Requirement of Podocalyxin in TGF-Beta Induced Epithelial Mesenchymal Transition

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeletion assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype

Ltd. „Tendo Management” competitiveness and perspectives in Kuldiga

Acquisition of a complex immune system during evolution provided organisms with the most effective defense mechanism against "foreign" or "non-self" invaders. This efficient protection against pathogens, however, has been achieved at the expense of a higher risk for "self"-directed reaction or autoimmunity. Establishment of self-tolerance and homeostasis in the immune system is regulated at different physiological stages of immune cells development. The breakdown in discrimination between "self" and "non-self" causes an aberrant immune response against autoantigens that promote damage to the "self" cells and tissue(s), resulting in various autoimmune phenotypes. Whereas activation and clonal proliferation of autoreactive T- and B-lymphocytes underlies the pathogenesis of autoimmune diseases, the mechanism by which self-tolerance is lost and autoimmune responses are induced is not clear yet. Autoimmunity is a multi-step process that occurs as a consequence of complex interaction between genetic susceptibility and non-genetic factors. Programmed cell death, as a key mechanism to regulate immune system function, has a crucial influence on both the selection process of immune cells and the maintenance of this immune tolerance in peripheral repertoire. Thus, defects in apoptotic death pathways may contribute to the development of autoimmune response in susceptible individuals in certain conditions

Autoimmunity and apoptosis - Therapeutic implications

Acquisition of a complex immune system during evolution provided organisms with the most effective defense mechanism against "foreign" or "non-self" invaders. This efficient protection against pathogens, however, has been achieved at the expense of a higher risk for "self"-directed reaction or autoimmunity. Establishment of self-tolerance and homeostasis in the immune system is regulated at different physiological stages of immune cells development. The breakdown in discrimination between "self" and "non-self" causes an aberrant immune response against autoantigens that promote damage to the "self" cells and tissue(s), resulting in various autoimmune phenotypes. Whereas activation and clonal proliferation of autoreactive T- and B-lymphocytes underlies the pathogenesis of autoimmune diseases, the mechanism by which self-tolerance is lost and autoimmune responses are induced is not clear yet. Autoimmunity is a multi-step process that occurs as a consequence of complex interaction between genetic susceptibility and non-genetic factors. Programmed cell death, as a key mechanism to regulate immune system function, has a crucial influence on both the selection process of immune cells and the maintenance of this immune tolerance in peripheral repertoire. Thus, defects in apoptotic death pathways may contribute to the development of autoimmune response in susceptible individuals in certain conditions

Induction of podocalyxin by TGF-β.

<p>A549 cells were incubated with TGF-β (2 ng/ml) for the indicated times and the cells were collected for western blot. The blots were established by mouse anti-podocalyxin and HRP-conjugated rabbit anti-mouse secondary antibody. GAPDH was also monitored on the same filter as control.</p

Podocalyxin is required for TGF-β induced EMT marker expressions.

<p>A549 and PODXL-KD cells were treated with TGF-β for 1–3 days, and monitored by western blot for their expressions of podocalyxin, E-cadherin and vimentin. TGF-β up-regulated the production of podocalyxin and vimentin in A549 cells, and down-regulated E-cadherin level to zero in a time dependent manner. Silencing of podocalyxin interfered with TGF-β mediated down-regulation of E-cadherin and up-regulation of vimentin.</p

Compositional analysis of Podocalyxin containing immunoprecipitates.

#<p>pep: number of peptides matched.</p><p>log(e): probability of mismatched.</p

Effects of podocalyxin silence on cell morphology and podocalyxin production.

<p><b>A</b>): Podocalyxin shRNA transduced cells (PODXL-KD) (lane 2,4,6,8) and control A549 cells (transduced with the same vector without shRNA insert) (lane 1,3,5,7) were cultured in the presence or absence of TGF-β for 1 to 3 days. Podocalyxin levels were examined by western blot. GAPDH were also determined as loading controls. <b>B</b>): Cell morphology under phase contrast microscope before and after silencing podocalyxin. Scale bar = 50 µm.</p