Inhibition of the proteasomal function in chondrocytes down-regulates growth plate chondrogenesis and longitudinal bone growth.

The proteasome is a large multiprotein complex that processes intracellular proteins functioning as cell cycle regulators and transcription factors. It has been shown that the chymotryptic component of the proteasome is an important regulator of osteoblast differentiation and bone formation, with inhibitors of the proteasome increasing osteoblast differentiation and bone formation. Yet, little is known about the effects of the proteasomal activity in the growth plate. In the present study, we cultured rat metatarsal bones in the presence of proteasome inhibitor I (PSI), a known inhibitor of the chymotrypsin-like activity of the 20S proteasome. PSI suppressed growth plate chondrocyte proliferation and hypertrophy/differentiation, and induced chondrocyte apoptosis. All these cellular effects led to reduced metatarsal linear growth. In cultured chondrocytes, PSI increased the expression of beta-catenin (a negative regulator of chondrogenesis) and reduced the DNA binding of nuclear factor kappaB, a transcription factor that stimulates growth plate chondrogenesis. In conclusion, our findings suggest that the proteasomal activity facilitates growth plate chondrogenesis and, in turn, longitudinal bone growth.

A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells.

Genetic diseases and knockout mice stress the importance of matrix metalloproteinases (MMPs) in skeletal turnover. Our study aims at clarifying which MMPs are expressed by osteoclasts. Previous analyses of this basic question led to conflicting reports in the literature. In the present study, we used a variety of approaches: PCR, Northern blots, Slot blots, in situ hybridization, and immunohistochemistry. We analyzed osteoclasts in culture as well as osteoclasts in native bone at different locations and compared mouse and rabbit osteoclasts. Osteoclasts express MMP-9 and -14 in all conditions, although to a variable extent, and they are able to synthesize MMP-3, -10, and -12, at least under some circumstances. The induction of a given MMP in osteoclasts is influenced by its environment (e.g., osteoclast culture vs. native bone, and various sites within the same bone) and depends on the species (e.g., mouse vs. rabbit). Osteoclasts show high amounts of MMP-2 and -13 protein presumably made to a large extent by other cells, thereby documenting how proteinases of nonosteoclastic origin may contribute to osteoclast activities and giving insight in why the resorptive activity of purified osteoclasts appears insensitive to MMP inhibitors. Our study shows that the confusion about osteoclastic MMPs in the literature reflects the remarkable ability of osteoclasts to adapt to their environment, as required by the structural or functional diversity of bone tissue. Our observations provide basic information needed for understanding the emerging role of MMPs in controlling cell signaling and bone resorption.

Intermittent compressive load stimulates osteogenesis and improves osteocyte viability in bones cultured "in vitro".

The effect of mechanical stresses on osteogenesis, the viability of osteocytes and their metabolic activity in organ culture of bones intermittently loaded "in vitro" are reported. Metatarsal bones, isolated from 12-day-old rats, were cultured in BGJb medium (with 10% foetal calf serum, 75 micrograms/ml of ascorbic acid, 100 U/ml of penicillin and 100 micrograms/ml of streptomycin), in humidified air enriched by 5% CO2 and 30% O2, and loaded in our original device for 1/2 an hour at 1 Hz. homotypic isolated and unloaded bones, cultured in the same medium, were taken as controls. The ALP (alkaline phophatase activity) increases in the media of loaded bones in comparison with the control bones. The percentage of viable osteocytes is significantly greater in loaded than in control bones. TEM observations demonstrate that in both loaded and control unloaded bones, osteocytes show well developed organelle machinery and several gap junctions with adjacent cellular processes. In the cells of loaded bones, however, a higher number of cytoplasmic organelles and gap junctions were found. In particular, RER increases twice, gap junctions three times. The induced osteogenesis and the TEM observations demonstrate the suitability of this experimental model and support the recent advanced hypothesis according to which the mechanical loading may exert a trophic function on osteocytes, stimulating both the proteic synthesis in the above-mentioned cells and the cell-to-cell communication. Furthermore, the loading is likely to exert a biological stimulus on osteoblasts via signalling molecules produced by osteocytes.

Endochondral bone formation in embryonic mouse pre-metatarsals.

Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.

The metatarsal cytochemical bioassay of parathyroid hormone: validation, specificity, and application to the study of pseudohypoparathyroidism type I.

The most sensitive method for assaying the bioactivity of PTH in unextracted plasma is the renal cytochemical bioassay. However, PTH acts on bone as well as kidney and clinical studies have suggested that the actions of circulating PTH level may be different at the two sites. We developed cytochemical bioassay for PTH based on the stimulation of glucose 6-phosphate dehydrogenase activity in the hypertrophic chondrocytes of the growth plate and the osteoblasts lining the metaphyseal trabeculae of rat metatarsal bones. The index of precision was 0.14 +/- 0.02 (SE) and the interassay variation was 31%. With this assay, plasma bioactive PTH levels in normal subjects and patients with primary hyperparathyroidism ranged from 0.5-18 ng/L and from 27-850 ng/L, respectively. Studies of patients with pseudohypoparathyroidism type I indicated that plasma PTH bioactivity in such patients is greater in the metatarsal bioassay than in the renal bioassay; no such differences were found in normal subjects or patients with primary hyperparathyroidism.