ABSTRACT
Palladin is a scaffold protein involved in the formation of actin-associated protein complexes. Gene expression array analysis on the poorly metastatic HCT116 colon cancer cell line and a metastatic derivative cell line (E1) with EMT (epithelial-mesenchymal transition) features showed a down-regulation of palladin gene expression in the latter. Knockdown of palladin expression in the HCT116 cells suppressed junctional localization of E-cadherin, reduced intercellular adhesion and collective cell migration, showing that palladin plays an important role in maintaining the integrity of adherens junctions. The acquisition of the EMT features by the E1 cell line was dependent on the Erk pathway. Inhibition of this pathway by U0126 treatment in E1 cells resulted in the re-expression of palladin, relocalization of E-cadherin to the adherens junctions and a reversal of EMT features. The re-establishment of intercellular adhesion was dependent on palladin expression. The down-regulation of palladin was also observed in poorly-differentiated tumor tubules and dissociated tumor cells that have undergone de-differentiation in human primary colon tumors. Our data show that palladin is an integral component of adherens junctions and plays a role in the localization of E-cadherin to the junctions. The loss of palladin may be an integral part of EMT, an early step in the metastatic spread of colon carcinoma.
Subject(s)
Adherens Junctions/metabolism , Cell Adhesion , Cell Movement , Colorectal Neoplasms/metabolism , Cytoskeletal Proteins/metabolism , Phosphoproteins/metabolism , Adherens Junctions/drug effects , Adherens Junctions/pathology , Animals , Antigens, CD , Cadherins/metabolism , Cell Adhesion/drug effects , Cell Dedifferentiation , Cell Movement/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Cytoskeletal Proteins/genetics , Epithelial-Mesenchymal Transition , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Mice , Mice, Nude , Neoplasm Invasiveness , Phenotype , Phosphoproteins/genetics , Protein Kinase Inhibitors/pharmacology , RNA Interference , Splenic Neoplasms/metabolism , Splenic Neoplasms/secondary , Time Factors , TransfectionABSTRACT
Microbial structures activate Toll-like receptors (TLRs) and TLR-mediated cell signalling elicits and regulates host immunity. Most TLRs are poorly expressed but the underlying expression mechanism is not clear. Examination TLR sequences revealed that most human TLR genes deviated from using major human codons. CD14 resembles TLRs in sequence but its gene preferentially uses major codons. Indeed, CD14 expression on monocytes was higher than expression of TLR1 and TLR2. The TLR9 gene is abundant in major codons and it also showed higher expression than TLR1, TLR2 and TLR7 in transfected 293T cells. Change of the 5'-end 302 base pairs of the TLR2 sequence into major human codons markedly increased TLR2 expression, which led to increased TLR2-mediated constitutive nuclear factor-kappaB activation. Change of the 5'-end 381 base pairs of the CD14 sequence into prevalent TLR codons markedly reduced CD14 expression. These results collectively show that the deviation of TLR sequences from using major codons dictates the low TLR expression and this may protect the host against excessive inflammation and tissue damages.
Subject(s)
Codon , Membrane Glycoproteins/genetics , Receptors, Cell Surface/genetics , Base Sequence , Cells, Cultured , Humans , Lipopolysaccharide Receptors/blood , Lipopolysaccharide Receptors/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Monocytes/immunology , NF-kappa B/metabolism , Receptors, Cell Surface/metabolism , Toll-Like Receptor 1 , Toll-Like Receptor 2 , Toll-Like Receptor 7 , Toll-Like Receptor 9 , Toll-Like Receptors , TransfectionABSTRACT
Toll-like receptors (TLRs) are activated by microbial structures. To investigate the mechanisms of TLR activation, the 10 human TLRs were expressed as chimeras with the integrin alphav and beta5 subunits. Co-expression of the alphav-TLR and beta5-TLR chimeras in 293T cells generated 10 TLR homodimers, but only TLR4/4 could effectively activate NF-kappaB. TLR4 monomers also activated NF-kappaB but it was enhanced upon homodimerization. The TLR homodimers showed differential surface/intracellular expression. In TLR heterodimers, only TLR2/1 and TLR2/6 were potent in NF-kappaB activation. NF-kappaB activation by TLR2/1, TLR2/6 and the TLR4 monomer, but not TLR4/4, was completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers could activate NF-kappaB through different mechanisms.
