Publications by authors named "Sohei Kitazawa"

130 Publications

Identification of calmodulin-like protein 5 as tumor-suppressor gene silenced during early stage of carcinogenesis in squamous cell carcinoma of uterine cervix.

Int J Cancer 2021 Sep 25;149(6):1358-1368. Epub 2021 May 25.

Division of Diagnostic Pathology, Ehime University Hospital, Toon City, Ehime, Japan.

In the course of identifying the molecular mechanism that is related to strong cell-cell adhesion in stratified structures of the squamous epithelium, calmodulin-like protein 5 (CALML5) was identified as a spinous structure-associated protein by producing monoclonal antibodies with the use of the crude intercellular portion of squamous tissue as an immunogen and by subsequent morphologic screening. By electrophoretic mobility shift assay (EMSA) and a series of mutagenesis studies, two transcription factors, ZNF750 and KLF4, by binding in line to the CALML5 gene promoter, were found to play a central role in CALML5 transcription. Knockdown of CALML5 by siRNA in the A431 cell line that expresses high levels of CALML5 resulted in the acceleration of wound confluence in a scratch assay, indicating that CALML5 functions as a tumor-suppressor in uterine cervical cancer. Immunohistochemical evaluation of squamous intraepithelial lesions, carcinoma in situ (CIS) and invasive uterine cancer, revealed a reduction in CALML5 expression during the stages of CIS through various molecular pathways including the blockage of the nuclear translocation of KLF4. Conversely, restoration of the nuclear translocation of KLF4 by inhibiting ERK-signaling reactivated CALML5 expression in ME180 cells expressing low levels of CALML5. Thus, alteration of the p63-ZNF750-KLF4 axis may result in critical functional loss of CALM-related genes during cancer progression. Although the morphological association of CALML5 with the spiny-structure in relation to cell motility is not clear, evaluation of CALML5 expression provides a useful diagnostic indicator of differentiating dysplasia, preinvasive and invasive cervical cancers.
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http://dx.doi.org/10.1002/ijc.33687DOI Listing
September 2021

New Insights into Development of Female Reproductive Tract-Hedgehog-Signal Response in Wolffian Tissues Directly Contributes to Uterus Development.

Int J Mol Sci 2021 Jan 26;22(3). Epub 2021 Jan 26.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

The reproductive tract in mammals emerges from two ductal systems during embryogenesis: Wolffian ducts (WDs) and Mullerian ducts (MDs). Most of the female reproductive tract (FRT) including the oviducts, uterine horn and cervix, originate from MDs. It is widely accepted that the formation of MDs depends on the preformed WDs within the urogenital primordia. Here, we found that the WD mesenchyme under the regulation of Hedgehog (Hh) signaling is closely related to the developmental processes of the FRT during embryonic and postnatal periods. Deficiency of Sonic hedgehog (Shh), the only Hh ligand expressed exclusively in WDs, prevents the MD mesenchyme from affecting uterine growth along the radial axis. The in vivo cell tracking approach revealed that after WD regression, distinct cells responding to WD-derived Hh signal continue to exist in the developing FRT and gradually contribute to the formation of various tissues such as smooth muscle, endometrial stroma and vascular vessel, in the mouse uterus. Our study thus provides a novel developmental mechanism of FRT relying on WD.
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http://dx.doi.org/10.3390/ijms22031211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865753PMC
January 2021

Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats.

Biochem Biophys Res Commun 2020 08 22;529(2):186-190. Epub 2020 Jun 22.

Laboratory of Veterinary Anatomy, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino City, Tokyo, 180-8602, Japan.

The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and β glycerophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.039DOI Listing
August 2020

Disseminated infection with novel human adenovirus (genotype 79) following allogeneic hematopoietic stem cell transplantation.

Ann Hematol 2020 Jun 18. Epub 2020 Jun 18.

Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.

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http://dx.doi.org/10.1007/s00277-020-04151-xDOI Listing
June 2020

New Insights into the Pathogenesis of Diabetic Nephropathy: Proximal Renal Tubules Are Primary Target of Oxidative Stress in Diabetic Kidney.

Acta Histochem Cytochem 2020 Apr 25;53(2):21-31. Epub 2020 Apr 25.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

Diabetic nephropathy is a major source of end-stage renal failure, affecting about one-third cases of diabetes mellitus. It has long been accepted that diabetic nephropathy is mainly characterized by glomerular defects, while clinical observations have implied that renal tubular damage is closely linked to kidney dysfunction at the early stages of diabetic nephropathy. In this study, we conducted pathohistological analyses focusing on renal tubular lesions in the early-stage diabetic kidney with the use of a streptozotocin (STZ)-induced diabetes mellitus mouse model. The results revealed that histological alterations in renal tubules, shown by a vacuolar nucleic structure, accumulations of PAS-positive substance, and accelerated restoration stress, occur initially without the presence of glomerular lesions in the early-stage diabetic kidney, and that these tubular defects are localized mainly in proximal renal tubules. Moreover, enhanced expression of RAGE, suggesting an aberrant activation of AGEs-RAGE signaling pathway, and accumulation of oxidative modified mitochondria through the impaired autophagy/lysosome system, were also seen in the damaged diabetic proximal renal tubules. Our findings indicate that proximal tubular defects are the initial pathological events increasingly linked to the progression of diabetic nephropathy, and that controlling renal tubular damage could be an effective therapeutic strategy for the clinical treatment of diabetic nephropathy.
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http://dx.doi.org/10.1267/ahc.20008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212204PMC
April 2020

A case of thoracic endometriosis-related pneumothorax in a menopausal woman.

Gen Thorac Cardiovasc Surg 2020 Dec 14;68(12):1584-1586. Epub 2020 May 14.

Center of Chest Medicine and Surgery, Ehime University, Toon, Ehime, Japan.

Thoracic endometriosis-related pneumothorax (TERP) or thoracic endometriosis syndrome (TES) usually occurs in women of childbearing age and affects the right thorax. Menopausal and left-sided cases are rare. A case of left-sided TERP in a postmenopausal woman after adjuvant endocrine therapy for breast cancer is reported. A 51-year-old woman underwent video-assisted thoracic surgery for recurrent left pneumothorax. Immunohistological examination of the resected specimen from the apical bleb and a diaphragmatic blueberry spot demonstrated thoracic endometriosis. Even in the case of a left-sided pneumothorax in a menopausal woman, clinicians should be aware of the possibility of TERP.
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http://dx.doi.org/10.1007/s11748-020-01381-8DOI Listing
December 2020

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.
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http://dx.doi.org/10.3390/ijms21082745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216259PMC
April 2020

Knockdown of Lrp1 in RAW264 cells inhibits osteoclast differentiation and osteoclast-osteoblast interactions in vitro.

Biochem Biophys Res Commun 2020 03 19;523(4):961-965. Epub 2020 Jan 19.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Low density lipoprotein receptor-related protein 1 (LRP1), a multifunctional cell surface protein, is expressed in bone marrow-derived macrophages. While LRP1 is thought to be a suppressor of osteoclast differentiation at late stages, its function at early stages remains unclear. Here we demonstrate that Lrp1 stable knockdown by lentiviral short hairpin RNA in macrophage cell line RAW264 cells inhibited RANKL-induced osteoclast formation and osteoclastic master transcription factor Nfatc1 mRNA expression as assessed by quantitative RT-PCR. Furthermore, knockdown of the Lrp1 gene suppressed not only differentiation, but also proliferation, and inhibitory effects on osteoblastic ALP activity by osteoclast-derived humoral factors. Thus, we propose that LRP1 in macrophages is required for both differentiation into osteoclasts and osteoclast-osteoblast interactions.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.065DOI Listing
March 2020

Recent Insights into Long Bone Development: Central Role of Hedgehog Signaling Pathway in Regulating Growth Plate.

Int J Mol Sci 2019 Nov 20;20(23). Epub 2019 Nov 20.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City 791-0295, Japan.

The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the "growth plate", is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.
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http://dx.doi.org/10.3390/ijms20235840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928971PMC
November 2019

Wilson Disease With Giant Splenic Artery Aneurysms Caused by Fibromuscular Dysplasia During Living Donor Liver Transplantation: A Case Report.

Transplant Proc 2019 Nov 11;51(9):3131-3135. Epub 2019 Oct 11.

Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Ehime, Japan.

Liver cirrhosis can cause splenic artery aneurysms (SAA) that pose a threat to patients undergoing liver transplantation. However, liver transplantation with multiple visceral artery aneurysms including giant SAA caused by arterial fragility has never been reported. We describe a 36-year-old man with decompensated liver cirrhosis due to Wilson disease that was complicated by giant SAA and multiple aneurysms in the bilateral renal arteries caused by fibromuscular dysplasia (FMD). The maximal diameter of the triple snowball-shaped SAA was 11 cm. We planned a 2-stage strategy consisting of a splenectomy with distal pancreatectomy to treat the SAA and subsequent living donor liver transplantation (LDLT) to address the liver cirrhosis. This strategy was selected to prevent fatal postoperative infectious complications caused by the potential development of pancreatic fistula during simultaneous procedures and to histopathologically diagnose the arterial lesion before LDLT to promote safe hepatic artery reconstruction. However, a postoperative pancreatic fistula did not develop after a splenectomy with distal pancreatectomy, and the pathologic findings of the artery indicated FMD. The patient underwent ABO-identical LDLT with a right lobe graft donated by his brother. Other than postoperative rupture of the aneurysm in the left renal artery requiring emergency interventional radiology, the patient has remained free of any other arterial complications and continues to do well at 2 years after LDLT.
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http://dx.doi.org/10.1016/j.transproceed.2019.06.005DOI Listing
November 2019

Modulation of αβ Integrin via Transactivation of β Integrin Gene on Murine Bone Marrow Macrophages by 1,25(OH)D, Retinoic Acid and Interleukin-4.

Acta Histochem Cytochem 2019 Aug 10;52(4):77-83. Epub 2019 Aug 10.

Department of Molecular Pathology, Ehime University Graduate School of Medicine.

The interleukin (IL)-4, 1,25(OH)D and retinoic acid, increase surface expression of functional integrin αβ on murine osteoclast precursors. All three agonists stimulate transcription of the β gene, leading to increased steady-state levels of mRNA this protein. By contrast, mRNA levels of α remain unchanged. In each instance, the increase in the surface expression of the integrin results in increased migration of the cells onto an αβ substrate. Because β subunit, except platelet where β subunit conform a dimer with α, associates solely with α subunit monogamously, while promiscuous α subunit combines with various subunit, our present data support the idea that the β subunit governs the surface-expressed functional integrin complex.
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http://dx.doi.org/10.1267/ahc.19015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773611PMC
August 2019

Activation of protein kinase C accelerates murine osteoclastogenesis partly via transactivation of RANK gene through functional AP-1 responsive element in RANK gene promoter.

Biochem Biophys Res Commun 2019 07 28;515(2):268-274. Epub 2019 May 28.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan; Division of Diagnostic Molecular Pathology, Kobe University Graduate School of Medicine, Kusunoki-cho 7-5-1, Kobe, 650-0017, Japan. Electronic address:

Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (-480), and MITF (-100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (-240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (-246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (-240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.144DOI Listing
July 2019

SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9.

J Cell Physiol 2019 08 19;234(10):17280-17294. Epub 2019 Feb 19.

Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime, Japan.

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.
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http://dx.doi.org/10.1002/jcp.28346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617759PMC
August 2019

Pulmonary Hemorrhaging as a Fatal Complication of IgA Vasculitis.

Intern Med 2018 Nov 6;57(21):3141-3147. Epub 2018 Jun 6.

Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, Japan.

A 64-year-old man was admitted to our hospital for purpuric rash, joint pain, and a fever. He had earlier undergone a follow-up examination for interstitial lung disease. At the current visit, the diagnosis was immunoglobulin A (IgA) vasculitis, based on skin and renal biopsy findings. He developed sudden breathlessness and hemoptysis. Chest computed tomography revealed ground glass opacity in the right lower lung fields, suggesting pulmonary hemorrhaging associated with IgA vasculitis. Despite steroid and cyclophosphamide therapy, and plasma exchange, he died 52 days after admission. Early aggressive therapies may be recommended for old patients with IgA vasculitis who have an additional comorbidities.
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http://dx.doi.org/10.2169/internalmedicine.0817-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262698PMC
November 2018

Morphology-oriented epigenetic research.

Histochem Cell Biol 2018 Jul 2;150(1):3-12. Epub 2018 May 2.

Division of Diagnostic Pathology, Ehime University Hospital, Toon City, Ehime, 791-0295, Japan.

Cytosine methylation plays a major role in the regulation of sequential and tissue-specific expression of genes. De novo aberrant DNA methylation and demethylation are also crucial processes in tumorigenesis and tumor progression. The mechanisms of how and when such aberrant methylation and demethylation occur in tumor cells are still obscure, however. To evaluate subtle epigenetic alteration among minor subclonal populations, morphology-oriented epigenetic analysis is requisite, especially where heterogeneity and flexibility are as notable as in the process of cancer progression and cellular differentiation at critical stages. Therefore, establishment of reliable morphology-oriented epigenetic studies has become increasingly important in not only the experimental but also the diagnostic field. By selecting a subset of cells based on characteristic morphological features disclosed by microdissection or in situ hybridization, we discovered how methylation at certain CpG sites outside of CpG islands would play a crucial epigenetic role in the versatility and flexibility of gene expression during cancer progression. In this review, we first introduce technical aspects of two morphology-oriented epigenetic studies: (1) histoendonuclease-linked detection of methylated sites of DNA (HELMET), and (2) padlock probe and rolling circle amplification (RCA) for in situ identification of methylated cytosine in a sequence-dependent manner. We then present our observation of a novel MeCP2-mediated gene-silencing mechanism through the addition of methylation to a single-CpG-locus upstream of the TATA-box of the receptor activator of NF-κB ligand (RANKL) and of secreted frizzled-related protein 4 (SFRP4) gene promoters.
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http://dx.doi.org/10.1007/s00418-018-1675-8DOI Listing
July 2018

In focus in HCB: Hard Tissue Biology.

Histochem Cell Biol 2018 Apr 6;149(4):287-288. Epub 2018 Mar 6.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.

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http://dx.doi.org/10.1007/s00418-018-1658-9DOI Listing
April 2018

Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas.

Diagn Pathol 2018 Jan 3;13(1). Epub 2018 Jan 3.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Background: A proper balance between the activator and the repressor form of GLI3, a zinc-finger transcription factor downstream of hedgehog signaling, is essential for proper development of various organs during development. Mutations in different domains of the GLI3 gene underlie several congenital diseases including Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS).

Case Presentation: Here, we describe the case of an overlapped phenotype of these syndromes with agenesis of the gallbladder and the pancreas, bearing a c.2155 C > T novel likely pathogenic variant of GLI3 gene by missense point mutation causing p.P719S at the proteolytic cleavage site.

Conclusions: Although agenesis of the gallbladder and the pancreas is uncommon in GLI3 morphopathy, a slight difference in the gradient or the balance between activator and repressor in this case may hinder sophisticated spatial and sequential hedgehog signaling that is essential for proper development of gallbladder and pancreas from endodermal buds.
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http://dx.doi.org/10.1186/s13000-017-0682-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389258PMC
January 2018

Pathologic conditions of hard tissue: role of osteoclasts in osteolytic lesion.

Histochem Cell Biol 2018 Apr 22;149(4):405-415. Epub 2018 Jan 22.

Department of Molecular Pathology, Graduate School of Medicine, Ehime University Graduate School of Medicine, 454 Shitsukawa, Toon, Ehime, 791-0295, Japan.

Hard tissue homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. This physiologic process allows adaptation to mechanical loading and calcium homeostasis. Under pathologic conditions, however, this process is ill-balanced resulting in either over-resorption or over-formation of hard tissue. Local over-resorption by osteoclasts is typically observed in osteolytic metastases of malignancies, autoimmune arthritis, and giant cell tumor of bone (GCTB). In tumor-related local osteolysis, tumor-derived osteoclast-activating factors induce bone resorption not by directly acting on osteoclasts but by indirectly upregulating receptor activator of NFκB ligand (RANKL) on osteoblastic cells. Similarly, synovial tissue in the autoimmune arthritis model does overexpress RANKL and contains numerous osteoclast precursors, and like a landing craft, when it comes in contact with eroded bone surfaces, osteoclast precursors are immediately polarized to become mature osteoclasts, inducing rapidly progressive bone destruction at a late stage of the disease. GCTB, on the other hand, is a common primary bone tumor, usually arising at the metaphysis of the long bone in young adults. After the discovery of RANKL, the concept of GCTB as a tumor of RANKL-expressing stromal cells was established, and comprehensive exosome studies finally disclosed the causative single-point mutation at histone H3.3 (H3F3A) in stromal cells. Thus, osteolytic lesions under various pathological conditions are ultimately attributable to the overexpression of RANKL, which opens up a common, practical and useful therapeutic target for diverse osteolytic conditions.
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http://dx.doi.org/10.1007/s00418-018-1639-zDOI Listing
April 2018

Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone.

Histochem Cell Biol 2018 Apr 22;149(4):365-373. Epub 2018 Jan 22.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.
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http://dx.doi.org/10.1007/s00418-018-1641-5DOI Listing
April 2018

Developmental Contribution of Wnt-signal-responsive Cells to Mouse Reproductive Tract Formation.

Acta Histochem Cytochem 2017 Aug 10;50(4):127-133. Epub 2017 Aug 10.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

In mammals, the müllerian duct (MD) is an embryonic tubular structure that gives rise to the female reproductive tract (FRT). The MD originates from the coelomic epithelium (CoE) and takes on a rostral to caudal shape to establish the primary structure of the FRT under the regulation of morphogenetic signals. During these developmental processes, the MD and its derivatives require proper regulation of the Wnt-signaling-pathway. Here, to investigate the developmental contribution of FRT primordia under the influence of the Wnt-signaling, genetic lineage tracing was carried out using TopCreER/Rosa-LacZ mice to follow the fate of Wnt-signal-responsive cells during reproductive tract formation. TopCreER-marked-LacZ+ cells, arising from the Wnt-signal-responsive progenitors in CoE, give rise to spatially restricted MD and the uterine luminal epithelium. Similarly, the progeny from LacZ+ mesenchymal cells surrounding the MD contribute to both the uterine smooth muscle and stroma. Furthermore, in males, the Wnt-signal-responsive MD mesenchyme develops into the epididymis. These results show, for the first time, evidence of the sequential involvement of reproductive tract progenitors under the influence of Wnt-signal throughout the developmental term. This study provides a precise outline for assessing the lineage relation between the reproductive tract and the cell fate of its primordia in a temporally regulated manner.
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http://dx.doi.org/10.1267/ahc.17017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593815PMC
August 2017

Primary Type3 (Non-ABC, Non-GCB) Subtype of Extranodal Diffuse Large B-Cell Lymphoma of the Thyroid Bearing No Mutation by Padlock Probe Hybridization.

Case Rep Oncol 2017 May-Aug;10(2):508-514. Epub 2017 Jun 14.

Department of Molecular Pathology, Ehime University, Graduate School of Medicine, Toon City, Japan.

Primary extranodal malignant lymphoma of the thyroid is a rare entity composed of mostly neoplastic transformation of germinal center-like B cells (GCB) or memory B cells. Other B-cell-type malignancies arising primarily in the thyroid have rarely been described. Immunohistochemical examination of autopsied primary malignant lymphoma of the thyroid in an 83-year-old Japanese female revealed the presence of a non-GCB subtype of diffuse large B-cell lymphoma (DLBCL) without the typical codon 206 or 265 missense mutation of . The lack of the highly oncogenic gene mutation, frequently observed in DLBCL of the activated B-cell (ABC) subtype, and the detection of an extremely aggressive yet local clinical phenotype demonstrated that the present case was an exceptional entity of the type3 (non-GCB and non-ABC) subtype.
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http://dx.doi.org/10.1159/000477489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498938PMC
June 2017

Histological assessment of the efficacy of drug-eluting beads in portal tumor thrombosis of hepatocellular carcinoma.

Radiol Case Rep 2017 Mar 27;12(1):179-184. Epub 2016 Dec 27.

Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.

A 58-year-old man was diagnosed with advanced hepatocellular carcinoma with portal vein tumor thrombosis (PVTT). The tumors were multiple and existed in both lobes. Drug-eluting beads transcatheter arterial chemoembolization (DEB-TACE) was performed for the tumors in the left lobe. Embosphere and Hepasphere were selected for embolization of the arterioportal shunt, followed by loaded epirubicin infusion into the left hepatic artery. Computed tomography showed reduction of PVTT. However, liver failure progressed, and the patient died 67 days after DEB-TACE. Autopsy showed that the beads reached the tumor thrombosis in the portal vein. The prognosis of hepatocellular carcinoma with PVTT is poor. Although there are no established treatments for unresectable PVTT, DEB-TACE might be a useful option for such cases.
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http://dx.doi.org/10.1016/j.radcr.2016.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310373PMC
March 2017

Two Young Women with Left-sided Pneumothorax Due to Thoracic Endometriosis.

Intern Med 2016;55(23):3491-3493. Epub 2016 Dec 1.

Department of Surgery, National Hospital Organization Ehime Medical Center, Japan.

Pneumothorax associated with thoracic endometriosis (TE) generally occurs in women around 30 years old and it usually affects the right pleural cavity. We herein report two cases of TE associated with left-sided pneumothorax in young women. The prevalence of TE in younger patients may be underestimated if these cases are treated as spontaneous pneumothorax. Pneumothorax occurring in younger patients has not been reported to show laterality. TE-related or catamenial pneumothorax in young women must therefore represent a different clinical entity from the condition seen in older patients.
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http://dx.doi.org/10.2169/internalmedicine.55.7187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216149PMC
January 2017

sFRP4-dependent Wnt signal modulation is critical for bone remodeling during postnatal development and age-related bone loss.

Sci Rep 2016 04 27;6:25198. Epub 2016 Apr 27.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

sFRP4 is an extracellular Wnt antagonist that fine-tunes its signal activity by direct binding to Wnts. Bone fragility under oxidative stress by diabetes and aging is partly related to the suppression of the Wnt signal through upregulated sFRP4. Here, to explore the functions of sFRP4 as a balancer molecule in bone development and remodeling, we analyzed the sFRP4 knock-in mouse strain. X-gal and immunohistochemically stained signals in sFRP4-LacZ heterozygous mice were detectable in restricted areas, mostly in osteoblasts and osteoclasts, of the femoral diaphysis after neonatal and postnatal stages. Histological and μCT analyses showed increased trabecular bone mass with alteration of the Wnt signal and osteogenic activity in sFRP4 mutants; this augmented the effect of the buildup of trabecular bone during the ageing period. Our results indicate that sFRP4 plays a critical role in bone development and remodeling by regulating osteoblasts and osteoclasts, and that its functional loss prevents age-related bone loss in the trabecular bone area. These findings imply that sFRP4 functions as a key potential endogenous balancer of the Wnt signaling pathway by efficiently having direct influence on both bone formation and bone absorption during skeletal bone development and maintenance through remodeling.
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http://dx.doi.org/10.1038/srep25198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846872PMC
April 2016

Phospholipase Cϵ Activates Nuclear Factor-κB Signaling by Causing Cytoplasmic Localization of Ribosomal S6 Kinase and Facilitating Its Phosphorylation of Inhibitor κB in Colon Epithelial Cells.

J Biol Chem 2016 Jun 6;291(24):12586-12600. Epub 2016 Apr 6.

Division of Molecular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Electronic address:

Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon.
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http://dx.doi.org/10.1074/jbc.M116.717561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933442PMC
June 2016

Parathyroid Hormone-related Peptide-producing Multiple Myeloma and Renal Impairment.

Intern Med 2015 1;54(23):3029-33. Epub 2015 Dec 1.

Department of Nephrology, Kurashiki Central Hospital, Japan.

A 68-year-old man was hospitalized and examined for renal impairment. A laboratory analysis showed hypercalcemia. Although the serum parathyroid hormone and serum 1-25(OH)2 vitamin D3 levels were not elevated, the serum parathyroid hormone-related peptide (PTHrP) level was increased. Immunoelectrophoresis of the urine and bone marrow aspiration indicated multiple myeloma (MM). He was diagnosed with the coexistence of cast nephropathy and light chain deposition disease by a renal biopsy. Notably, PTHrP expression was detected in the myeloma cells based on immunohistochemistry and in situ hybridization. It is therefore important to examine the PTHrP concentration in MM patients with hypercalcemia.
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http://dx.doi.org/10.2169/internalmedicine.54.5085DOI Listing
August 2016

Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory.

Acta Histochem Cytochem 2015 Aug 20;48(4):115-24. Epub 2015 Aug 20.

Department of Molecular Pathology, Ehime University Graduate School of Medicine.

To explore the epigenetic mechanism that reactivates CDX2 (a homeobox transcription factor that serves as a tumor-suppressor gene) in intestinal-type gastric cancer during cancer progression, we examined the methylation status of the CDX2 gene promoter and the expression pattern of methyl-CpG binding protein-2 (MeCP2). From archives of the pathology records of surgically excised advanced stomach cancer cases in the Department of Molecular Pathology, Ehime University in a past decate (n=265), 10 cases of intestinal-type tubular adenocarcinoma, well-differentiated type (wel) with minor poorly-differentiated adenocarcinoma (por) components were selected. The expression pattern of CDX2, MUC2 and MeCP2 in these 10 cases was analyzed by immunohistochemistry. The cancerous and non-cancerous areas were selectively obtained by microdissection, and the methylation status of the CDX2 promoter of each area was assessed by methylation-specific polymerase chain reaction (MSP). In all 10 cases, CDX2 expression was clearly observed in the nucleus of the non-cancerous background of the intestinal metaplasic area, where the unmethylation pattern of the CDX2 gene promoter prevailed with reduced MeCP2 expression. In this metaplastic area, CDX2 expression was co-localized with its target gene, MUC2. CDX2 expression then disappeared from the deep invasive wel area. Reflecting the reduced CDX2 expression, microdissected samples from all the wel areas showed hypermethylation of the CDX2 gene promoter by MSP, with prominent MeCP2 expression. Interestingly, while hypermethylation of the CDX2 gene promoter was maintained in the por area in 8 of the 10 cases, CDX2 expression was restored in por areas where MeCP2 expression was markedly and selectively reduced. The other two cases, however, showed a constant MeCP2 expression level comparable to the surrounding deep invasive wel area with negative CDX2 expression. Therefore, gene silencing by hypermethylation may be overcome by the reduction of methyl-CpG binding proteins, resulting in apparent but non-functional reactivation of CDX2 as a mere molecular mark for gene silencing memory.
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http://dx.doi.org/10.1267/ahc.15014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564377PMC
August 2015

A Case of Mediastinal Granular Cell Tumor with Horner's Syndrome.

Ann Thorac Cardiovasc Surg 2015 2;21(6):567-9. Epub 2015 Jun 2.

Center of Chest Medicine and Surgery, Ehime University, Toon, Ehime, Japan.

Granular cell tumor (GCT) is found in various organs but is rare in the mediastinum. We report a case of mediastinal GCT in a 19-year-old woman who presented with left ptosis and miosis. CT and MRI revealed a 29-mm well-circumscribed tumor located close to the first thoracic vertebra with features suggesting a neurogenic tumor. The tumor was completely excised using single-port video-assisted thoracoscopic surgery. Histopathological and immunohistochemical analysis revealed that the tumor was a benign GCT. Postoperatively, left ptosis and miosis had improved slightly. To our knowledge, this is the first report regarding mediastinal GCT presenting with preoperative Horner's syndrome.
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http://dx.doi.org/10.5761/atcs.cr.15-00112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905036PMC
October 2016

Epigenetic regulation of Tbx18 gene expression during endochondral bone formation.

Cell Tissue Res 2015 Feb 8;359(2):503-512. Epub 2014 Nov 8.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Endochondral bone formation is tightly regulated by the spatial and sequential expression of a series of transcription factors. To disclose the roles of TBX18, a member of the T-box transcription factor family, during endochondral bone formation, its spatial and temporal expression patterns were characterized in the limb skeletal region of the developing mouse together with those of established osteochondrogenic markers Sox9, Col2a1, and Runx2. TBX18 expression first appeared in condensed mesenchymal cells (chondro-progenitors) in embryonic-day-10.5 (E10.5) limb bud and was co-localized with Sox9 expression, whereas at E11.5 and E12.5, it became undetectable in mesenchymal cells committed to the chondrocyte lineage. From E13.5 to E18.5, TBX18 expression reappeared in chondrocytes, correlating strongly with Col2a1 expression; furthermore, low level TBX18 expression was found in the Runx2-positive perichondral osteoblastic cell lineage. At the postnatal stage, TBX18 expression was observed in epiphyseal chondrocytes and osteocytes within the lacunae of mature trabecular bone. On the assumption that such characteristic Tbx18 gene expression is epigenetically regulated during mouse limb development, we examined the methylation status of the CpG-island in the mouse Tbx18 gene by methylation-specific polymerase chain reaction. Hypermethylation of the Tbx18 gene promoter became evident at an early embryonic stage in TBX18-negative cells and then disappeared at a late embryonic stage in TBX18-positive cells. Therefore, the temporal suppression of Tbx18 gene expression by the hypermethylation of its promoter seems to trigger the differentiation of mesenchymal cells into hypertrophic chondrocytes in the early stages of endochondral ossification.
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http://dx.doi.org/10.1007/s00441-014-2028-0DOI Listing
February 2015
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