IntroductionRheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts.MethodsEndoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability.ResultsRA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells.ConclusionsTNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.

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Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.

c-Kit is a receptor tyrosine kinase and a member of the subfamily that includes the PDGF, 1 CSF-1, and flt-3/flk-2 receptors. Together with its ligand steel factor (SLF), c-Kit is a key controlling receptor for a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells. c-Kit is the gene product of the W locus in mice (1), and its ligand SLF is the product of the sl locus (2, 3). Mutations in either locus severely affect the growth and survival of stem cells of these tissues.As with other receptor tyrosine kinases, stimulation of the c-Kit receptor with SLF results in the recruitment and tyrosine phosphorylation of SH2-containing second messenger-generating enzymes (4) such as phospholipase C-␥ and PI3-kinase (5, 6). The phosphorylated lipid products of these enzymes stimulate a variety of intracellular processes including Ca 2ϩ mobilization and actin reorganization (7-9). Coincident with second messenger generation, the process of receptor internalization is also initiated. Within minutes following ligand binding, receptors cluster in dimers or oligomers and internalize by endocytosis, likely through clathrin-coated pits (10 -12). Eventually, clathrin coats are removed, and the remaining vesicles fuse with endosomes, late endosomes, and ultimately lysosomes, resulting in receptor degradation.Numerous deletion and mutagenesis studies have been carried out to map regions of receptor tyrosine kinases required for ligand-stimulated internalization (13-22). Tyrosine kinase activity (23, 24), autophosphorylation sites (17, 18), and interactions with second messenger-generating enzymes have been implicated. In particular, recruitment and activation of PI3-kinase have been associated with ligand-stimulated internalization of the PDGF receptor (14). Receptors carrying mutations within the cytoplasmic domain that disrupt the PI3-kinase-binding site are impaired in the later stages of endocytosis (25). However, conflicting results with a deletion mutant encompassing this binding site demonstrate no impairment in any stage of endocytosis (16).Ligand-stimulated endocytosis of c-Kit was investigated by Yee et al. (26). They reported that a c-Kit mutant with no kinase activity was impaired for ligand-stimulated internalization. Individual mutations converting tyrosines 719 and 821 to phenylalanines, however, failed to affect internalization, although Phe-821 was impaired for mitogenesis (27). Tyr-719 is located in the kinase insert region of the c-Kit catalytic domain. In its phosphorylated form, it forms part of a consensus binding site for the p85 subunit of PI3-kinase and has been shown to mediate this interaction in vitro and in vivo (6).We have also examined the role of PI3-kinase activation on ligand-stimulated internalization of c-Kit. We found that in the absence of PI3-kinase activation, the c-Kit receptor internalizes but remains localized near the inner aspect of the plasma membrane. However, when both PI3-kinase and Ca 2ϩ influx are inhibited, clathrin fails to co-immunoprec...