This study addressed the hypothesis that duration and magnitude of applied intermittent hydrostatic pressure (IHP) are critical parameters in regulation of normal human articular chondrocyte aggrecan and type I1 collagen expression. Articular chondrocytes were isolated from knee cartilage and maintained as primary, high-density monolayer cultures. IHP was applied at magnitudes of 1, 5 and 10 MPa at I Hz for durations of either 4 h per day for one day (4 x 1) or 4 h per day for four days (4 x 4). Total cellular RNA was isolated and analyzed for aggrecan and type I1 collagen mRNA signal levels using specific primers and reverse transcription polymerase chain reaction (RT-PCR) nested with beta-actin primers as internal controls. With a 4 x 1 loading regimen, aggrecan mRNA signal levels increased 1.3-and 1.5-fold at 5 and 10 MPa, respectively, relative to beta-actin mRNA when compared to unloaded cultures. Changing the duration of loading to a 4 x 4 regimen increased aggrecan mRNA signal levels by 1.4-, 1.8-and 1.9-fold at loads of 1, 5 and 10 MPa, respectively. In contrast to the effects of IHP on aggrecan, type I1 collagen mRNA signal levels were only upregulated at loads of 5 and 10 MPa with the 4 x 4 loading regimen. Analysis of cell-associated protein by western blotting confirmed that IHP increased aggrecan and type I1 collagen in chondrocyte extracts. These data demonstrate that duration and magnitude of applied IHP differentially alter chondrocyte matrix protein expression. The results show that IHP provides an important stimulus for increasing cartilage matrix anabolism and may contribute to repair and regeneration of damaged or diseased cartilage.

Whether non-steroidal anti-inflammatory drug (NSA1D)-induced suppression of bone ingrowth is due to cyclooxygenase-1 (COX-I ) inhibition, cyclooxygenase-2 (COX-?) inhibition, or through a yet unidentified pathway is unknown. In this study, the effects of a non-specific COX-1 and COX-2 inhibitor, versus a specific COX-2 inhibitor on bone ingrowth and tissue differentiation are examined in vivo. Harvest chambers were implanted unilaterally in the tibiae of eight mature, New Zealand white rabbits. After a 6-week period for osseointegration of the chamber, the following oral treatments were given for 4 weeks each, followed by a harvest in each case: drinking water with no NSAID (control l), Naproxen sodium-a COX-1 and COX-2 non-specific inhibitor at a dose of 110 mglkglday in the drinking water, drinking water with no NSAID (control 2), and Rofecoxib-a COX-2 inhibitor at a dose of 12.5 mglday inserted directly into the rabbit's mouth. Harvested specimens were snap frozen, cut into serial 6 prn sections and stained with hematoxylin and eosin for general morphological characterization, and alkaline phosphatase (osteoblast marker). Sections were also processed for immunoperoxidase staining using monoclonal antibodies to identify cells expressing the vitronectin receptor (osteoclast-like cells). With drinking water alone, the percentage of bone ingrowth averaged 24.8 f 2.9% and 29.9 f 4.5% respectively. Naproxen sodium in the drinking water and oral Rofecoxib decreased bone ingrowth significantly (1 5.9 f 3.3%, p = 0.031 and 18.5 i 2.4%, p = 0.035 compared to drinking water respectively). Both Naproxen sodium ( p = 0.016) and Rofecoxib ( p = 0.02) decreased the number of CD51 positive osteoclast-like cells per section compared with drinking water alone. Rofecoxib decreased the area of osteoblasts per section area ( p = 0.014) compared to controls, although the value for Naproxen sodium did not reach statistical significance. The results of the present study suggest that bone formation is suppressed by oral administration of an NSAID which contains a COX-' inhibitor. COX-2 inhibitors currently taken for arthritis and other conditions may potentially delay fracture healing and bone ingrowth.

Outer mitochondrial membrane cytochrome b 5 (OMb), which is an isoform of cytochrome b 5 (cyt b 5 ) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b 5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b 5 , in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxylterminal portion of these proteins determine their locations in the cell.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTHlPTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTHlPTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTHlPTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine andlor paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification. IntroductionDuring fracture healing, several cellular events occur simultaneously and/or sequentially: inflammatory reaction, chondrogenesis, intramembranous ossification and endochondral ossification, resulting in the formation of a fracture callus followed by bony union. These dynamic events of bone metabolism are biologically regulated and can be investigated representatively in animal fracture models [15]. It has been reported that various local regulators such as cytokines and growth factors are expressed during fracture healing suggesting they regulate proliferation and/or differentiation of cells involved in fracture healing [11,39]. Since many growth factors regulate biological reaction of cells in co-operation with other factors or modulate the function of other factors [9,10,36], these interaction between different local regulators may be important for the well-organized repair process of fracture healing.Parathyroid hormone-related peptide (PTHrP) was originally isolated from tumors associated with humoral hypercalcemia of malignancy [34]. It has been reported that PTHrP is expressed in a wide variety of fetal and neonatal tissues [8,37], and acts in an autocrine/paracrine fashion binding with the same receptor as parathyroid hormone (PTH) does [16]. During embryonal cartilage development, PTHrP is expressed in the perichondrium induced by Indian ...

Japan 1 (7)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166), a novel oral hypoglycaemic agent is a non-sulphonylurea insulin secretagogue. 2 We investigated the insulin-releasing action and hypoglycaemic e ect of A-4166 compared to sulphonylureas in vitro and in vivo. 3 A-4166 stimulated insulin secretion from rat freshly isolated pancreatic islets at concentrations from 3610 -6 M to 3610 -4 M in the presence of 2.8 mM glucose. There was no obvious di erence in glucose dependency between the insulinotropic e ect of A-4166 and that of glibenclamide, and no additive or synergistic e ect was observed between these two drugs. 4 A-4166 displaced [ 3 H]-glibenclamide bound to intact HIT-T15 cells in a concentration-dependent manner. The K i value was 4.34+0.04610 77 M, and the displacement potency of A-4166 was between that of glibenclamide and tolbutamide, being similar to that of gliclazide. 5 In fasted beagle dogs, A-4166 showed a dose-dependent hypoglycaemic e ect after oral administration over the range 1 to 10 mg kg 71 . The hypoglycaemic action of A-4166 showed an earlier onset and a shorter duration than that of sulphonylureas. 6 Simultaneous measurement of plasma insulin levels revealed that the hypoglycaemic e ect of A-4166 was caused by a rapid-onset and brief burst of insulin secretion. 7 The pharmacokinetic pro®le of A-4166 was consistent with the changes of the blood glucose and plasma insulin levels. 8 Although the in vitro insulin-releasing e ect of A-4166 was similar to that of sulphonylureas, its hypoglycaemic e ect was more rapid and shorter-lasting, associated with rapid absorption and clearance. Thus, A-4166 may be useful in suppressing postprandial hyperglycaemia in patients with non-insulindependent diabetes mellitus.

Onset and progression of cartilage degeneration is associated with shear stress occurring in diarthrodial joints subjected to inappropriate loading. This study tested the hypothesis that shear stress induced nitric oxide is associated with altered expression of regulatory onco-proteins, bcl-2, and Fas (APO-1/CD95) and apoptosis in primary human osteoarthritic chondrocyte cultures. Shear stress induced membrane phosphatidylserine and nucleosomal degradation were taken as evidence of chondrocyte apoptosis. Application of shear stress upregulated nitric oxide in a dose-dependent manner and was associated with increases in membrane phosphatidylserine and nucleosomal degradation. Increasing levels of shear stress decreased expression of the anti-apoptotic factor, bcl-2, from 44 to 10 U/ml. Addition of the nitric oxide antagonists, L-N(5)-(1-iminoethyl) ornithine and Nomega-nitro-L-arginine methyl ester (L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%, respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coincided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to shear stress in the absence of L-NAME. These data suggest that shear stress induced nitric oxide is associated with changes in apoptotic regulatory factors that alter chondrocyte metabolism and may contribute to joint degeneration.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

Mechanical loading alters articular cartilage metabolism. However, mechanisms underlying intracellular signaling and communication between cells in response to mechanical stresses remain enigmatic. This study tested the hypothesis that shear stressinduced nitric oxide (NO) production participates in the regulation of matrix protein gene expression. The data presented here demonstrate that exposure of human osteoarthritic chondrocytes to a continuously applied shear stress (1.64 Pa) upregulated NO synthase gene expression and increased N O release by 1.8-, 2.4-, and 3.5-fold at 2, 6, and 24 h, respectively. Exposure of chondrocytes to a short duration of shear stress for 2 h resulted in the release of accumulation of NO in the culture medium. Exposure of chondrocytes to shear stress for 2, 6, and 24 h inhibited type I1 collagen mRNA signal levels by 27%, 18% and 20% after a constant post-shear incubation period of 24 h. Aggrecan mRNA signal levels were inhibited by 30%, 32% and 41% under identical conditions. Addition of an NO antagonist increased type I1 collagen mRNA signal levels by an average of 1.8-fold (137% of the un-sheared control) and reestablished the aggrecan mRNA signal levels by an average of 1 &fold after shear stress (92% of the un-sheared control) (ANOVAp < 0.05). These data support the hypothesis that shear stress-induced NO release may influence the development of degenerative joint diseases by inhibiting matrix macromolecule synthesis.