• PMCA as a regulator of calcium/calmodulin-dependent signal transduction pathways

      Armesilla, Angel Luis; Holton, Marylouisa (University of Wolverhampton, 2009)
      Plasma membrane calcium/calmodulin-dependent calcium ATPases (PMCAs) are high affinity calcium pumps regulating many calcium-dependent processes and advances in its characterisation have discovered that it may play a novel role in signal transduction pathways. It was the aim of this work to further characterise and confirm the role PMCA plays in regulating calcium/calmodulin-dependent signal transduction pathways. PMCA4 has already been shown to inhibit the NFAT family of transcription factors by its interaction with calcineurin A in mammalian cells when ectopically expressed. This prompted the investigation into other isoforms of PMCA that may interact with the calcium/calmodulin-dependent calcineurin, to determine if this interaction was isoform-specific in a variety of cell lines. Endogenous proteins were isolated by immunoprecipitation with calcineurin A antibody and the presence of PMCA isoforms was determined by western blot using isoform-specific antibodies. This work has demonstrated that the PMCA and calcineurin interaction occurs in vitro at endogenous levels in MCF-7 human breast adenocarcinoma cells and endothelial cells and is isoform specific, predominantly for PMCA2. The characterisation of the PMCA2b-calcineurin A interactive domain was performed and it was demonstrated that PMCA2b significantly inhibits the NFAT/calcineurin pathway. These results indicate that PMCA2 is important in regulating the calcineurin/NFAT pathway in tissues where it is highly expressed. This work also demonstrates that the Flag-tagged, characterised interaction domain of PMCA2 with calcineurin, F-PMCA(462-684) when overexpressed, can disrupt the inhibitory PMCA2/calcineurin interaction in endothelial cells and significantly increase calcineurin activity. The expression of PMCA in endothelial cells prompted the investigation of calcium/calmodulin-dependent proteins in endothelial cells as evidence for the important role of PMCA in regulating signal transduction pathways. Nitric oxide synthases have been shown to be regulated by PMCA4 in cardiac cells. To further characterise the regulation of NOS by PMCA, this work shows that there is a novel molecular interaction between endogenous eNOS and the plasma membrane calcium ATPase (PMCA) in HUVEC primary endothelial cells. PMCA2 has been identified as the major isoform interacting with eNOS in endothelial cells. The interaction between the two proteins has been mapped to the region 735-934 of eNOS and 462-684 of human PMCA2b. NO production was found to be inhibited by ectopic expression of PMCA2b in HUVEC cells. Moreover, disruption of the interaction between endogenous PMCA and eNOS by overexpression of theFlag-tagged, PMCA2b interaction domain, F-PMCA2(462-684), significantly increased NO levels in activated HUVEC endothelial cells. In summary, these results offer strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA2 as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. Overall this is a novel discovery which clearly demonstrates that PMCA is an important regulator of calcium/calmodulin-dependent signal transduction pathways in various cell types. Parts of this work have been published; ‘Holton, M., Yang, D., Wang, W., Mohamed, T.M., Neyses, L. and Armesilla, A. (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS letter. 581(21), 4115-4119.’ and presented at ‘The 14th congress of calcium binding proteins, La Palma, Canary Islands, Spain. 2007’ and ‘The 25th Conference of the European Society on Microcirculation (August 26-29, 2008, Budapest, Hungary).’
    • Role of the Plasma Membrane Calcium ATPase as a Negative Regulator of Angiogenesis

      Baggott, Rhiannon Rebecca (University of Wolverhampton, 2014)
      Angiogenesis is the formation of new blood vessels from pre-existing ones. Unregulated angiogenesis is associated with several diseases such as diabetic retinopathy and tumour growth. Many signal transduction pathways have been implicated in the regulation of angiogenesis such as p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), extracellular signal-related kinase 1/2 (Erk1/2) and of particular interest the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. Inhibition of calcineurin activity by the drug cyclopsorin A (CsA) has been shown to inhibit processes required for successful angiogenesis such as in vitro cell migration, tube formation and additionally attenuates corneal angiogenesis in vivo. CsA is associated with severe side effects and therefore the identification of an endogenous regulator of this pathway would be beneficial. One possibility is the plasma membrane calcium ATPases (PMCAs). These high affinity calcium extrusion pumps have been shown to interact with calcineurin in mammalian cells and cardiomyocytes and down-regulate the calcineurin/NFAT pathway. This is hypothesised to be due to the interaction between the two proteins which maintains calcineurin in a low calcium micro-environment generated by the calcium removal function of the pump. Interestingly, PMCA4 has been shown to interact with calcineurin in endothelial cells. The aim of our study was to further our understanding of PMCA4s regulation of the calcineurin/NFAT pathway specifically in endothelial cells and establish if PMCA4 has a role in the regulation of angiogenesis. ‘Gain of function’ by adenoviral over-expression of PMCA4 and ‘loss of function’ by either si-RNA mediated knockdown of PMCA4 or isolation of PMCA4-/- MLEC were used as models. Over-expression of PMCA4 in HUVEC resulted in inhibition of the calcineurin/NFAT pathway with the opposite result occurring in the case of the knockout of PMCA4, identifying PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells. Over-expression of PMCA4 significantly attenuated VEGF-induced protein and mRNA expression of the pro-angiogenic proteins RCAN1.4 and Cox-2, endothelial cell migration and in vitro and in vivo tube formation with the opposite result occurring in knockdown or knockout studies, confirming PMCA4 as a down-regulator of angiogenesis. Interestingly, over-expression or knockdown of PMCA4 had no effect on VEGF-induced HUVEC proliferation or Erk1/2 phopshorylation proposing PMCA4 may be a potential inhibitor of angiogenesis without compromising cell survival. Disruption of the interaction between PMCA4 and calcineurin by generation and ectopic expression of an adenovirus encoding the region of PMCA4 that interacts with calcineurin (428-651) (Ad-ID4) resulted in an increase in NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify the mechanism of PMCA4s inhibitory effect of the calcineurin/NFAT pathway and consequently angiogenesis is a result of the interaction between the two proteins. The novel findings of this study establish PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells and angiogenesis. These results are far reaching and highlight a potential role for PMCA4 as a therapeutic target in a variety of diseases that are associated with pathological angiogenesis.
    • The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells

      Holton, Marylouisa; Yang, Di; Wang, Weiguang; Mohamed, Tamer M. A.; Neyses, Ludwig; Armesilla, Angel (Elsevier, 2007)
      Plasma membrane calcium/calmodulin-dependent ATPases (PMCAs) are high affinity calcium pumps that extrude calcium from the cell. Emerging evidence suggests a novel role for PMCAs as regulators of calcium/calmodulin-dependent signal transduction pathways via interaction with specific partner proteins. In this work, we demonstrate that endogenous human PMCA2 and -4 both interact with the signal transduction phosphatase, calcineurin, whereas, no interaction was detected with PMCA1. The strongest interaction was observed between PMCA2 and calcineurin. The domain of PMCA2 involved in the interaction is equivalent to that reported for PMCA4b. PMCA2-calcineurin interaction results in inhibition of the calcineurin/nuclear factor of activated T-cells signalling pathway.