Yasuyuki Katayama

Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures

We examined the mode of action of bisphosphonates on osteoclastic cell recruitment using mouse marrow cultures with or without osteoblastic cells. Tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] formed in cultures were determined to be osteoclastic cells. In marrow cultures, TRAP(+) MNC formation in the presence of 10(-8) mol/L 1,25(OH)2D3 was not affected by the addition of 10(-6) mol/L dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175). However, it was inhibited in cocultures of marrow cells with osteoblastic cells. The inhibitory effect was evident throughout the entire culture period. YM175 dose dependently inhibited TRAP(+) MNC formation, and other bisphosphonates--pamidronate and alendronate--also inhibited TRAP(+) MNC formation in the coculture. Similar observations were also made in the coculture of spleen cells with osteoblastic cells. The conditioned media of osteoblastic cells treated with 10(-6) mol/L YM175 inhibited TRAP(+) MNC formation in marrow cultures. The presence of YM175 in methylcellulose cultures affected neither the colony formation of monocyte-macrophage lineage, nor TRAP(+) MNC formation in the succeeding cocultures of recovered cells with osteoblastic cells. These results indicate that YM175 and probably other bisphosphonates as well preferentially inhibit the later stage of osteoclastogenesis through its action on osteoblastic cells. Our findings suggest that part of the inhibitory action by osteoblastic cells in the presence of bisphosphonates is mediated through soluble factor(s).

Urinary type IV collagen as a marker for early diabetic nephropathy

We investigated the urinary secretion of type IV collagen in 115 subjects with non-insulin-dependent diabetes mellitus (NIDDM) without macroproteinuria, 34 normal healthy subjects and 19 subjects with chronic glomerulonephritis (CGN). We examined the relation between the urinary level of type IV collagen and various clinical parameters. The urinary level of type IV collagen was significantly elevated in NIDDM subjects compared with normal subjects (4.88 +/- 3.12 vs. 1.7 +/- 1.25 micrograms/gCr, P < 0.001). The urinary level of type IV collagen was increased even in NIDDM subjects with normoalbuminuria. The ratio of urinary type IV collagen was significantly lower in subjects with chronic glomerulonephritis (CGN) than those in NIDDM subjects (P < 0.001), although there was no significant difference in the urinary level of type IV collagen between NIDDM and CGN subjects. The ratio of urinary type IV collagen to albumin was under 10.0 x 10(-6) in all subjects with CGN. Our results suggest that measurement of the urinary level of type IV collagen is useful for detection of early diabetic nephropathy and for the differential diagnosis of diabetic nephropathy and chronic glomerulonephritis.

The Mouse Mammary Tumor Cell Line, MMT060562, Produces Prostaglandin E2 and Leukemia Inhibitory Factor and Supports Osteoclast Formation In Vitro Via a Stromal Cell–Dependent Pathway

Osteoclastic bone resorption increases at the site of bone metastasis, but little is known about how tumor cells induce osteoclast (OC) recruitment in the bone marrow microenvironment. To clarify this point, we examined the effects of various mouse tumor cells on OC recruitment using cocultures of tumor cells and mouse marrow cells. The mouse mammary tumor cell lines, MMT060562 (MMT), BALB/c‐MC, Jyg‐MC(A), or other nonmammary tumor cell lines, LLC and B16, were cocultured with mouse marrow cells, and OC recruitment from marrow cells was determined by counting the number of tartrate‐resistant acid phosphatase–positive multinucleated cells (TRAP(+) MNCs) formed. Of the tumor cells examined, MMT and BALB/c‐MC stimulated OC formation, but other tumor cells did not. OC formation with MMT was dependent on the number of MMTs inoculated, and only ten cells per well were sufficient to induce OC development. OCs appeared on day 4, and the number reached a maximum on days 5–8 and decreased thereafter. TRAP(+) MNCs induced by MMT satisfied the major criteria of OCs, such as the presence of calcitonin receptors and the ability to resorb calcified tissues. The majority of OCs were formed adjacent to the stromal cells, which were positive for alkaline phosphatase. When spleen cells were cocultured with MMT, no OCs were formed. In contrast, when osteoblastic cells were added to cocultures of spleen cells and MMT, many OCs were formed. The cultured media (CM) of MMT induced OC formation in mouse marrow cultures. Neither parathyroid hormone–like nor interleukin 1‐like activity was present in the CM. MMT constitutively produced prostaglandin E2 (PGE2) and OC formation in cocultures was completely inhibited by indomethacin. Fractionation of the CM of MMT by ultrafiltration indicated that the OC‐inducing activities were present not only in the fraction with molecular weight below 3 kDa but also in the fraction with molecular weight above 3 kDa. OC‐inducing activity with high molecular weight was eluted around 50 kDa by Bio‐Gel P‐60 column chromatography. The active fractions also possessed leukemia inhibitory factor (LIF) activity, and OC‐inducing activity of the peak fraction was inhibited in the presence of anti‐LIF neutralizing antibody. The results of this study indicated that MMTs release PGE2 and LIF, which in turn stimulate OC formation via a stromal cell–dependent pathway. These culture systems will help to clarify the mechanisms by which tumor cells induce OC formation in a bone marrow microenvironment.

Homozygous missense mutation (band 3 Fukuoka: G130R): a mild form of hereditary spherocytosis with near-normal band 3 content and minimal changes of membrane ultrastructure despite moderate protein 4.2 deficiency

The characteristics of phenotypic expression were studied in a Japanese family with hereditary spherocytosis and an extremely rare homozygous missense mutation of the band 3 gene (band 3 Fukuoka: G130R). The homozygous unsplenectomized proband was a 29‐year‐old male with compensated haemolytic anaemia (red cell count 4.21 × 1012/l, reticulocytes 278 × 109/l, and indirect bilirubin 44 μmol/l). His red cell band 3 (B3) protein demonstrated a 9.3% reduction and his protein 4.2 (P4.2) level was substantially reduced (45.0%), compared to normal subjects. P4.2 protein was composed mostly of a wild type (72 kD) with a trace of 68 kD peptide. The binding properties of the mutated B3 to normal P4.2 were significantly impaired, which probably resulted in the substantial reduction of P4.2 in this proband, since no abnormalities were detected on the P4.2 gene. Electron microscopy (EM) using the freeze‐fracture method demonstrated a mild decrease in intramembrane particles (IMPs) of near‐normal size (8 nm in diameter) with no substantial increases in their oligomerization. Their distribution on the membrane P face was almost normal, although most of the IMPs could represent the homozygously mutated B3 protein. EM (quick‐freeze deep‐etching method) disclosed a skeletal network of near‐normal size and size distribution of the skeletal units, suggesting that the mutated B3 protein itself did not have much effect on the skeletal network in situ. Therefore the reduced P4.2 content (45% of that of normal subjects), which remained on the red cell membrane of this proband, appeared to be nearly sufficient for maintaining the normal structure of the skeletal network and IMPs in situ, contrary to the marked abnormalities in both IMPs and the skeletal network in complete P4.2 deficiencies.

Chinese Hamster Ovary Cells Expressing α4β1 Integrin Stimulate Osteoclast Formation In Vitro

It is reported that Chinese hamster ovary cells transfected with human α4 cDNA (α4CHOs) and expressing functional α4β1 integrin developed bone metasasis in nude mice. To clarify the role of α4β1 integrin in bone metastasis, in terms of tumor‐mediated bone destruction, we examined whether α4CHOs stimulate osteoclast formation in cocultures with mouse bone marrow cells. The number of osteoclast‐like cells identified as tartrate‐resistant acid phosphatase positive multinucleated cells (TRAP(+) MNCs) formed from bone marrow cells increased with the increasing number of α4CHOs cocultured. The effects of 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) and prostaglandin E2 (PGE2) on TRAP(+) MNC formation were enhanced in cocultures with α4CHOs. TRAP(+) MNCs induced by α4CHOs possessed calcitonin receptors and resorbed calcified tissues. In cocultures, α4CHOs and bone marrow stromal cells were in contact with each other and bone marrow stromal cells expressed vascular cell adhesion molecule‐1 (VCAM‐1), which is one of the ligands for α4β1 integrin. TRAP(+) MNC formation was not stimulated in cocultures where direct contact between α4CHOs and bone marrow cells was inhibited by membrane filters. α4CHOs do not support TRAP(+) MNC formation in cocultures with spleen cells but do support TRAP(+) mononuclear cell and MNC formation from spleen cells in the presence of osteoblastic cells. Cultured media from α4CHOs, bone marrow cells, and cocultures of α4CHOs and bone marrow cells did not stimulate TRAP(+) MNC formation or enhance the effects of 1,25(OH)2D3 and PGE2 in bone marrow cultures. The concentrations of PGE2 and interleukin‐6 (IL‐6) in cultured media were not different between the cultures of bone marrow cells and the cocultures of bone marrow cells and α4CHOs. Anti‐human α4 and anti‐mouse VCAM‐1 antibodies inhibited TRAP(+) MNC formation induced by α4CHOs. These results indicate that α4CHOs stimulated TRAP(+) MNC formation through direct cell‐to‐cell interaction between α4β1 and VCAM‐1. It is suggested that in addition to various soluble factors regulating osteoclast formation, cell‐to‐cell interaction between tumor cells and bone marrow cells is important for inducing osteoclasts at the site of bone metastasis and leading to bone destruction.