Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Production and characterisation of monoclonal antibodies for the detection of AOZ, a tissue bound metabolite of furazolidone

3-amino-2-oxazolidinone (AOZ) is a tissue bound toxic metabolite derived from the nitrofuran antibiotic, furazolidone. AOZ is detected in the derivatised form of 3-{[(2-nitrophenyl) methylene]amino}-2-oxazolidinone (NP AOZ). 3-{[( 3- carboxyphenyl)-methylene]amino-2-oxazolidinone (CP AOZ) was used as the immunising hapten for the production of monoclonal antibodies against NP AOZ. Monoclonal antibodies were produced using hybridomas from the fusion of murine myeloma cells and spleen cells isolated from BALB/c mice immunised with CP AOZ-ethylenediamine-human serum albumin (CP AOZ-ed-HSA). The antibody production in ascitic fluids from clones 3B8/2B9 and 2D11/A4 was monitored during a 16 month period. Repeated cultures of these hybridomas, followed by injection into mice and cloning did not change the assay parameters. Clone 2D11/A4 exhibited long term stability in antibody production throughout the experiment whereas clone 3B8/2B9 demonstrated variability in particular antibody yields whilst retaining assay sensitivity. Reasons for this production variability in clones are discussed. In an optimised direct ELISA format, the antibodies exhibited a 50% binding inhibition in the range of 0.52-1.15 ng/ml with NP AOZ (0.22-0.50 ng/ml, respective AOZ equivalents) and showed high specificity towards this analyte. The sensitivity of monoclonal antibodies incorporated into the ELISA is compatible with the European Union MRLP and is currently in use for routine analysis.