Benjamin V. Treadwell

Expression of IL-1 genes in human and bovine chondrocytes: a mechanism for autocrine control of cartilage matrix degradation.

In this report we describe the presence of interleukin-1 activity in medium conditioned by bovine articular cartilage. Preparations partially purified by Sephacryl S200 chromatography (Mr 18000-25000) stimulate murine thymocyte proliferation in the lymphocyte activation factor assay. Furthermore, the factor(s) activate cartilage tissue to secrete a protease which is essential for the activity of purified synovial collagenase. We also demonstrate the presence of mRNA coding for IL-1 alpha and beta in human articular chondrocytes and conclude that the human monocytic and chondrocytic mRNAs are identical. Our results demonstrating cartilage expression of IL-1 genes suggest the possibility of an autocrine mechanism whereby chondrocyte production of matrix degrading proteases is initiated by chondrocyte derived IL-1.

Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible autocrine/paracrine role in pathogenesis

The interleukin-1 (IL-1) cytokines stimulate the synthesis of degradative enzymes in joint tissues and may play a role in the pathological joint destruction in osteoarthritis (OA). In this study, we have used immunohistochemistry and Western blot analysis to identify IL-1 in human OA cartilage. IL-1 alpha and IL-1 beta were evident in chondrocytes at the articular surface, as well as distributed throughout the cartilage. In many specimens, IL-1 beta but not IL-1 alpha was detected as a diffuse staining of the extracellular matrix especially surrounding superficial zone chondrocytes. Although chondrocyte-associated IL-1 alpha and IL-1 beta were detected in most specimens, cartilages exhibiting early osteoarthritic changes had the highest intensity of staining and the highest frequency of positive cells. Western blot analysis revealed intense immunoreactive bands corresponding to the 35 kDa precursor form of IL-1 alpha in all four chondrocyte lysates tested. The processed 18 kDa IL-1 beta species was present in only one of four chondrocyte lysates, and there was no clear evidence of precursor form within these cells. The results of this study indicate increased IL-1 alpha in cartilage showing early degenerative changes, suggesting an autocrine/paracrine role for this cytokine in OA pathogenesis.

Biochemical and Physiological Events During Closure of the Stapled Distal Femoral Epiphyseal Plate in Rats

The biochemical, histochemical, and histological changes occurring in the surgically stapled closing distal femoral growth plate of rats have been described and the changes demonstrated were: 1. An initial stimulation of growth, probably due to the surgical trauma of stapling, followed by a gradual decline in cell division which produces narrowing of the plate. An earlier narrowing occurs, however, which appears to be caused by disturbances in cell column alignment. 2. Despite decreased cell division, matrix production continues at a high rate, and hexosamines accumulate, perhaps to a greater extent than they normally do. This accumulation may be related to a reduction in lysosomal enzymes.

Purification and characterization of collagenase activator protein synthesized by articular cartilage

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.

Acid Hydrolase Activity in Osteoarthritic and Normal Human Cartilage

As an index of lysosomal enzyme activity in cartilage from twenty osteoarthritic and twelve normal joints, the acid phosphatase activity was determined and correlated with the histochemical-histological grading of the severity of the arthritis. The activity was increased four to five times normal in the arthritic cartilage with a positive correlation between activity and severity of the disease and was also increased in the cartilage of the osteophytes, possibly due to immaturity. This finding supports current concepts of the role of lysosomal enzymes in osteoarthritis.

Insulin promoted decrease in the phosphorylation of protein synthesis initiation factor eIF-2

Insulin stimulates cellular protein synthesis in calf chondrocytes in suspension culture. This enhanced synthetic activity is seen in association with a decrease in phosphorylation of the alpha subunit of protein synthesis initiation factor eIF-2. [32P] associated with the alpha subunit is reduced approximately 50% by insulin treatment of chondrocytes incubated in [32P] containing media. Identical or closely located amino acids in the eIF-2 alpha subunit are phosphorylated by the chondrocyte kinase(s) and the rabbit reticulocyte hemin regulated kinase as indicated by comparative peptide fragment analysis of [32P] labeled alpha subunits.

A plasminogen-related gene is expressed in cancer cells

The breakdown of blood clots requires the fibrinolytic action of the serine proteinase plasmin, a two-chain polypeptide derived posttranslationally from its precursor zymogen, plasminogen. While investigating plasminogen gene expression in human extrahepatic tissues, a cDNA sequence was obtained which closely resembled the plasminogen cDNA, yet appeared to represent a distinct gene product. This sequence, which represents the transcript of the recently characterized plasminogen-related gene B, encodes a putative polypeptide of Mr 8800 and is expressed most prominently in malignant cancer cells.

Stimulation of the synthesis of collagenase activator protein in cartilage by a factor present in synovial-conditioned medium.

We have purified a low molecular weight protein from medium conditioned by calf synovium with physical and biological properties similar to the leukocyte cytokine interleukin 1 (IL-1). The factor is active in stimulating the synthesis (three- to fivefold) of collagenase activator protein (CAP) by the surface (1-2 mm) of articular cartilage while CAP synthesis in the deeper zones of articular cartilage is not affected. Recombinant mouse IL-1 and commercially available purified human IL-1 are also capable of stimulating cartilage to synthesize and secrete CAP. The synthesis of other proteins, including collagenase, appeared to be unaffected by either the synovial factors or the human and mouse IL-1.

Degradative enzyme systems in cartilage.

There appears to be a final common pathway in the pathogenesis of osteoarthritis, regardless of the initiating cause. This involves an increase in degradative enzymes that arise from the cartilage. Both proteoglycan-and collagen-degrading enzymes, active at a neutral pH, increase in proportion to the severity of the arthritis until a final end-stage state is reached. This increase in enzyme activity may be triggered by release of a synovial messenger protein similar to interleukin-1. It is suggested by studies in an animal model that inhibition of these enzymes could lead to treatment of osteoarthritis.

Alternatively spliced annexin XI transcripts encode proteins that differ near the amino-terminus☆

Annexins are a family of structurally related calcium-dependent phospholipid binding proteins. We recently described a new member of this family, bovine annexin XI [1]. Two kinds of cDNAs were identified corresponding to annexin XI mRNA variants A and B, which are generated by alternative splicing of identical primary transcripts. Annexin XI isoforms are predicted to differ at the amino-terminus, suggesting that they may have distinct biological roles.