ca
2+
e. This stimulatory effect was abolished
by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an
alkaline phosphatase inhibitor. In addition, probenecid (2mM),
an inhibitor of Ank, suppressed matrix calcification in both
control and high Ca
2+
e-treated group, suggesting the possible
role of Ank in matrix calcification by osteoblasts. Taken
together, these results showed that high Ca
2+
e stimulates expression of Ank, PC-1 and osteopontin as well as matrix
calcification in late differentiation stage of osteoblasts and
that p44/42 MAPK activation is involved in high Ca
2+
e-
induced expression of Ank, PC-1 and osteopontin
Tricho-dento-osseous Syndrome Mutant Dlx3 Shows Lower Transactivation Potential but Has Longer Half-life than Wild-type Dlx3
Dlx3 is a homeodomain protein and is known to play a role
in development and differentiation of many tissues. Deletion
of four base pairs in DLX3 (NT3198) is causally related to
tricho-dento-osseous (TDO) syndrome (OMIM #190320), a
genetic disorder manifested by taurodontism, hair abnormalities,
and increased bone density in the cranium. The
molecular mechanisms that explain the phenotypic characteristics
of TDO syndrome have not been clearly determined.
In this study, we examined phenotypic characteristics of
wild type DLX3 (wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3)
in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3
differentially regulate osteoblastic differentiation, reporter
assays were performed by using luciferase reporters containing
the promoters of alkaline phosphatase, bone sialoprotein or
osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced
significantly all the reporter activities but the effect of
mtDlx3 was much weaker than that of wtDlx3. In spite of
these differences in reporter activity, electrophoretic mobility
shift assay showed that both wtDlx3 and TDO mtDlx3
formed similar amounts of DNA binding complexes with
Dlx3 binding consensus sequence or with ALP promoter
oligonucleotide bearing the Dlx3 binding core sequence.
TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it
corresponds to PESTfind analysis result showing that
potential PEST sequence was missed in carboxy terminal of
TDO mtDlx3. In addition, co-immunoprecipitation demonstrated
that TDO mtDlx3 binds to Msx2 more strongly than
wtDlx3. Taken together, though TDO mtDlx3 acted as a
weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast
differentiation TDO mtDlx3 may antagonize transcriptional
repressor activity of Msx2 more effectively and for longer
period than wtDlx3, resulting in enhancement of osteoblast
differentiation
The Expression of Matrix Metalloprotease 20 is Stimulated by Wild Type but not by 4 bp- or 2 bp- Deletion Mutant DLX3
Mutations in DLX3 are associated with both autosomal
dominant hypoplastic hypomaturation amelogenesis
imperfecta (ADHHAI) and tricho-dento-osseous (TDO)
syndrome. ADHHAI is caused by a c.561_562delCT (2bpdel
DLX3) mutation whereas TDO syndrome is associated
with a c.571_574delGGGG (4bp-del DLX3) mutation.
However, although the causal relationships between DLX3
and an enamel phenotype have been established, the
pathophysiological role of DLX3 mutations in enamel
development has not yet been clarified. In our current study,
we prepared expression vectors for wild type and deletion
mutant DLX3 products (4bp-del DLX3, 2bp-del DLX3) and
examined the effects of their overexpression on the
expression of the enamel matrix proteins and proteases.
Wild type DLX3 enhanced the expression of matrix
metalloprotease 20 (MMP20) mRNA and protein in murine
ameloblast-like cells. However, neither a 4bp-del nor 2bpdel
DLX3 increased MMP20 expression. Wild type DLX3,
but not the above DLX3 mutants, also increased the activity
of reporters containing 1.5 kb or 0.5 kb of the MMP20
promoter. An examination of protein stability showed that
the half-life of wild type DLX3 protein was less than 12 h
whilst that of both deletion mutants was longer than 24 h.
Endogenous Dlx3 was also found to be continuously
expressed during ameloblast differentiation. Since
inactivating mutations in the gene encoding MMP20 are
associated with amelogenesis imperfecta, the inability of
4bp-del or 2bp-del DLX3 to induce MMP20 expression
suggests a possible involvement of such mutations in the enamel phenotype associated with TDO syndrome or
ADHHAI
Growth differentiation factor 11 locally controls anterior-posterior patterning of the axial skeleton.
Growth and differentiation factor 11 (GDF11) is a transforming growth factor β family member that has been identified as the central player of anterior-posterior (A-P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A-P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11-/- mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning
Identification of genes modulated by high extracellular calcium in coculuture of mouse osteoblasts and bone marrow cells by oligo chip assay
Calcium concentration in the bone resorption lacunae is
high and is in the mM concentration range. Both osteoblast
and osteoclast have calcium sensing receptor in the cell
surface, suggesting the regulatory role of high extracellular
calcium in bone metabolism. In vitro, high extracellular
calcium stimulated osteoclastogenesis in coculture of mouse
osteoblasts and bone marrow cells. Therefore we examined
the genes that were commonly regulated by both high
extracellular calcium and 1,25(OH)2vitaminD3 (VD3) by
using mouse oligo 11 K gene chip. In the presence of 10 mM
[Ca2+]e or 10 nM VD3, mouse calvarial osteoblasts and bone
marrow cells were co-cultured for 4 days when tartrate
resistant acid phosphatase-positive multinucleated cells
start to appear. Of 11,000 genes examined, the genes
commonly regulated both by high extracellular calcium and
by VD3 were as follows; 1) the expression of genes which
were osteoclast differentiation markers or were associated
with osteoclastogenesis were up-regulated both by high
extracellular calcium and by VD3; trap, mmp9, car2, ctsk,
ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and
chemokine receptor genes such as sdf1, scya2, scyb5, scya6,
scya8, scya9, and ccr1 were up-regulated both by high
extracellular calcium and by VD3, 3) the genes such as
mmp1b, mmp3 and c3 which possibly stimulate bone
resorption by osteoclast, were commonly up-regulated, 4)
the gene such as c1q and msr2 which were related with
macrophage function, were commonly down-regulated, 5)
the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were
commonly down-regulated; slc8a1, admr, plod2, lox, fosb, 6)
the genes which possibly suppress osteoblast differentiation
and/or mineralization of extracellular matrix, were commonly
up-regulated; s100a4, npr3, mme, 7) the genes such
as calponin 1 and tgfbi which possibly suppress osteoblast
differentiation and/or mineralization of extracellular matrix,
were up-regulated by high extracellular calcium but were
down-regulated by VD3. These results suggest that in
coculture condition, both high extracellular calcium and
VD3 commonly induce osteoclastogenesis but suppress
osteoblast differentiation/mineralization by regulating the
expression of related genes.본 연구는 보건복지부 보건의료기술진흥사업의 지원에
의하여 이루어진 것임(03-PJ1-PG3-20500-0013)
Comparative Evaluation of Nanofibrous Scaffolding for Bone Regeneration in Critical-Size Calvarial Defects
In a previous study we found that nanofibrous poly(l-lactic acid) (PLLA) scaffolds mimicking collagen fibers in size were superior to solid-walled scaffolds in promoting osteoblast differentiation and bone formation in vitro. In this study we used an in vivo model to confirm the biological properties of nanofibrous PLLA scaffolds and to evaluate how effectively they support bone regeneration against solid-walled scaffolds. The scaffolds were implanted in critical-size defects made on rat calvarial bones. Compared with solid-walled scaffolds, nanofibrous scaffolds supported substantially more new bone tissue formation, which was confirmed by micro-computed tomography measurement and von Kossa staining. Goldner's trichrome staining showed abundant collagen deposition in nanofibrous scaffolds but not in the control solid-walled scaffolds. The cells in these scaffolds were immuno-stained strongly for Runx2 and bone sialoprotein (BSP). In contrast, solid-walled scaffolds implanted in the defects were stained weakly with trichrome, Runx2, and BSP. These in vivo results demonstrate that nanofibrous architecture enhances osteoblast differentiation and bone formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78127/1/ten.tea.2008.0433.pd
Blood-testis barrier integrity depends on Pin1 expression in Sertoli cells
The conformation and function of a subset of serine and threonine-phosphorylated proteins are regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Pin1 is intensely expressed in Sertoli cells, but its function in this post mitotic cell remains unclear. Our aim was to investigate the role of Pin1 in the Sertoli cells. Lack of Pin1 caused disruption of the blood-testis barrier. We next investigated if the activin pathways in the Sertoli cells were affected by lack of Pin1 through immunostaining for Smad3 protein in testis tissue. Indeed, lack of Pin1 caused reduced Smad3 expression in the testis tissue, as well as a reduction in the level of N-Cadherin, a known target of Smad3. Pin1−/− testes express Sertoli cell marker mRNAs in a pattern similar to that seen in Smad3+/− mice, except for an increase in Wt1 expression. The resulting dysregulation of N-Cadherin, connexin 43, and Wt1 targets caused by lack of Pin1 might affect the mesenchymal–epithelial balance in the Sertoli cells and perturb the blood-testis barrier. The effect of Pin1 dosage in Sertoli cells might be useful in the study of toxicant-mediated infertility, gonadal cancer, and for designing male contraceptives
Gene expression profile of the skin in the 'hairpoor' (HrHp) mice by microarray analysis
<p>Abstract</p> <p>Background</p> <p>The transcriptional cofactor, Hairless (HR), acts as one of the key regulators of hair follicle cycling; the loss of function mutations is the cause of the expression of the hairless phenotype in humans and mice. Recently, we reported a new <it>Hr </it>mutant mouse called 'Hairpoor' (<it>Hr<sup>Hp</sup></it>). These mutants harbor a gain of the function mutation, T403A, in the <it>Hr </it>gene. This confers the overexpression of HR and <it>Hr<sup>Hp </sup></it>is an animal model of Marie Unna hereditary hypotrichosis in humans. In the present study, the expression profile of <it>Hr<sup>Hp</sup>/Hr<sup>Hp </sup></it>skin was investigated using microarray analysis to identify genes whose expression was affected by the overexpression of HR.</p> <p>Results</p> <p>From 45,282 mouse probes, differential expressions in 43 (>2-fold), 306 (>1.5-fold), and 1861 genes (>1.2-fold) in skin from <it>Hr<sup>Hp</sup>/Hr<sup>Hp </sup></it>mice were discovered and compared with skin from wild-type mice. Among the 1861 genes with a > 1.2-fold increase in expression, further analysis showed that the expression of eight genes known to have a close relationship with hair follicle development, ascertained by conducting real-time PCR on skin RNA produced during hair follicle morphogenesis (P0-P14), indicated that four genes, <it>Wif1</it>, <it>Casp14</it>, <it>Krt71</it>, and <it>Sfrp1</it>, showed a consistent expression pattern with respect to HR overexpression in vivo.</p> <p>Conclusion</p> <p><it>Wif1 </it>and <it>Casp14 </it>were found to be upregulated, whereas <it>Krt71 </it>and <it>Sfrp1 </it>were downregulated in cells overexpressing HR in transient transfection experiments on keratinocytes, suggesting that HR may transcriptionally regulate these genes. Further studies are required to understand the mechanism of this regulation by the HR cofactor.</p
The role of primary tumor resection in colorectal cancer patients with asymptomatic, synchronous unresectable metastasis: Study protocol for a randomized controlled trial
BACKGROUND: Approximately 20 % of all patients with colorectal cancer are diagnosed as having Stage IV cancer; 80 % of these present with unresectable metastatic lesions. It is controversial whether chemotherapy with or without primary tumor resection (PTR) is effective for the treatment of patients with colorectal cancer with unresectable metastasis. Primary tumor resection could prevent tumor-related complications such as intestinal obstruction, perforation, bleeding, or fistula. Moreover, it may be associated with an increase in overall survival. However, surgery delays the use of systemic chemotherapy and affects the systemic spread of malignancy. METHODS/DESIGN: Patients with colon and upper rectal cancer patients with asymptomatic, synchronous, unresectable metastasis will be included after screening. They will be randomized and assigned to receive chemotherapy with or without PTR. The primary endpoint measure is 2-year overall survival rate and the secondary endpoint measures are primary tumor-related complications, quality of life, surgery-related morbidity and mortality, interventions with curative intent, chemotherapy-related toxicity, and total cost until death or study closing day. The authors hypothesize that the group receiving PTR following chemotherapy would show a 10 % improvement in 2-year overall survival, compared with the group receiving chemotherapy alone. The accrual period is 3 years and the follow-up period is 2 years. Based on the inequality design, a two-sided log-rank test with α-error of 0.05 and a power of 80 % was conducted. Allowing for a drop-out rate of 10 %, 480 patients (240 per group) will need to be recruited. Patients will be followed up at every 3 months for 3 years and then every 6 months for 2 years after the last patient has been randomized. DISCUSSION: This randomized controlled trial aims to investigate whether PTR with chemotherapy shows better overall survival than chemotherapy alone for patients with asymptomatic, synchronous unresectable metastasis. This trial is expected to provide evidence so support clear treatment guidelines for patients with colorectal cancer with asymptomatic, synchronous unresectable metastasis. TRIAL REGISTRATION: Clinicaltrials.gov NCT01978249
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