BBA - Molecular Cell Research (v.1589, #1)

Toll-like receptors as adjuvant receptors by Tsuneyasu Kaisho; Shizuo Akira (1-13).
The mammalian Toll-like receptors (TLRs) are expressed on macrophages and dendritic cells, which are primarily involved in innate immunity. At present, ligands for several of the TLRs, such as TLR2, TLR3, TLR4, TLR5, TLR6, and TLR9, have been identified. Most of these ligands are derived from pathogens, but not found in the host, suggesting that the TLRs are critical to sensing invading microorganisms. Pathogen recognition by TLRs provokes rapid activation of innate immunity by inducing production of proinflammatory cytokines and upregulation of costimulatory molecules. Activated innate immunity subsequently leads to effective adaptive immunity. In this regard, the TLRs are considered to be adjuvant receptors. Distinct TLRs can exert distinct, but overlapping sets of biological effects. Accumulating evidence indicates that this can be attributed to both the common and unique aspects of the signaling mechanisms that mediate TLR family responses. For example, TLR2 and TLR9 require MyD88 as an essential signal transducer, whereas TLR4 can induce costimulatory molecule upregulation in a MyD88-independent manner. Understanding the TLR system should offer invaluable opportunity for manipulating host immune responses.
Keywords: Toll-like receptor; Adjuvant; Dendritic cell; Lipopolysaccharide; MyD88; Innate immunity;

Upregulation by retinoic acid of transforming growth factor-β-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases by Daijiro Hatakeyama; Osamu Kozawa; Masayuki Niwa; Hiroyuki Matsuno; Hidenori Ito; Kanefusa Kato; Norichika Tatematsu; Toshiyuki Shibata; Toshihiko Uematsu (15-30).
We investigated whether transforming growth factor-β (TGF-β) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-β increased the level of HSP27 but had no effect on the HSP70 level. TGF-β stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-β induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-β was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-β-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-β. Retinoic acid enhanced the TGF-β-induced increase of mRNA for HSP27. The amplification of TGF-β-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-β-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-β-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-β-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.
Keywords: Transforming growth factor-β; Heat shock protein; Osteoblast; Mitogen-activated protein kinase; Retinoic acid;

The alkylation of amino groups of the mineralocorticoid receptor (MR) with pyridoxal 5′-phosphate or 2,4,6-trinitrobenzenesulphonate (TNBS) under controlled conditions modifies only one lysyl residue, which accounts for a 70% inhibition of steroid binding capacity. The K d of aldosterone for MR is not affected by the treatment, but the total number of binding sites is greatly decreased. The modified receptor is capable of dynamically conserving its association with the hsp90-based heterocomplex. Importantly, the binding of natural agonists protects the hormone binding capacity of the MR from the inactivating action of alkylating agents. In contrast, antagonistic steroids are totally incapable of providing such protection. Like the antagonistic ligands, and despite its potent mineralocorticoid biological effect, the sole MR specific synthetic agonist known to date, 11,19-oxidoprogesterone (11-OP), shows no protective effect upon treatment of the MR with pyridoxal 5′-phosphate or TNBS. Limited digestion of the MR with α-chymotrypsin generates a 34 kDa fragment, which becomes totally resistant to digestion upon binding of natural agonists, but not upon binding of antagonists. Interestingly, the synthetic 21-deoxypregnanesteroid 11-OP exhibits an intermediate pattern of proteolytic degradation, suggesting that the conformational change generated in the MR is not equivalent to that induced by antagonists or natural agonists. We conclude that in the first steps of activation, the MR changes its conformation upon binding of the ligand. However, the nature of this conformational change depends on the nature of the ligand. The experimental evidence shown in this work suggests that a single lysyl group can determine the hormone specificity of the MR.
Keywords: Aldosterone; Sodium retention; Pyridoxal 5′-phosphate; 2,4,6-Trinitrobenzenesulfonate; hsp90 heterocomplex;

Modulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by cell cycle inhibitors by Maria Wartenberg; Kerstin Fischer; Jürgen Hescheler; Heinrich Sauer (49-62).
The effects of cell cycle inhibition on the expression of the multidrug resistance transporter P-glycoprotein (P-gp) as well as of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p21WAF-1 were investigated in DU-145 prostate tumor spheroids. With increasing spheroid size the number of cells in the G0/G1 phase augmented, whereas the number of cells in the G2/M phase and the S phase of the cell cycle declined. The number of G0/G1 cells was elevated after incubation with either mimosine, staurosporine or serum-free medium. Mitomycin C and roscovitine increased the number of S phase cells. Roscovitine additionally increased cells in the G2/M phase. Incubation in serum-free medium upregulated p21WAF-1, p27Kip1 and P-gp. Mimosine treatment resulted in upregulation of p27Kip1 and P-gp, whereas p21WAF-1 remained unchanged. Upon roscovitine treatment p27Kip1 and p21WAF-1 were downregulated, whereas P-gp was unaltered. Mitomycin C treatment resulted in downregulation of p27Kip1 and p21WAF-1; no significant change in P-gp levels was observed. Staurosporine induced upregulation of p21WAF-1 whereas p27Kip1 remained unaltered. P-gp was downregulated upon staurosporine treatment, which was owing to an elevation of intracellular reactive oxygen species by this compound. It is concluded that upregulation of P-gp in G0/G1 phase cells requires coexpression of the CDK inhibitor p27Kip1 but not the CDK inhibitor p21WAF-1.
Keywords: Multicellular tumor spheroid; Multidrug resistance; P-glycoprotein; Cell cycle; p27Kip1; p21WAF-1;

Cytotoxic effects of metal complexes of biogenic polyamines. I. Platinum(II) spermidine compounds: prediction of their antitumour activity by M.P.M Marques; T Girão; Maria C Pedroso De Lima; A Gameiro; E Pereira; P Garcia (63-70).
Cytotoxicity and cell growth inhibition studies were performed for three distinct polynuclear platinum(II) complexes of spermidine, which showed to have significant cytotoxic and antiproliferative properties on the HeLa cancer cell line. The chemical environment of the metal centres in the drugs, as well as the coordination pattern of the ligand, were found to be strongly determinant of their cytotoxic ability. In the light of the results gathered, the most effective anticancer compound among the ones tested (IC50=5 μM) was found to be the one displaying three difunctional (PtCl2N2) moieties ((PtCl2)3(spd)2). Both the cytotoxic activity and the antiproliferative properties of the complexes studied showed to be irreversible for all the concentrations tested.
Keywords: Platinum complex; Spermidine; HeLa cell; Cytotoxicity; Antitumor activity;

Identification of interaction between MEK2 and A-Raf-1 by Xiang L. Yin; She. Chen; Jun. Yan; Yun Hu; Jian X. Gu (71-76).
Mitogen-activated protein (MAP) kinases are activated by dual-specificity kinases, termed MEKs. Using MEK2 as bait in yeast two-hybrid screening, besides c-Raf and KSR, A-Raf was identified as a novel partner that interacts with MEK2. This interaction was confirmed by in vitro binding assay. Further investigation indicates that regions critical for this interaction were located between residues 255 and 606 that represent the kinase domain of A-Raf.
Keywords: MEK2; A-Raf kinase; Interaction; Two-hybrid system;

Hyperosmotic stress activates Na+-K+-2Cl cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl/OH(HCO3 ) exchange carriers. Hyperosmolarity decreased intracellular volume (V i) after 10 min from 7.8 to 5.4 μl/mg protein and increased intracellular Cl (Cl i) from 353 to 532 nmol/mg protein. Treatment with an α-adrenergic agent rapidly increased Cl i and V i in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-δ and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-δ and ERK.
Keywords: Hypertonicity; Cell shrinkage; Mitogen-activated protein kinase; Na+-K+-2Cl cotransport; Cystic fibrosis;