'Special K' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine

Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention

A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR↓STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors

TGF-{beta}1 and laminin-111 cooperate in the induction of IL-16 expression in synovial fibroblasts from rheumatoid arthritis patients

OBJECTIVES: In synovial tissues of rheumatoid arthritis (RA) patients strong expression of laminins and integrins co-localizes with elevated expression of inflammatory cytokines. Synovial fibroblasts (SF) contribute to the pathogenesis of RA through elevated expression of cytokines and chemoattractant factors, one of which is IL-16. We therefore investigated regulatory pathways of IL-16 in SF from RA and osteoarthritis (OA) patients. METHODS: SF were seeded in laminin-coated flasks and activated by the addition of cytokines. Expression of IL-16 was investigated by quantitative RT-PCR, immunoblotting, and ELISA; its biological activity was determined by a cell migration assay. Cell - matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signaling pathways were studied by immunoblotting and with pharmacological blocking reagents. RESULTS: The stimulation of SF with TGF-beta1 and growth on laminin-111 (LM-111) significantly increased the expression of IL-16. In RA-SF, binding to LM-111 induced significantly more IL-16 mRNA than in OA-SF (p<0.05). The IL-16 cytokine was detected in supernatants of TGF-beta1-activated and in LM-111 plus TGF-beta1-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL-16 regulation involved p38MAPK, ERK1/2 and SMAD2 signaling, but not NFkappaB. CONCLUSIONS: Binding of RA-SF to LM-111 in the presence of TGF-beta1 triggers a significant IL-16 response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL-16 induction represents a novel pathway leading to IL-16 production in RA-SF but not in OA-SF, and it operates independently of NFkappaB signaling