Osteoarthritic (OA) chondrocytes are metabolically active, displaying increased synthesis of type II collagen. Here, we show by immunohistochemistry and polymerase chain reaction that in comparison with healthy cartilage, OA articular chondrocytes exhibit increased in vivo synthesis of collagen prolyl-4-hydroxylase type II, a pivotal enzyme in collagen triple helix formation. Exposure of primary human articular chondrocytes to 1% oxygen enhanced accumulation of native type II collagen and stabilized hypoxia-inducible factor-1␣ (HIF-1␣). This effect was abolished by addition of the HIF-1 inhibitor 2-methoxyestradiol. Real-time polymerase chain reaction analyses of mRNAs from these cultures revealed increased transcript levels of both ␣-subunits of prolyl-4-hydroxylase (P4HA1, ϳ2-fold; P4HA2, ϳ2.3-fold) and of classical HIF-1 target genes (glucosetransporter-1, ϳ2.1-fold; phosphoglyceratekinase-1, ϳ2.2-fold). Treatment of hypoxic chondrocytes with 2-methoxyestradiol reduced transcriptional activity of HIF-1 and synthesis of ␣(II), and to a lesser extent ␣(I), subunits of collagen prolyl-4-hydroxylases. mRNA levels of type II collagen (Col2A1) and the ␤-subunit (P4HB) of prolyl-4-hydroxylase, however, displayed only modest changes at 1% oxygen. From these results and our in vivo data, we inferred that besides increased Articular hyaline cartilage is the most important component in synovial joints for frictionless joint motion. During movement, the articular cartilage is compressed by load and is then able to recover from this deformity. These unique biomechanical properties depend on the specific zonal architecture of articular cartilage as well as the extracellular matrix (ECM), which consists of two phases: solid and fluid phases. The main ECM components are proteoglycans, glycoproteins, and collagens, mainly type II collagen.1-3 Type II collagen is synthesized as a procollagen molecule with noncollagenous amino and carboxy extension peptides, by articular chondrocytes which represent the only living elements within hyaline cartilage. 4 Large type II collagen fibrils form a network with embedded proteoglycans, glycoproteins, water, and soluble ions. Since this collagen network is established in adults, the half-life of collagens is estimated to be over decades.Proper function of a load-bearing joint depends on the structural integrity of this highly specialized cartilage tissue and its ability to absorb and respond to mechanical stress. During the course of osteoarthritis, increased degradation processes of the ECM by mechanical factors as well as activity of metalloproteinases and aggrecanases have been observed. 5,6 Ultimately, destruction of large parts of the collagen network and ion-binding capacity via loss of negatively charged proteoglycans lead to a significantly decreased water-binding capability of the ECM. These biochemical changes are responsible for the devel-

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Increase of glomerular mesangial cells (MCs) is a prominent histopathological finding in many types of glomerulonephritis. We have shown previously that expression of the zinc-finger transcription factor, early growth response gene-1 (egr-1), is closely correlated with the proliferation of cultured MCs. To elucidate whether Egr-1 is required for MC proliferation, we inhibited serum-induced Egr-1 expression by phosphothioate-modified antisense oligonucleotides (ODNs Mesangial hypercellularity is a prominent histological characteristic of many types of human glomerular kidney disease, such as IgA nephropathy, membranoproliferative glomerulonephritis, focal glomerulosclerosis, and lupus nephritis. Growth factors leading to a proliferative response of MCs 1 may be derived from intrinsic glomerular cells or from infiltrating leukocytes or platelets. Inadequate control of MC proliferation may be an initial step leading to progressive glomerular alterations and end stage renal disease. Potentially detrimental effects of MC hypercellularity include mechanical obstruction of glomerular capillaries, increased production of proinflammatory mediators, and disturbance of production and removal of extracellular matrix proteins promoting mesangial and glomerular sclerosis.The immediate early gene, early growth response gene-1 (egr-1), encodes a 75-80-kDa transcriptional regulator that binds DNA through three zinc-finger domains (1-6). Egr-1, also known as zif 268, Krox 24, TIS 8, and NGFI-A, is rapidly and transiently induced in response to multiple mitogenic signals (7,8). The induction of Egr-1 occurs mainly at the transcriptional level (9 -11). This activation is mediated through serum response elements in the egr-1 promoter in fibroblasts and rat MCs (9, 12, 13). Egr-1 modulates specific gene expression (14 -18).So far, only a few specific biological functions can be ascribed to Egr-1. Egr-1 is involved in differentiation processes. It was shown to be essential and restrictive for differentiation of myeloblastic HL60 cells along the macrophage lineage (19). Egr-1 has been found to suppress v-sis-dependent transformation of NIH 3T3 cells (20). In addition, Egr-1 has recently also been shown to be involved in the regulation of proliferation of T lymphocytes (21) and astrocytes (22).In previous studies, we observed a very close correlation between the stimulation of MC growth and induction of egr-1 mRNA expression by mitogens such as platelet-derived growth factor, serotonin, and arginine vasopressin. Angiotensin II, which was not mitogenic for cultured rat MCs, failed to induce egr-1 mRNA expression (7). Here, we report that different antisense oligonucleotides directed against egr-1 mRNA block mitogen-induced Egr-1 expression and inhibit proliferation of cultured MCs. This indicates that Egr-1 is an important component of the mitogenic signal transduction cascade in glomerular MCs. MATERIALS AND METHODSReagents-Phosphothioate-modified antisense and sense, scrambled, or mismatched control oligodeoxynucleotides (ODNs) were fr...

Objective. To investigate crosslinks between catabolic and anabolic pathways in articular cartilage by examining the synthesis and distribution pattern of microsomal prostaglandin E synthase 1 (mPGES-1) in healthy and osteoarthritic (OA) cartilage and analyzing its functional relationship to hypoxia-inducible factor 1␣ (HIF-1␣) in primary articular chondrocytes.Methods. Normal cartilage and OA cartilage were subjected to immunohistochemical staining for mPGES-1 and HIF-1␣. Isolated chondrocytes were cultivated under 21% or 1% O 2 . Microarray analysis and quantitative reverse transcriptase-polymerase chain reaction were used to detect genes differentially expressed in chondrocytes cultured under normoxic compared with hypoxic conditions. Immunoblotting was conducted to evaluate intracellular protein levels of mPGES and nuclear accumulation of HIF-1␣ under different oxygen tension levels and with different stimulatory or inhibitory chemical agents.Results. We found enhanced levels of expression of the mPGES-1 gene and an increased number of OA chondrocytes showing staining for mPGES-1 in OA cartilage. Microarray analysis demonstrated that mPGES-1 was among the genes that were up-regulated to the greatest degree in primary chondrocytes exposed to 1% O 2 . In vitro, hypoxia led to an enhanced synthesis of mPGES-1, coinciding with a nuclear accumulation of the transcription factor HIF-1␣. In chondrocyte culture, stimulation with dimethyloxaloylglycine promoted the expression of mPGES-1, phosphoglycerate kinase 1, and cyclooxygenase 2 (COX-2) by stabilizing HIF-1␣ protein levels. A reduction of mPGES-1 synthesis was detected after treatment with 2-methoxyestradiol, correlating with lower HIF-1␣ activity. In contrast, synthesis of mPGES-1 was not influenced by treatment with the specific COX-2 inhibitor NS398.Conclusion. These findings suggest that the transcription factor HIF-1␣ is involved in the up-regulation of mPGES-1 and may therefore play an important role in the metabolism of OA cartilage.The regulation of the metabolism of articular cartilage involves a complex network of a multitude of signaling pathways. In osteoarthritis (OA), the usually well-regulated maintenance between extracellular matrix synthesis and degradation is greatly disturbed. Inflammatory cytokines promote catabolism by inducing expression of matrix-degrading enzymes, which lead to the breakdown of the collagen network and the release of cartilage proteoglycans (1). The biologic effects of interleukin-1␤ (IL-1␤), one of the key agents in the pathophysiology of OA, are mediated by signaling pathways involving NF-B, JNK, p38, or ERK, by nitric oxide, and by cytokines as well as by prostaglandins (2). Prostaglandin E 2 (PGE 2 ), the most abundant prostaglandin in the skeletal system (3), induces a number of matrix-degrading enzymes, thus stimulating the cataboSupported by the Interdisciplinary Center of Clinical Research Erlangen (Project C2) and the DFG (PF 383/4-1).

Castor bean (Ricinus communis L.) plants were grown for 5-7 weeks in a controlled environment at 350 &mgr;l l(-1) or 700 &mgr;l l(-1) CO(2). Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO(2) concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1) CO(2). The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1) CO(2). On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1). Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO(2) treatment. Respiration was 25% higher in high-CO(2) plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 &mgr;l l(-1) and 700 &mgr;l l(-1) CO(2). During the night the low-CO(2) plants had only 50% of the daytime export rate, in contrast to the high-CO(2) plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO(2) regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO(2) concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This "assimilation efficiency" was the same for both CO(2) regimes. It is speculated that under 350 &mgr;l l(-1) CO(2) the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night.