MinQi Li - Academia.edu (original) (raw)

Papers by MinQi Li

Archives of Histology and Cytology, 2005

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain ... more Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

Biomedical Research-tokyo, 2007

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteo... more The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

Febs Letters, 2006

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells... more Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic b-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIFK/K) mice. Compared with wild-type (WT) mice, MIFK/K mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIFK/K mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIFK/K mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIFK/K mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIFK/K mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIFK/K mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIFK/K mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Micron, 2005

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast form... more Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colonystimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization. q

PLOS One, 2010

Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and ce... more Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells.

Bone, 2011

To elucidate the histological events that follow administration of eldecalcitol, a second-generat... more To elucidate the histological events that follow administration of eldecalcitol, a second-generation of vitamin D analog currently awaiting approval as a drug for treatment of osteoporosis, we employed the ovariectomy (OVX) rat model. OVX rats received vehicle or 30 ng/kg of eldecalcitol, and sham-operated animals received vehicle only. Rats were sacrificed after 12 weeks and had their femora and tibiae removed and processed for histochemical and histomorphometrical analyses. When compared with OVX group, osteoclastic number and bone resorption parameters were significantly reduced in eldecalcitol-treated rats, accompanied by decreased bone formation parameters. The preosteoblastic layer, with which osteoclastic precursors interact for mutual differentiation, was poorly developed in the eldecalcitol group, indicating less cell-to-cell contact between preosteoblasts and osteoclast precursors. Interestingly, eldecalcitol did promote a type of focal bone formation that is independent of bone resorption, a process known as bone minimodeling. While the number of ED-1positive macrophages was higher in the bone marrow of treated rats, though osteoclastic number was deceased. Taken together, our findings suggest that eldecalcitol stimulates preosteoblastic differentiation rather than their proliferation, which in turn may prevent or diminish cell-to-cell contact between preosteoblasts and osteoclastic precursors, and therefore, lead to lower osteoclast numbers and decreased bone resorption.

Biomedical Research-tokyo, 2005

Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenes... more Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, appears to facilitate proliferation of tumor cells after the onset of bone metastasis. In this study, we examined metastatic lesions in the femora of BALB/c nu/nu mice two weeks after intracardiac injection with human breast carcinoma MDA-231 cells. Histopathological observations showed the metastatic lesions close to the chondro-osseous junction, and revealed MDA-231 cells loosely intermingled with different cell types such as osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts accumulated in direct contact with or were close to alkaline phosphatase (ALPase)- or receptor activator of NF-kappaB ligand (RANKL)-positive osteoblastic cells. It seems likely that osteoclastogenesis is mediated through cell-to-cell contacts with ALPase- and RANKL-expressing osteoblastic cells. Formation of many capillaries lacking complete basal membranes and pericytes ratified the results of in situ hybridization, which revealed intense expression of VEGF in tumor nests, and therefore, indicated ongoing tumor-induced angiogenesis. The tumor cells possessed matrix metallo-proteinases (MMPs)-1 and -9, and frequently extended their stout cytoplasmic processes into fragmented fibrillar components of the growth plate cartilage, implicating degradation of cartilaginous matrix. Thus, osteolytic bone metastasis has demonstrated pathological features as tumor-induced angiogenesis and degradation of extracellular matrix, in addition to osteoclastogenesis. This complex interplay between tumor cells and host tissues may enable and nourish the establishment of a microenvironment that facilitates tumor progression.

Journal of Bone and Mineral Research, 2006

The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular... more The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP-3, -9, and -13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo.

Microscopy Research and Technique, 2006

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of u... more The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25–30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc. Res. Tech. 69:73–83, 2006. © 2006 Wiley-Liss, Inc.

Molecular Endocrinology, 2005

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves a... more Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR ؊/؊ ) were signifi-cantly lower than those of wild-type (GIPR ؉/؉ ) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR ؊/؊ mice, indicating that GIPR ؊/؊ mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR ؊/؊ mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone. (Molecular Endo-

Journal of Bone and Mineral Metabolism, 2007

Giving the complexity that characterizes the mechanisms of bone remodeling and the number of even... more Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoen's silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.

Microscopy Research and Technique, 2008

This study aimed to evaluate the histological characteristics of the new bone formed at dental im... more This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R®). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R®. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R®-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R® surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R® are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc.

Biomedical Research-tokyo, 2007

We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem) can induce cortic... more We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem) can induce cortical bone formation, which is essential for long-lasting bone augmentation. Standardized bone defects in rat calvariae were covered with a convexly-shaped DeltaSystem plate, and then processed for histological observations. At 1 week, alkaline phosphatase-positive osteoblasts were seen in the newly-formed bone extending from the cavity's bottom, indicating accelerated osteogenesis. A thick layer of soft connective tissue positive for periostin, a hallmark of periosteum, covered this new bone. At 2 weeks, a spongy bone had filled the cavity up to half its height. The inner layer of the soft tissue facing the spongy bone revealed abundant periostin and osteopontin, and had many tartrate-resistant acid phosphatase-positive osteoclasts. At 4 weeks, this layer had given rise to thin new bony matrices without relation to the spongy bone arising from the cavity. These bone matrices had been thickened by 8 weeks, and turned into a thick cortical bone outlining the regenerated bone at 12 weeks. Thus, our study has provided histological evidences of cortical osteogenesis when DeltaSystem plates are used for bone augmentation procedures.

Journal of Bone and Mineral Research, 2009

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after inter... more This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.

Biomedical Research-tokyo, 2008

A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PT... more A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), i.e., PTH-R, has been discovered in human Blomstrand's lethal chondrodysplasia. As skeletal deformities in this type of chondrodysplasia appear to compromise the receptor binding to its ligands, we examined the possibility that rat PTH-R carrying P132L mutation (PTH-R(P132L)) would result in abnormal intracellular localization. Osteoblastic MC3T3-E1 cells were transfected with expression vectors containing cDNAs encoding either wild-type PTH-R or mutant PTH-R(P132L). The cells expressing the wild-type PTH-R produced a receptor protein with a molecular mass of 66.3 kDa, which localized its immunoreactivity mainly on the cell surfaces. In contrast, the PTH-R(P132L) was hardly detected on the cell surfaces, but accumulated within the rough-surfaced endoplasmic reticulum. Consistent with this localization, the cells expressing the mutant receptor failed to generate cyclic AMP in response to PTH. Furthermore, a remarkably weaker intensity of the 66.3 kDa band compared with the wild-type counterpart suggests that PTH-R(P132L) is prone to degradation in the transfected cells. In summary, these findings indicate that defective transport of PTH-R(P132L) to the cell surface would be a molecular basis for Blomstrand's chondrodysplasia.

Microscopy Research and Technique, 2004

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesi... more We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the “cambium layer” of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration. Microsc. Res. Tech. 64:330–342, 2004. © 2004 Wiley-Liss, Inc.

Microscopy Research and Technique, 2005

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralizat... more The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the “articular zone,” “articular layer,” and “resting zone” from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles. Microsc. Res. Tech. 67:325–335, 2005. © 2005 Wiley-Liss, Inc.

Bone, 2006

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of cer... more Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.

Cell Metabolism, 2007

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone i... more Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%-80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these ''osteocyte-less'' mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.

Journal of Oral Biosciences, 2004

ABSTRACT

Archives of Histology and Cytology, 2005

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain ... more Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

Biomedical Research-tokyo, 2007

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteo... more The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

Febs Letters, 2006

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells... more Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic b-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIFK/K) mice. Compared with wild-type (WT) mice, MIFK/K mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIFK/K mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIFK/K mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIFK/K mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIFK/K mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIFK/K mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIFK/K mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Micron, 2005

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast form... more Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colonystimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization. q

PLOS One, 2010

Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and ce... more Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells.

Bone, 2011

To elucidate the histological events that follow administration of eldecalcitol, a second-generat... more To elucidate the histological events that follow administration of eldecalcitol, a second-generation of vitamin D analog currently awaiting approval as a drug for treatment of osteoporosis, we employed the ovariectomy (OVX) rat model. OVX rats received vehicle or 30 ng/kg of eldecalcitol, and sham-operated animals received vehicle only. Rats were sacrificed after 12 weeks and had their femora and tibiae removed and processed for histochemical and histomorphometrical analyses. When compared with OVX group, osteoclastic number and bone resorption parameters were significantly reduced in eldecalcitol-treated rats, accompanied by decreased bone formation parameters. The preosteoblastic layer, with which osteoclastic precursors interact for mutual differentiation, was poorly developed in the eldecalcitol group, indicating less cell-to-cell contact between preosteoblasts and osteoclast precursors. Interestingly, eldecalcitol did promote a type of focal bone formation that is independent of bone resorption, a process known as bone minimodeling. While the number of ED-1positive macrophages was higher in the bone marrow of treated rats, though osteoclastic number was deceased. Taken together, our findings suggest that eldecalcitol stimulates preosteoblastic differentiation rather than their proliferation, which in turn may prevent or diminish cell-to-cell contact between preosteoblasts and osteoclastic precursors, and therefore, lead to lower osteoclast numbers and decreased bone resorption.

Biomedical Research-tokyo, 2005

Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenes... more Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, appears to facilitate proliferation of tumor cells after the onset of bone metastasis. In this study, we examined metastatic lesions in the femora of BALB/c nu/nu mice two weeks after intracardiac injection with human breast carcinoma MDA-231 cells. Histopathological observations showed the metastatic lesions close to the chondro-osseous junction, and revealed MDA-231 cells loosely intermingled with different cell types such as osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts accumulated in direct contact with or were close to alkaline phosphatase (ALPase)- or receptor activator of NF-kappaB ligand (RANKL)-positive osteoblastic cells. It seems likely that osteoclastogenesis is mediated through cell-to-cell contacts with ALPase- and RANKL-expressing osteoblastic cells. Formation of many capillaries lacking complete basal membranes and pericytes ratified the results of in situ hybridization, which revealed intense expression of VEGF in tumor nests, and therefore, indicated ongoing tumor-induced angiogenesis. The tumor cells possessed matrix metallo-proteinases (MMPs)-1 and -9, and frequently extended their stout cytoplasmic processes into fragmented fibrillar components of the growth plate cartilage, implicating degradation of cartilaginous matrix. Thus, osteolytic bone metastasis has demonstrated pathological features as tumor-induced angiogenesis and degradation of extracellular matrix, in addition to osteoclastogenesis. This complex interplay between tumor cells and host tissues may enable and nourish the establishment of a microenvironment that facilitates tumor progression.

Journal of Bone and Mineral Research, 2006

The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular... more The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP-3, -9, and -13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo.

Microscopy Research and Technique, 2006

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of u... more The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25–30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc. Res. Tech. 69:73–83, 2006. © 2006 Wiley-Liss, Inc.

Molecular Endocrinology, 2005

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves a... more Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR ؊/؊ ) were signifi-cantly lower than those of wild-type (GIPR ؉/؉ ) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR ؊/؊ mice, indicating that GIPR ؊/؊ mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR ؊/؊ mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone. (Molecular Endo-

Journal of Bone and Mineral Metabolism, 2007

Giving the complexity that characterizes the mechanisms of bone remodeling and the number of even... more Giving the complexity that characterizes the mechanisms of bone remodeling and the number of events that have to be in absolute harmony for it to occur flawlessly, the postulation that temporospatial distribution of osteocytes and their lacunar canalicular system might influence and be influenced by bone remodeling can be regarded, at least, as feasible. In this study, using Schoen's silver staining, we have examined the distribution of the osteocytic lacunar canalicular system (OLCS) in bones of developing mice. Trabecular bones of 3-day-old, 2-week-old, and 3-week-old mice displayed osteocytic cytoplasmic processes without any perceptible alignment. Also, many plump osteocytes were embedded in the mineralized bone matrix in a disorderly manner. At 4 weeks of age, however, mice bones showed some osteocytic processes that reached the bone surface on a right angle, while other osteocytes displayed the same features seen on 3-week specimens. Samples at 8 weeks of age featured osteocytes with their usual spindle shape, organized so as to parallel the longitudinal axis of trabecular bone. They also extended their cytoplasmic processes perpendicularly to the bone surface. However, several osteocytes immersed in older bone, i.e., a residual mix of cartilage and bone matrices, still showed a random pattern of distribution of their cytoplasmic processes. Up to 12 weeks of age, the majority of the osteocytes became flattened and were shown to be aligned with their long axis paralleling the bone surface. This tendency for such a gradual arrangement was also observed in cortical bones. We have further demonstrated that 8-week-old osteoprotegerin-deficient mice, which demonstrated histological evidence of higher than average bone turnover, revealed a disorganized OLCS. Given the data gathered in this work, the OLCS appears to assume an organized, probably function-related spatial distribution as normal bone remodeling goes on.

Microscopy Research and Technique, 2008

This study aimed to evaluate the histological characteristics of the new bone formed at dental im... more This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R®). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R®. Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R®-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R® surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R® are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc.

Biomedical Research-tokyo, 2007

We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem) can induce cortic... more We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem) can induce cortical bone formation, which is essential for long-lasting bone augmentation. Standardized bone defects in rat calvariae were covered with a convexly-shaped DeltaSystem plate, and then processed for histological observations. At 1 week, alkaline phosphatase-positive osteoblasts were seen in the newly-formed bone extending from the cavity's bottom, indicating accelerated osteogenesis. A thick layer of soft connective tissue positive for periostin, a hallmark of periosteum, covered this new bone. At 2 weeks, a spongy bone had filled the cavity up to half its height. The inner layer of the soft tissue facing the spongy bone revealed abundant periostin and osteopontin, and had many tartrate-resistant acid phosphatase-positive osteoclasts. At 4 weeks, this layer had given rise to thin new bony matrices without relation to the spongy bone arising from the cavity. These bone matrices had been thickened by 8 weeks, and turned into a thick cortical bone outlining the regenerated bone at 12 weeks. Thus, our study has provided histological evidences of cortical osteogenesis when DeltaSystem plates are used for bone augmentation procedures.

Journal of Bone and Mineral Research, 2009

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after inter... more This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.

Biomedical Research-tokyo, 2008

A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PT... more A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), i.e., PTH-R, has been discovered in human Blomstrand's lethal chondrodysplasia. As skeletal deformities in this type of chondrodysplasia appear to compromise the receptor binding to its ligands, we examined the possibility that rat PTH-R carrying P132L mutation (PTH-R(P132L)) would result in abnormal intracellular localization. Osteoblastic MC3T3-E1 cells were transfected with expression vectors containing cDNAs encoding either wild-type PTH-R or mutant PTH-R(P132L). The cells expressing the wild-type PTH-R produced a receptor protein with a molecular mass of 66.3 kDa, which localized its immunoreactivity mainly on the cell surfaces. In contrast, the PTH-R(P132L) was hardly detected on the cell surfaces, but accumulated within the rough-surfaced endoplasmic reticulum. Consistent with this localization, the cells expressing the mutant receptor failed to generate cyclic AMP in response to PTH. Furthermore, a remarkably weaker intensity of the 66.3 kDa band compared with the wild-type counterpart suggests that PTH-R(P132L) is prone to degradation in the transfected cells. In summary, these findings indicate that defective transport of PTH-R(P132L) to the cell surface would be a molecular basis for Blomstrand's chondrodysplasia.

Microscopy Research and Technique, 2004

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesi... more We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the “cambium layer” of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration. Microsc. Res. Tech. 64:330–342, 2004. © 2004 Wiley-Liss, Inc.

Microscopy Research and Technique, 2005

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralizat... more The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the “articular zone,” “articular layer,” and “resting zone” from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles. Microsc. Res. Tech. 67:325–335, 2005. © 2005 Wiley-Liss, Inc.

Bone, 2006

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of cer... more Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.

Cell Metabolism, 2007

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone i... more Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%-80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these ''osteocyte-less'' mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.

Journal of Oral Biosciences, 2004

ABSTRACT