Pannus invasion into cartilage and bone in rheumatoid arthritis

Rheumatoid arthritis is the most common of all types of arthritis and despite of intensive research etiology of the disease remains unclear. Distinctive features of rheumatic arthritis comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus tissue formation. Pannus formation, appearance of proteolytic enzymes and osteoclast formation cause articular cartilage and bone destruction, which lead to erosions and permanent joint damage. Proteolytic pathways play major roles in the development of tissue lesions in rheumatoid arthritis. Degradation of extracellular matrix proteins is essential to pannus formation and invasion. Matrix metalloproteinases (MMP) form a large proteolytic enzyme family and in rheumatoid arthritis they contribute to pannus invasion by degrading extracellular matrix and to joint destruction by directly degrading the cartilage. MMP-1 and MMP-3 are shown to be increased during cell invasion and also involved in cartilage destruction. Increase of many cytokines has been observed in rheumatoid arthritis, especially TNF-α and IL-1β are studied in synovial tissue and are involved in rheumatoid inflammation and degradation of cartilage. Underlying bone resorption requires first demineralization of bone matrix with acid secreted by osteoclasts, which exposes the collagen-rich matrix for degradation. Cathepsin K is the best known enzyme involved in bone matrix degradation, however deficiency of this protein in pycnodysostosis patient did not prevent bone erosion and on the contrary pannus tissue invading to bone did not expressed much cathepsin K. These indicate that other proteinases are involved in bone degradation, perhaps also via their capability to replace the role of other enzymes especially in diseases like pycnodysostosis or during medication e.g. using cathepsin K inhibitors. Multinuclear osteoclasts are formed also in pannus tissue, which enable the invasion into underlying bone matrix. Pannus tissue express a receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclast differentiation and a disintegrin and a metalloproteinase 8 (ADAM8), an osteoclast-activating factors, involved in formation of osteoclast-like giant cells by promoting fusion of mononuclear precursor cells. The understanding of pannus invasion and degradation of extracellular matrix in rheumatic arthritis will open us new more specific methods to prevent this destructive joint disease.

Golgi proteomics : Identification of a novel cartilage-specific Golgi protein GoPro49

The Golgi complex is a central organelle of the secretory pathway, responsible for a range of post-translational modifications, as well as for membrane traffic to the plasma membrane and to the endosomal-lysosomal pathway. In addition, this organelle has roles in cell migration, in the regulation of traffic, and as a mitotic check point. The structure of the Golgi complex is highly dynamic and able to respond to the amount of cargo being transported and the stage of the cell cycle. The Golgi proteome reflects the functions and structure of this organelle, and can be divided into three major groups: the Golgi resident proteins (e.g. modification enzymes), the Golgi matrix proteins (involved in structure and tethering events), and trafficking proteins (e.g. vesicle coat proteins and Rabs). The Golgi proteome has been studied on several occasions, from both rat liver and mammary gland Golgi membranes using proteomic approaches, but still little more than half of the estimated Golgi proteome is known. Nevertheless, methodological improvements and introduction of shotgun proteomics have increased the number of identified proteins, and especially the number of identified transmembrane proteins. Cartilage, even though not a typical tissue in which to study membrane traffic, secretes large amounts of extracellular matrix proteins that are extensively modified, especially by amino acid hydroxylation, glycosylation and sulfation. Furthermore, the cartilage ECM contains several, large oligomeric proteins (such as collagen II) that are difficult to assemble and transport. Indeed, cartilage has been shown to be susceptible to changes both in secretory pathway (e.g. the COPII coat assembly) and in post-translational modifications (e.g. heparan sulfate formation). Dental follicle, and the periodontal ligament (PDL) that it forms, are another type of connective tissue, and they have a role in anchoring teeth to bone. This anchorage is achieved by numerous matrix fibres that connect the bone matrix with the cementum. These tissues have in common the secretion of large matrix molecules. In this study the Golgi proteome was analysed from purified, stacked Golgi membranes isolated from rat liver. The identified, extensive proteome included a protein similar to Ab2-095, or Golgi protein 49kDa (GoPro49), which was shown to localise to the Golgi complex as an EGFP fusion protein. Surprisingly, in situ hybridisation showed the GoPro49 expression to be highly restricted to different mesenchymal tissues, especially in cartilage, and this expression pattern was clearly developmentally regulated. In addition to cartilage, GoPro49 was also expressed in the dental follicle, but was not observed in the mature PDL. Importantly, GoPro49 is the first specific marker for the dental follicle. Endogenous GoPro49 protein co-localised with β-COP in both chondrosarcoma and primary dental follicle cell lines. The COPI staining in these cells was highly dynamic, showing a number of tubules. This may reflect the type of secretory cargo they secrete. Currently GoPro49 is the only Golgi protein with such a restricted expression pattern.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.