Shaheen Mowla

Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

The Highly Homologous T-Box Transcription Factors, TBX2 and TBX3, Have Distinct Roles in the Oncogenic Process

The T-box transcription factors TBX2 and TBX3 are overexpressed in several cancers and are able to bypass senescence by repressing ARF and p21(WAF1/CIP1/SDII). Although these studies suggest that they may both contribute to the oncogenic process by repressing common targets, whether they have redundant or distinct roles in cancers where they are both overexpressed remains to be elucidated. Importantly, when Tbx2 function is inhibited in melanoma cells lacking Tbx3, the cells senesce, but whether this is possible in melanoma cells overexpressing both proteins is not known. An understanding of this issue may have important implications for the design of an effective pro-senescence therapy. In this study, the authors used a sh-RNA approach to knock down TBX2 and TBX3 individually in 2 human melanoma cell lines that overexpress both these factors and then examined their specific involvement in the oncogenic process. They demonstrate, using in vitro and in vivo cell proliferation, as well as colony- and tumor-forming ability and cell motility assays, that TBX2 and TBX3 have distinct roles in melanoma progression. In the tested lines, although TBX2 could promote proliferation and transformation and was required by primary melanoma cells for immortality, TBX3 was required for tumor formation and cell migration. These findings were reproducible in a human breast cancer cell line, which confirms that TBX2 and TBX3, although highly homologous, do not have redundant roles in the transformation process of cancers where they are both overexpressed. These results have important implications for the development of new cancer treatments and in particular for melanoma, which is a highly aggressive and intractable cancer.

A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.

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 p19ARF and p21WAF1/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 p21WAF1/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.

PMA-induced up-regulation of TBX3 is mediated by AP-1 and contributes to breast cancer cell migration

The T-box transcription factor TBX3 provides an important link between embryonic development and cancer. TBX3 mediates limb, mammary gland and heart development and, in humans, mutations resulting in haplo-insufficiency of TBX3 lead to ulnar-mammary syndrome. Importantly, the de-regulation of TBX3 gene expression has been linked to several cancers, where it acts to suppress senescence and promotes proliferation and tumour invasion. Despite the negative impact of de-regulated TBX3 expression as seen by developmental defects and cancer, surprisingly little is known about the regulation of the TBX3 gene. In the present paper, we show that the phorbol ester PMA increases TBX3 protein and mRNA levels in a protein kinase C-dependent manner via the AP-1 (activator protein 1) transcription factors c-Jun and JunB. Furthermore, these AP-1 factors are shown to mediate the activation of the TBX3 gene by binding a non-consensus PMA-response element in the TBX3 promoter in vitro and in vivo. We also demonstrate that TBX3 contributes to the PMA-induced migration previously observed for the MCF-7 breast epithelium cancer cell line. Our present results reveal a previously unidentified pathway that up-regulates TBX3 expression and provides additional evidence that increased levels of TBX3 contribute to metastasis.

A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease

Aim: To report on molecular mechanisms of fetal hemoglobin (HbF) induction by hydroxyurea (HU) for the treatment of sickle cell disease. Study Design: Systematic review. Results: Studies have provided consistent associations between genomic variations in HbF-promoting loci and variable HbF level in response to HU. Numerous signal transduction pathways have been implicated, through the identification of key genomic variants in BCL11A, HBS1L-MYB, SAR1 or XmnI polymorphism that predispose the response to the treatment, and signal transduction pathways that modulate γ-globin expression (cAMP/cGMP; Giα/c-Jun N-terminal kinase/Jun; methylation and miRNA). Three main molecular pathways have been reported: i) Epigenetic modifications, transcriptional events and signaling pathways involved in HU-mediated response, ii) Signaling pathways involving HU-mediated response and iii) Post-transcriptional pathways (regulation by miRNAs). Conclusions: The complete picture of HU-mediated mechanisms of HbF production in Sickle Cell Disease remains elusive. Research on post-transcriptional mechanisms could lead to therapeutic targets that may minimize alterations to the cellular transcriptome.

A role for Tbx2 in the regulation of the α2(1) collagen gene in human fibroblasts

The T‐box gene family encodes highly conserved transcription factors that play important roles in embryonic development and have been implicated in carcinogenesis. One member of the family, Tbx2, is generally regarded as a transcriptional repressor but appears to be capable of functioning as an activator depending on the cellular context. This study shows that Tbx2 is expressed in normal human fibroblasts but is drastically reduced in several transformed fibroblast cell lines. This pattern of Tbx2 expression correlated with that observed for the human α2(1) collagen gene (COL1A2). Interestingly, stable expression of transfected Tbx2 in transformed fibroblast cell lines further reduces expression of the human endogenous COL1A2 gene. This ability of Tbx2 to repress the human COL1A2 gene was confirmed in luciferase reporter assays and shown to be independent of the consensus T‐box binding element. J. Cell. Biochem. 102: 618–625, 2007.

Phosphorylation of histone H3 by protein kinase C signaling plays a critical role in the regulation of the developmentally important TBX2 gene.

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

The African −387 C>T TGFB1 variant is functional and associates with the ophthalmoplegic complication in juvenile myasthenia gravis

Although extraocular muscles are commonly affected by myasthenia gravis (MG) at presentation, a treatment-resistant ophthalmoplegic complication of MG (OP-MG) occurs in younger patients with African-genetic ancestry. In MG, pathogenic antibodies activate complement-mediated muscle damage and this may be potentiated in some OP-MG cases because of relative deficiency of decay-accelerating factor/CD55. Extending this argument, we hypothesized that OP-MG individuals may harbor African-specific polymorphisms in key genes influencing extraocular muscle remodeling. We screened the regulatory region of the transforming growth factor beta-1 (TGFB1) gene encoding the cytokine pivotal in muscle healing responses. We show the frequency of an African-specific polymorphism TGFB1 c.−387 T (rs11466316) among South Africans with African-genetic ancestry is higher than 1000 Genomes African controls (17.2% vs 4.8%; P<1 × 10−7), and associates with juvenile OP-MG (28%; P=0.043). Further, TGFB1 −387 C>T is functional because it represses the TGFB1 promoter construct basal activity by fivefold, and OP-MG fibroblasts (−387 C/T or T/T) have lower basal TGFB1 mRNA transcripts compared with controls (−387 C/C)(P=0.001). Co-transfections with Sp1 show less responsiveness of the −387 T promoter compared with wild-type −387 C (P=0.015). Our findings suggest that population-specific alleles may lower TGFB1 expression, thereby influencing OP-MG susceptibility by inhibiting extraocular muscle CD55 upregulation and/or altered endplate remodeling.

The T-Box factor TBX3 is important in S-phase and is regulated by c-Myc and cyclin A-CDK2.

The transcription factor, TBX3, is critical for the formation of, among other structures, the heart, limbs and mammary glands and haploinsufficiency of the human TBX3 gene result in ulnar-mammary syndrome which is characterized by hypoplasia of these structures. On the other hand, the overexpression of TBX3 is a feature of a wide range of cancers and it has been implicated in several aspects of the oncogenic process. This includes its ability to function as an immortalizing gene and to promote proliferation through actively repressing negative cell cycle regulators. Together this suggests that TBX3 levels may need to be tightly regulated during the cell cycle. Here we demonstrate that this is indeed the case and that TBX3 mRNA and protein levels peak at S-phase and that the TBX3 protein is predominantly localized to the nucleus of S-phase cells. The increased levels of TBX3 in S-phase are shown to occur transcriptionally through activation by c-Myc at E-box motifs located at −1210 and −701 bps and post-translationally by cyclin A-CDK2 phosphorylation. Importantly, when TBX3 is depleted by shRNA the cells accumulate in S-phase. These results suggest that TBX3 is required for cells to transit through S-phase and that this function may be linked to its role as a pro-proliferative factor.