Immunological effects of photodynamic therapy in the treatment of actinic keratosis and squamous cell carcinoma.

The use of photodynamic therapy is extensive, due to its antitumoral, antibacterial and photorejuvenation effects. It destroys tumor via direct cell destruction and indirectly via vascular shutdown, induction of acute local inflammatory response and activation of the immune system. Both innate and adaptive immune cells are involved in the immunological effects of photodynamic therapy. In addition to UV-induced DNA damage, inflammation and immunosuppression are also essential elements in the pathogenesis of actinic keratosis. Both immunosuppression induced by UV and defective immune response to dysplastic keratinocytes may be the target of photodynamic therapy to eliminate actinic keratosis. These elements are discussed in the present review, highlighting the possible mechanism of photodynamic therapy to effectively treat actinic keratosis.

Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells.

Merlin (moesin-ezrin-radixin like protein), the product of neurofibromatosis type 2 gene, was primarily recognized as a tumor suppressor, but it also functions as a membrane-cytoskeletal linker and regulator of multiple signaling pathways. The activity and localization of merlin is regulated by head to tail folding that is controlled by phosphorylation of the Ser518 side chain. Merlin localizes in the nucleus when the Ser518 side chain is not phosphorylated, while the phosphorylated form is present in the cytoplasm and the plasma membrane. In this work interactions and their impact on the subcellular localization and phosphorylation state of the Ser518 side chain of merlin were investigated in endothelial cells. It is shown that merlin (dephospho-Ser518 form) interacts in the nucleus of endothelial cells with the scaffolding protein EBP50, a member of the Na+/H+exchanger regulatory factor family. Upon EBP50 depletion, merlin translocated from the nucleus, suggesting that binding of merlin to EBP50 is critical in the nuclear localization of merlin. Along with the translocation, the phosphorylation level of phospho-Ser518-merlin was increased in EBP50 depleted cells. TIMAP (TGFß-inhibited membrane-associated protein), a type 1 protein phosphatase (PP1) regulatory subunit, was newly recognized as an interacting partner for merlin. Domain mapping using truncated mutant forms in GST pull down revealed that the N-terminal half of TIMAP (aa 1-290) and the FERM domain of merlin are the regions responsible for the interaction.The catalytic subunit of PP1 (PP1c) was present in all merlin-TIMAP pull down or immunoprecipitation samples demonstrating that merlin actually interacts with the PP1c-TIMAP holoenzyme. On the other hand, from TIMAP depleted cells, without its targeting protein, PP1c could not bind to merlin. Also, when the phosphatase activity of PP1c-TIMAP was inhibited either with depletion of TIMAP or by treatment of the cells with specific PP1 inhibitor, there was an increase in the amount of phospho-Ser518 form of merlin in the membrane of the cells. These data strongly suggest that the PP1c-TIMAP- complex dephosphorylates phospho-Ser518-merlin. ECIS measurements indicate that phospho-merlin accelerates in vitro wound healing of the endothelial monolayer. In conclusion, in endothelial cells, EBP50 is required for the nuclear localization of merlin and the PP1c-TIMAP holoenzyme plays an important role in the dephosphorylation of merlin on its Ser518 side chain, which influence cell migration and proliferation.

Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

TIMAP (TGF-ß inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements.