LFA-1 Integrin beta2 Chain Phosphorylation Regulates Protein Interactions and Mediates Signals in T Cells

Integrins are heterodimeric transmembrane adhesion receptors composed of alpha- and beta-subunits and they are vital for the function of multicellular organisms. Integrin-mediated adhesion is a complex process involving both affinity regulation and coupling to the actin cytoskeleton. Integrins also function as bidirectional signaling devices, regulating cell adhesion and migration after inside-out signaling, but also signal into the cell to regulate growth, differentiation and apoptosis after ligand binding. The LFA-1 integrin is exclusively expressed in leukocytes and is of fundamental importance for the function of the immune system. The LFA-1 integrins have short intracellular tails, which are devoid of catalytic activity. These cytoplasmic domains are important for integrin regulation and both the alpha and beta chain become phosphorylated. The alpha chain is constitutively phosphorylated, but the beta chain becomes phosphorylated on serine and functionally important threonine residues only after cell activation. The cytoplasmic tails of LFA-1 bind to many cytoskeletal and signaling proteins regulating numerous cell functions. However, the molecular mechanisms behind these interactions have been poorly understood. Phosphorylation of the cytoplasmic tails of the LFA-1 integrin could provide a mechanism to regulate integrin-mediated cytoskeletal interactions and take part in T cell signaling. In this study, the effects of phosphorylation of LFA-1 integrin cytoplasmic tails on different cellular functions were examined. Site-specific phosphorylation of both the alpha- and beta-chains of the LFA-1 was shown to have a role in the regulation of the LFA-1 integrin.Alpha-chain Ser1140 is needed for integrin conformational changes after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1, whereas beta-chain binds to 14-3-3 proteins through the phosphorylated Thr758 and mediates cytoskeletal reorganization. Thr758 phosphorylation also acts as a molecular switch to inhibit filamin binding and allows 14-3-3 protein binding to integrin cytoplasmic domain, and it was also shown to lead to T cell adhesion, Rac-1/Cdc42 activation and expression of the T cell activation marker CD69, indicating a signaling function for Thr758 phosphorylation in T cells. Thus, phosphorylation of the cytoplasmic tails of LFA-1 plays an important role in different functions of the LFA-1 integrin in T cells. It is of vital importance to study the mechanisms and components of integrin regulation since leukocyte adhesion is involved in many functions of the immune system and defects in the regulation of LFA-1 contributes to auto-immune diseases and fundamental defects in the immune system.

Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

Lebanese Plants and Plant-Derived Compounds Against Colon Cancer

Colorectal cancer (CRC) is a major health concern and demands long-term efforts in developing strategies for screening and prevention. CRC has become a preventable disease as a consequence of a better understanding of colorectal carcinogenesis. However, current therapy is unsatisfactory and necessitates the exploration of other approaches for the prevention and treatment of cancer. Plant based products have been recognized as preventive with regard to the development of colon cancer. Therefore, the potential chemopreventive use and mechanism of action of Lebanese natural product were evaluated. Towards this aim the antitumor activity of Onopordum cynarocephalum and Centaurea ainetensis has been studied using in vitro and in vivo models. In vitro, both crude extracts were non cytotoxic to normal intestinal cells and inhibited the proliferation of colon cancer cells in a dose-dependent manner. In vivo, both crude extracts reduced the number of tumors by an average of 65% at weeks 20 (adenomas stage) and 30 (adenocarcinomas stage). The activity of the C. ainetensis extract was attributed to Salograviolide A, a guaianolide-type sesquiterpene lactone, which was isolated and identified through bio-guided fractionation. The mechanism of action of thymoquinone (TQ), the active component of Nigella sativa, was established in colon cancer cells using in vitro models. By the use of N-acetyl cysteine, a radical scavenger, the direct involvement of reactive oxygen species in TQ-induced apoptotic cells was established. The analytical detection of TQ from spiked serum and its protein binding were evaluated. The average recovery of TQ from spiked serum subjected to several extraction procedures was 2.5% proving the inability of conventional methods to analyze TQ from serum. This has been explained by the extensive binding (>98%) of TQ to serum and major serum components such as bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AGP). Using mass spectrometry analysis, TQ was confirmed to bind covalently to the free cysteine in position 34 and 147 of the amino acid sequence of BSA and AGP, respectively. The results of this work put at the disposal for future development new plants with anti-cancer activities and enhance the understanding of the pharmaceutical properties of TQ, a prerequisite for its future clinical development.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.