Regulation of the anti-senescence factor, TBX2, by the UV stress signalling pathway and the mitotic cyclin dependent kinases

The T-box gene family has achieved great prominence in the field of developmental biology because its members have been demonstrated to play important roles in embryonic development and mutations within several T-box genes are associated with a number of human congenital diseases. Several lines of evidence have also implicated members of the T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors, Tbx2 and Tbx3, can suppress senescence through repressing the cyclin dependent kinase inhibitors, p21wAF11 c1P118011 and p19ARF. Both Tbx2 and Tbx3 have also been linked to several cancers primarily because their expression levels have been found to be deregulated in these cancers. However, despite the pivotal role that members of the T-box family play in a wide variety of biological processes, very little is known about the biochemical pathways that regulate their levels and transcriptional activity. In view of the detrimental consequences resulting from altered levels of T-box proteins, as seen both in developmental disorders and in certain cancers, the need to identify such pathways is important. The aim of this study was therefore to identify kinases that phosphorylate and regulate the levels and activity of Tbx2 with the view to understanding its role in cell cycle regulation and cancer. This study shows that the p38 stress mitogen-activated protein kinase, and the mitotic cyclin A/Cdk2 and cyclin B1/Cdk1, are direct regulators of Tbx2 both in vitro and in vivo. It is possible that Tbx2 and Tbx3 may contribute towards the oncogenic process through their anti-senescence function, especially since a dominant negative form of Tbx2 induces senescence in melanoma cells overexpressing Tbx2. However, very little is known about whether Tbx2 is indeed regulated during replicative- or stress-induced senescence. In this study, using a breast cancer cell line known to overexpress Tbx2, the Tbx2 protein is shown to be specifically phosphorylated by the p38 kinase in response to stress induced by ultraviolet irradiation. Using site-directed mutagenesis and in vitro kinase assays, serine residues 336, 623 and 675 in the Tbx2 protein were identified as p38 target sites. These sites are also shown to be phosphorylated in vivo. Importantly, western blotting, immunofluorescence and reporter assays reveal that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localisation of the protein, and an increase in the ability of Tbx2 to repress the p21wAF11 c/P118011 promoter. These results show, for the first time, that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway. This leads to a better understanding of the anti-senescence function of Tbx2. The ability of Tbx2 to function as an anti-senescence factor, as well as its altered regulation being associated with certain cancers, suggests that"its levels may need to be tightly regulated during the cell cycle. Indeed, the Tbx2 protein was previously shown to be regulated during the various phases of the cell cycle, peaking at G2. The changes in the Tbx2 protein levels did not match changes in Tbx2 mRNA levels, suggesting that the protein may be regulated by posttranslational modifications such as phosphorylation. This study shows that the phosphorylation status of Tbx2 is regulated during the cell cycle with levels of phosphorylation peaking in G2 and M, in mouse and human cells respectively. Phosphorylation was shown to be specifically mediated by the mitotic kinases as demonstrated in experiments when the mitotic kinase inhibitor, olomoucine, was included. This study provides data to suggest that Tbx2 may be regulated differently during the cell cycle in mouse and human cells. Using site-directed mutagenesis and in vitro kinase assays, Tbx2 was found to be specifically phosphorylated at serine residues 192 and 336 by cyclin A/Cdk2 and serine residues 336 and 342 by cyclin 81/Cdk1. These sites are also targets for phosphorylation in vivo since mutating them altered the phosphorylation status ofTbx2. Moreover, both cyclin A and 81 were shown to bind Tbx2 in vitro and in vivo and the minimal region required for binding was mapped to its DNA-binding domain. Importantly, immunofluorescence demonstrates that the Tbx2 protein localises specifically to the nucleus at G2; this translocation was shown to be blocked in the presence of olomoucine. Furthermore, western blot analyses and reporter assays showed that pseudo-phosphorylation by cyclin 81/ Cdk1, but not cyclin A/Cdk2, leads to increased Tbx2 protein levels and an increase in the ability of Tbx2 to repress the p21wAFtiCIPt!Sou promoter. These results disclose, for the first time, that phosphorylation by cyclin A/Cdk2 and cyclin 81/Cdk1 of the Tbx2 protein regulates its activity. This data provide additional evidence to support a role for Tbx2 in the G2 and/or M phase of the cell cycle

UV-mediated Regulation of the anti-senescence factor Tbx2

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2

What happens to misunderstandings of biomedical concepts across a medical curriculum?

Research on the extent and nature of commonly misunderstood fundamental biomedical concepts across a medical curriculum is scarce. These misunderstandings could point toward robust misconceptions. We examined first whether common misunderstandings persist throughout a medical curriculum, followed by a fine-grained analysis to identify their nature. We designed and administered a 2-tier test to 987 medical students across our curriculum, with 8 questions covering the respiratory and cardiovascular systems, cell div

Integrating targeted gene expression and a skin model system to identify functional inhibitors of the UV activated p38 MAP kinase

International audienceThe stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds

Building a community of practice in an extended curriculum: the intervention programme

Chapter in bookThis chapter describes how the pedagogic approach underpinning the extended curriculum offering in a Health Sciences faculty attempts to offset the discomfort students often feel when they have to join this intervention. Their reflective enquiry is able to shed light on the extent to which the internal pedagogic model, which is built around the notion of supporting a community of practice, has been effective in mediating and supporting students’ transition to this extended curriculum programme.ECP Unit, Fundani: Centre for Higher Education Development, at Cape Peninsula University of Technology, and Head of Department, James Garrawa