It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.

In this study, the effects of fragmentation of the glycosoaminoglycans of the cell-associated matrix by hyaluronidase (HAase) on the expression of CD44 receptor and matrix metalloproteinase (MMP) mRNAs in cultured articular chondrocytes were examined. Chondrocytes, isolated from rabbit and bovine articular cartilage, were treated with bovine testicular HAase (0-200 units/ml) in the presence or absence of an antibody for CD44. The mRNA levels of CD44, CD44 variant (CD44v), MMPs (MMP-1, -3 and -9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined by RT-PCR. The treatment of cultured chondrocytes with HAase resulted in the production of low molecular weight fragments of hyaluronan (HA). The expression of CD44, CD44v and MMP (MMP-1, -3 and -9) mRNAs, but not TIMP-1 or TIMP-2 mRNA, was up-regulated in the cultures treated with HAase, whereas this expression was not affected by treatment with purified HA of 1.0 x 10(5) Da. Furthermore, the induction of CD44 and MMPs on treatment with HAase was suppressed by an anti-CD44 antibody. The results suggest that the fragmentation of HA may lead to cartilage destruction in terms of the enhanced expression of MMPs as well as the upregulation of CD44.

Hyaluronan (HA) synthesis in the synovial membrane is affected by various chemical mediators. It is hypothesized that transforming growth factor-beta 1 (TGF-beta 1) would be a mediator to modulate HA synthesis in cultured synovial membrane fibroblasts of the temporomandibular joint (TMJ). Fibroblasts were extracted from the TMJ synovial membrane of four-week-old Japanese white rabbits. The amount of HA and expression levels of HA synthase (HAS) mRNAs induced by TGF-beta 1 treatment were analyzed by means of high-performance liquid chromatography and real-time polymerase chain-reaction, respectively. Both medium and large amounts of HA were enhanced by the stimulation of TGF-beta 1. HAS2 mRNA expression was enhanced 13-fold after six-hour stimulation with TGF-beta 1 (10 ng/mL), whereas HAS3 mRNA expression was not changed significantly. These results suggest that TGF-beta 1 enhances the expression of HAS2 mRNA in the TMJ synovial membrane fibroblasts and may contribute to the production of high-molecular-weight HA in the joint fluid.

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4+ and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

During endochondral ossification, the production of hyaluronan (HA) is strictly and selectively regulated by chondrocytes, with a temporal peak at the hypertrophic stage. This study was conducted to clarify the effects of HA on expression and activity of runt-related gene 2 (Runx2), a potent transcription factor for chondrocyte differentiation in hypertrophic chondrocytes. Immature chondrocytes from an ATDC5 cell line were cultured and differentiated in DMEM/Ham's F12 with pre-defined supplements. Using real-time PCR, the gene expressions of type II collagen, MMP-13, HAS2, and Runx2 in cultured chondrocytes were analysed from days 0 to 18 of cell differentiation. The activity and expression of Runx2 in hypertrophic chondrocytes were analysed after the treatment with HA oligosaccharide (HAoligo) using AML-3/Runx2 binding, real-time PCR and Western blot analysis. The effects of pre-incubation of anti-CD44 antibody on Runx2 expression were also examined. Expression of type X collagen and Runx2 mRNAs reached a maximum at the terminal differentiation of chondrocytes. The activity and expression of Runx2 was significantly inhibited in hypertrophic chondrocytes treated with HAoligo compared to the untreated controls. High molecular weight-HA did not affect the expression or activity of Runx2. The expression of Runx2 mRNA was significantly decreased in hypertrophic chondrocytes treated with anti-CD44 antibody. These results suggest that HAoligo may affect the terminal differentiation of chondrocytes during the endochondral ossification by inhibiting the expression and activity of Runx2.