Akitoshi Jikko

Collagen Integrin Receptors Regulate Early Osteoblast Differentiation Induced by BMP‐2

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the α1β1 and α2β1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)‐2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP‐2 promoter driving SV40 T‐antigen. These cells require exogenous BMP‐2, as well as ascorbic acid and β‐glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen‐I affect BMP‐2 signaling, function‐perturbing anti‐rat α1 and/or α2 integrin subunit, or anti‐type I collagen (Col‐I), antibodies were added to human recombinant (hr)BMP‐2–treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti‐Collagen‐I or both anti–integrin‐α1 and ‐α2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5–1.8% control) of these cells. In all cases, adding anti‐α1 or anti‐α2 antibodies separately produced partial effects, while their combined effect approached that of anti‐Collagen‐I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR‐IB) showed elevated ALP activity without hrBMP‐2; this constitutive activity was also suppressed by α1 and α2 integrin antibodies and by anti‐Col‐I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR‐IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.

Cemento-osseous dysplasia of the jaws in 54 Japanese patients: A radiographic study

OBJECTIVE The aim of this study was to describe the radiographic patterns of cemento-osseous dysplasia. STUDY DESIGN Fifty-four patients affected with benign fibro-osseous jaw lesions that showed periapical radiopacities and/or radiolucencies in a focal or a multiplex form were studied. The clinical, radiographic, and histopathologic features of the patients with cemento-osseous dysplasia were retrospectively studied. Radiographic features of the cemento-osseous dysplasia lesions were classified according to the appearance of calcified bodies. Radiographic visibility of periodontal ligament spaces of related teeth was assessed. RESULTS Forty-nine (91 %) of the 54 patients were women. The mean age of the total group was 50.8 years, and that of the male group was 64.6 years. The cemento-osseous dysplasia lesions could be classified into 6 types radiographically. Eighteen patients had at least 2 or more types of cemento-osseous dysplasia lesions. Of 147 related teeth, 142 had periodontal ligament spaces clearly visible. Six of 9 patients who had a total of 25 teeth with active hypercementosis showed concomitant occurrence of other types of cemento-osseous dysplasia lesions. Biopsy specimens showed various amounts of bonelike and cementumlike tissues. CONCLUSIONS It is likely that cemento-osseous dysplasia consists of 3 variations of a single entity, all with the same unknown cause. In one variation, the entity originates from the periodontium; in another, it is of medullary bone origin; and in the third it results from the simultaneous involvement of both tissues.

Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor‐β1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it “cartducin”. Cartducin shares a similar modular organization to adipocyte‐derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole‐mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein‐2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo‐oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS‐2/8 cells in a dose‐dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes. J. Cell. Physiol. 206: 537–544, 2006.

EFFECTS OF INTERLEUKIN-6 ON PROLIFERATION AND PROTEOGLYCAN METABOLISM IN ARTICULAR CHONDROCYTE CULTURES

Interleukin‐6 (IL‐6) levels are markedly increased in the synovial fluid of patients with rheumatoid arthritis or osteoarthritis. However, the effects of IL‐6 on proliferation and proteoglycan metabolism in articular cartilage are not known. We demonstrated here the effects of human recombinant (hr) IL‐6 on proliferation and proteoglycan metabolism in rabbit articular chondrocyte cultures. In vitro, these cells proliferated and produced abundant extracellular matrices. We found that 1–10ng/ml of hrIL‐6 inhibited proliferation to approximately 65% of control levels and suppressed colony formation induced by bFGF in soft agarose. The same concentration of hrIL‐6 depressed proteoglycan synthesis to approximately 60% of control levels. Moreover, hrIL‐6 significantly enhanced proteoglycan degradation induced by hrIL‐1β, although hrIL‐6 alone did not affect proteoglycan degradation. These findings suggest that IL‐6 is a negative regulator for chondrocyte proliferation and articular cartilage metabolism.

EFFECT OF X-RAY IRRADIATION ON PROLIFERATION AND DIFFERENTIATION OF OSTEOBLAST

Summary We exposed the osteoblast-like cell line, MC3T3-E1, to 1-to 10-Gy X-ray. Irradiation at doses of 5-Gy dose or more decreased the DNA content of cells at the proliferation stage, confluence, and post-proliferation stages. The alkaline phosphatase activity, conversely, was increased by irradiation, and the calcium content of irradiated cells was greater than that of nonirradiated. These findings suggest that irradiation induces terminal differentiation and calcification of osteoblasts.

Inhibition of Chondrocyte Terminal Differentiation and Matrix Calcification by Soluble Factors Released by Articular Chondrocytes

Abstract. Chondrocytes do not undergo terminal differentiation in normal articular cartilage, whereas growth plate chondrocytes synthesize ALPase and induce matrix calcification terminally. Articular chondrocytes in osteoarthritic joints have been reported to express the terminal differentiation phenotypes, suggesting that terminal differentiation of articular chondrocytes is inhibited in normal joints. In the present study, we investigated the underlying inhibitory mechanism of the terminal differentiation in articular cartilage using a culture on type II collagen-coated dishes or a novel culture model on Millipore filters. ALPase activity increased from day 7 to day 8 in growth plate chondrocyte cultures on the collagen-coated dishes, but not in articular chondrocyte cultures. The ALPase expression of growth plate chondrocytes on the collagen-coated dish was completely inhibited when the same number of articular chondrocytes was mixed in the growth plate chondrocyte cultures. When articular chondrocytes or growth plate chondrocytes were maintained on Millipore filters held in 16-mm dishes, they started to synthesize ALPase. The ALPase expression of the chondrocytes on Millipore filters was inhibited by the presence of articular chondrocytes maintained on the bottom collagen-coated substratum in the same dishes. These results indicate that factors that diffused into the medium through the Millipore filters are involved in the inhibition of terminal differentiation. Since the conditioned medium from articular chondrocyte cultures did not affect the ALPase expression, it is considered that the soluble factors, which are continuously released from articular chondrocytes, are responsible for the inhibition of terminal differentiation.

Establishment of a novel chondrocytic cell line N1511 derived from p53-null mice

We established a clonal chondrocytic cell line N1511 derived from rib cartilage of a p53‐null mouse. N1511 cells proliferated in polygonal shape and elicited differentiation at confluence when treated with combination of bone morphogenetic protein (BMP) 2 and insulin or parathyroid hormone (PTH) and dexamethasone. BMP‐2/insulin‐treated cells became refractile without forming cartilaginous nodules and reached terminal differentiation, became positive for alizarin red staining, and developed considerable ALP activity. In contrast, PTH/dexamethasone‐treated cells formed Alcian blue‐positive nodules but remained negative for alizarin red staining and ALP activity. Northern blot analysis revealed that BMP‐2/insulin‐treated cells sequentially expressed type II, IX, and X collagens, whereas PTH/dexamethasone‐treated cells slowly expressed type II collagen and then type IX, and they did not exhibit type X collagen expression. These results show that BMP‐2/insulin treatment induces full differentiation toward hypertrophy, whereas treatment with PTH/dexamethasone slows and limits differentiation. Recovery of p53 expression in N1511 cells by transient transfection inhibited cell proliferation, suggesting that cell proliferation could be regulated with p53 in this cell line. These results indicate that N1511 is the only cell line with known genetic mutation, which undergoes multiple steps of chondrocyte differentiation toward hypertrophy, and because proliferation could be regulated by expression of p53, N1511 could be an excellent model for studies of chondrogenesis, the function of p53, and genetic engineering of cartilage tissue.

Analysis of genomic instability in squamous cell carcinoma of the head and neck using the random amplified polymorphic DNA method

Using the random amplified polymorphic DNA (RAPD) method, we identified genomic instability in head and neck squamous cell carcinoma (HNSCC) tissues. We extracted DNA from tumor and corresponding normal tissues of 30 HNSCC patients and amplified with ten random 10-mer arbitrary primers by the RAPD method. Genomic instabilities, which appeared as banding pattern changes between normal and tumor DNA, were detected by at least one primer in all tumor tissues. Moreover, there was significant correlation between the frequency of genomic instability and the degree of tumor differentiation. These results indicate a possible association of genomic instability with malignant potential of head and neck cancer.

Changes in phenotypic expression of osteoblasts after X irradiation.

Changes in the phenotypic expression of osteoblasts after X irradiation were investigated. Osteoblast-like MC3T3-E1 cells at the actively proliferating, confluent and postproliferation stages were subjected to 10 Gy X irradiation. Irradiation at the confluent stage enhanced accumulation of type I collagen normalized to the DNA content. Irradiation at all stages down-regulated the expression of osteocalcin, but the levels of osteopontin and osteonectin mRNAs were unchanged from the control level. After irradiation at the later stages, the time-dependent increase in alkaline phosphatase activity per cell exceeded that in the control cells. The localization of alkaline phosphatase-positive cells was concordant with that of calcification. In addition, the quality of the calcium deposits was found to be similar to that in control cells as determined by energy dispersive spectrometry and the ratio of calcium to phosphorus, even if the cells were not exactly the same morphologically. The changes in phenotypic expression observed here are closely related to the enhancement of calcification observed in a previous study.

Effects of X irradiation on metabolism of proteoglycans.

The effects of X irradiation on matrix formation by growth-plate and articular chondrocytes, as reflected by metabolism of proteoglycans and type II collagen, were examined in a rabbit chondrocyte culture system. Irradiation with 1 to 10 Gy selectively inhibited synthesis of proteoglycans (incorporation of [35S]sulfate) depending on the stage of differentiation of the irradiated cells; however, synthesis of type II collagen was not affected. Irradiation of an immature culture, in which chondrocytes had just reached confluence, suppressed incorporation of [35S]sulfate into the glycosaminoglycan, 10 Gy inducing approximately 45-50% inhibition. In contrast, the irradiation of mature cultures, in which chondrocytes had already secreted extensive cartilage matrix, did not affect the rate of synthesis of proteoglycans (incorporation of [35S]sulfate). We also found that here irradiation stimulated the degradation of proteoglycans, but with the effect differing in growth-plate chondrocytes and articular chondrocytes. In growth-plate chondrocytes, cleavage from a site close to the G1 globular domain induced by 10 Gy enhanced the release of 35S-labeled proteoglycans into the medium, whereas in articular chondrocytes, irradiation had only marginal effects on the release of 35S-labeled proteoglycans. Our results show that irradiation with 1-10 Gy impaired proteoglycan metabolism in cartilage, with differing effects according to the stage of cell differentiation and the type of chondrocyte.