Angiogenesis, the formation of new blood vessels from a pre-existing vasculature, is crucial during development and for many diseases including cancer. Despite tremendous progress in the understanding of the angiogenic process, many aspects are still not fully elucidated. Several attempts have been made to identify novel genes involved in endothelial cell biology and angiogenesis. Here we focused on Pald1, a recently identified, vascular-enriched gene encoding paladin. Our in vitro studies indicate that paladin is a lipid phosphatase catalyzing dephosphorylation of phosphatidylinositol phosphates, a process essential for endocytosis and intracellular vesicle trafficking. We confirmed paladin’s vascular expression pattern and revealed a shift from a broad endothelial cell expression during development to an arterial mural cell-restricted expression in several vascular beds in adult mice. Paladin expression in the lung, however, was not restricted to the vasculature, but was also observed in pneumocytes and myofibroblasts. Lungs of female, but not male, Pald1 null mice displayed an obstructive lung phenotype with increased alveolar air sacs that were already apparent early in the alveolarization process. Only endothelial cells, but not other main lung cell types, were affected by loss of paladin. Endothelial cell number was reduced in 4-week old mice, possibly due to increased endothelial turnover in Pald1 deficient lungs. Vascular defects were also found in the retina. Loss of paladin led to reduced retinal vascular outgrowth accompanied by a hyperdense and hypersprouting vascular front. Downstream signaling of the major angiogenic driver, vascular endothelial growth factor receptor 2 (VEGFR2) was sustained in Pald1 null mice, and VEGFR2 degradation was impaired. Furthermore, paladin inhibited endothelial cell junction stability and loss of paladin led to reduced vascular permeability. Whether the differences in VEGFR2 signaling and adherens junction stability are connected remains to be fully explored. The newly identified lipid phosphatase activity of paladin and its specific effects on VEGFR2 signaling and adherens junction stability indicate that paladin may be controlling the endocytic pathway.

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No description available.

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INTRODUCTION: In this study, we identified the association of Vitamin D with orthodontic tooth movement and the impact of Vitamin D 1,25OHD and 25OHD forms on osteoblast function. MATERIALS AND METHODS: This study is comprised of two parts; a clinical and a laboratory part. In part I, saliva samples were collected from orthodontic patients each month for the first six months of orthodontic treatment along with casts at the beginning and the end of the study period. The samples were measured for Vitamin D binding protein (VitDBP) and alkaline phosphatase (ALP) and correlated with clinical tooth movement using absolute change in irregularity index (II). In part II, osteoblasts were collected from the calvaria of 3-5 day old healthy wild-type mice and cultured with differing concentrations of 1,25OHD (1, 10 and 100nmol) and 25OHD (100, 200, 400 nmol). ALP, OPG, and RANKL were measured as outcomes of Vitamin D treatment of osteoblasts. Intracellular signaling in response to Vitamin D was assessed by identifying the phosphorylation of ERK 1/2, p38 and NLK in primary osteoblasts. RESULTS: Measurement of salivary Vitamin D binding protein (VitDBP) showed that both low (<2.75 ng/ml) and high (>6.48 ng/ml) logVitDBP were associated with reduced tooth movement. There was no significant correlation between ALP levels and orthodontic treatment. Significant seasonal changes in VitDBP using a two-season year model were found with lower levels noticed in the summer (Mar-Sept) than in the winter (Oct-Feb) at p<0.05. A decrease in OPG production with higher concentrations of 1,25OHD and 25OHD with a corresponding increase in RANKL levels in primary osteoblast cultures was found. Similar to the clinical findings, ALP levels were not significantly affected by increasing concentrations of both 1,25OHD and 25OHD. The ERK 1/2 showed upregulation in response to treatment with 1,25OHD and downregulation in response to treatment with 25OHD concentrations. Meanwhile, p38 and NLK were affected by 1,25OHD and not by 25OHD. CONCLUSIONS: Clinical outcomes of orthodontic treatment are associated with a range of optimal Vitamin D binding protein (VitDBP) as detected in saliva. Different forms of Vitamin D affect osteoblast response and signaling differently.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Doctor of Philosophy. Johannesburg, 2004 / Alkaline phosphatase (ALP) is a ubiquitously expressed enzyme, that has been shown to play a role in cell differentiation and organogenesis. One study has also demonstrated ALP activity in rat adipocytes. The purpose of the present study was therefore to determine whether ALP is expressed in preadipocytes and what role it may have in adipogenesis. ALP activity was detected in the murine preadipocyte cell line, 3T3-L1, and in human preadipocytes isolated from mammary tissue, and from subcutaneous abdominal fat depots. In all the cell types studied ALP activity increased in parallel with adipogenesis. In the 3T3 -L1 cell line the tissue- non -specific ALP inhibitors, levamisole and histidine inhibited ALP activity, and adipogenesis, whereas the tissue specific ALP inhibitor Phe- Gly-Gly did not inhibit ALP or adipogenesis. In human preadipocytes, histidine inhibited adipogenesis and ALP activity, whereas levamisole inhibited adipogenesis, but did not block ALP activity in intact cells. However, levamisole did inhibit ALP activity by 50% in cell extracts. Levamisole was able to inhibit adipogenesis in human preadipocytes. The tissue specific ALP inhibitor, Phe Gly Gly, did not inhibit ALP activity or adipogenesis in human preadipocytes. ALP activity and adipogenesis, were compared in preadipocytes isolated from mammary tissue taken from black (13) and white (15) female subjects. Both ALP activity and adipogenesis, were lower in white compared to black female subjects. iii Immunocytochemical, analysis of the 3T3-L1 cell line and human preadipocytes demonstrated that ALP activity was restricted to the lipid droplets of these cells. ALP activity was also measured in serum samples obtained from 100 African subjects (74 females and 26 males) of varying BMI. ALP activity was found to be higher in obese than lean subjects, whereas, the other liver enzymes or products measured in serum were not. In fact these variables correlated to varying degrees with waist-hip ratio, whereas ALP levels did not. This suggest that liver function is predominantly influenced by abdominal obesity whereas serum ALP levels are more influenced by overall body adiposity. In conclusion, ALP, may be involved in the control of adipogenesis, in the 3T3- L1 preadipocyte cell line and in human preadipocytes isolated from mammary adipose tissue and subcutaneous abdominal adipose tisssue. The presence of ALP activity in lipid droplets in 3T3-L1 cells and human preadipocytes, and the ability of ALP inhibitors to block adipogenesis strongly suggest that ALP plays a role in the control of adipogenesis. / IT2017

Reversible protein phosphorylation is an essential component of metazoan signaling and cardiovascular physiology. Protein kinase activity is required for regulation of cardiac ion channel and membrane receptor function, metabolism, and transcription, and aberrant kinase function is widely observed across disparate cardiac pathologies. In fact, multiple generations of cardiac therapies (eg. beta-adrenergic receptor blockers) have targeted cardiac kinase regulatory cascades. In contrast, essentially nothing is known regarding the mechanisms that regulate cardiac phosphatase activity at baseline or in cardiovascular disease. Protein phosphatase 2A (PP2A) is a key phosphatase with multiple roles in cardiac physiology. Here we demonstrate the surprisingly complex regulatory platforms that control PP2A holoenzyme activity in heart. We present the first full characterization of the expression and regulation of the PP2A family of polypeptides in heart. We identify the expression of seventeen different PP2A genes in human heart and define their differential expression and distribution across species and in different cardiac chambers. We show unique subcellular distributions of PP2A regulatory subunits in myocytes, strongly implicating the regulatory subunit in conferring PP2A target specificity in vivo. We report striking differential regulation of PP2A scaffolding, regulatory, and catalytic subunit expression in multiple models of cardiovascular disease as well as in human heart failure samples. Importantly, we demonstrate that PP2A regulation in disease extends far beyond expression and subcellular location, by identifying and describing differential post-translational modifications of the PP2A holoenzyme in human heart failure. Furthermore, we go to characterize a mechanism for this method of post-translational modification that may represent a pathway capable of being therapeutically manipulated in human heart failure. Lastly we provide evidence that dysregulation of phosphatase activity contributes to the cellular pathology associated with a previously described inheritable human arrhythmia syndrome, highlighting the importance of the PP2A in cardiovascular physiology and disease. Together, our findings provide new insight into the functional complexity of PP2A expression, activity, and regulation in heart and in human cardiovascular disease and identify potentially new and specific gene and subcellular targets for the treatment of human arrhythmia and heart failure.

This research investigated the mechanism of action in bacteria of 3,4-methylenedioxy-β-nitropropene (BDM-I), a very broad spectrum antimicrobial lead compound in development as an anti-infective drug. The thesis proposes that BDM-I inhibits bacterial protein tyrosine phosphatases, a novel mechanism of action for an antimicrobial agent and a new target in microorganisms. This very open investigation was directed by considerable biological information on the effects of BDM-I in microorganisms and animals which provided insights into possible and improbable cellular targets. The biological effects of BDM-I were investigated using biochemical and cell-based assays, transmission electron microscopy and whole genome DNA microarray analysis. The specific experiments and order of execution were largely dependent on information gained as the project progressed. BDM-I was shown not to target the metabolic pathways of the major classes of antibacterial drugs, which supports a novel mechanism of action. Investigation of several species-specific effects suggested that cell signalling pathways were a possible target. Based on the structure of BDM-I and review of the scientific literature on cell signalling in bacteria, the hypothesis that BDM-I acted by inhibition of protein tyrosine phosphatases (PTP) was supported by demonstrating inhibition of human and bacterial PTP's in an enzyme assay. This mechanism was consistent with other demonstrated effects: inhibition of the intracellular pathogen, Chlamydia trachomatis; inhibition of swarming in Proteus spp. and inhibition of pigment production in Serratia marcescens; and with kill kinetics in bacteria and yeast. A pilot global genome analysis of BDM-I treated Bacillus subtilis did not detect differential expression of PTP genes but has provided many avenues for further investigation. This research further supports the development of BDM-I as a broad spectrum anti-infective drug.

The proteins tyrosine kinases (PTKs) and the proteins tyrosine phosphatases (PTPs) are very important proteins implicated in the regulation of the cell cycle and numerous human diseases including cancer. VHR is a dual-specific phosphatase whose principles substrats are the MAPKs ERK and JNK. Previous studies of our laboratory showed that this phosphatase is regulated during the cell cycle. Its level is low during the G1 phase and increases during S and G2 phases to reach a top at the G2/M phases. The low level of VHR during the G1 phase is probably due to an alteration of the protein stability demonstrated by a decreased half-life (after treatment of the cell with cycloheximide). In addition, VHR deletion by RNA interference in HeLa cells induces a cell cycle arrest during G1/S and G2/M transitions [1]. In the first part of our work, we show that the dual-specificity phosphatase VHR is overexpressed in cervix cancer cell lines compared to primary keratinocytes. These cell lines are infected (HeLa, CaSki and SiHa) or not (C33 and HT3) by HPV, suggesting that VHR overexpression is HPV independent, virus which is responsible of cervix cancer. We show that VHR overexpression is associated with a differential subcellular localization. Indeed, VHR is localized in the cytoplasm of normal keratinocytes while it localizes in both cytoplasm and nucleus of the cell lines studied. This observed overexpression is not associated with an increased expression of its mRNA but with a stabilization of the protein. CHX chase showed us that VHR half life is about 2 hours in primary keratinocytes and longer than 8 hours in cervix cancer cell lines. The TMA technique allowed us to study a large number of preneoplasic and neoplasic cervical lesions. Interestingly, we observe that VHR is significantly overexpressed in CIN III (Cervical intraepithelial Lesions III) (n=18) and in SCCs (Squamous Cell Carcinoma) (n=12) compared to normal exocols (n=16) and although in ADCs (Adenocarcinoma) (n=12) and AISs (Adenocarcinoma in situ) (n=9) compared to normal endocols (n=19). The differential subcellular localization is also observed in CIN III and SCCs compared to normal exocols but not in ADCs and AISs compared to normal endocols. In the second part of our work, we analyzed the effect of small selectif inhibitors of VHR developped by Dr. L. Tautz from Burnham Institute in La Jolla, CA on cervical cell lines, HeLa and CaSki. We show that these small sulfonic acids induced a decreased number and a decreased proliferation of HeLa and CaSki cells. These effects are similar to those induced by RNA interference. We also show that these inhibitors induced an increased level of ERK phosphorylation. Since ERK is a specific substrat of VHR, these results suggest that the small inhibitors developped by L. Tautz et al. are specific for VHR.

Reversible protein phosphorylation is an essential component of metazoan signaling and cardiovascular physiology. Protein kinase activity is required for regulation of cardiac ion channel and membrane receptor function, metabolism, and transcription, and aberrant kinase function is widely observed across disparate cardiac pathologies. In fact, multiple generations of cardiac therapies (eg. beta-adrenergic receptor blockers) have targeted cardiac kinase regulatory cascades. In contrast, essentially nothing is known regarding the mechanisms that regulate cardiac phosphatase activity at baseline or in cardiovascular disease. Protein phosphatase 2A (PP2A) is a key phosphatase with multiple roles in cardiac physiology. Here we demonstrate the surprisingly complex regulatory platforms that control PP2A holoenzyme activity in heart. We present the first full characterization of the expression and regulation of the PP2A family of polypeptides in heart. We identify the expression of seventeen different PP2A genes in human heart and define their differential expression and distribution across species and in different cardiac chambers. We show unique subcellular distributions of PP2A regulatory subunits in myocytes, strongly implicating the regulatory subunit in conferring PP2A target specificity in vivo. We report striking differential regulation of PP2A scaffolding, regulatory, and catalytic subunit expression in multiple models of cardiovascular disease as well as in human heart failure samples. Importantly, we demonstrate that PP2A regulation in disease extends far beyond expression and subcellular location, by identifying and describing differential post-translational modifications of the PP2A holoenzyme in human heart failure. Furthermore, we go to characterize a mechanism for this method of post-translational modification that may represent a pathway capable of being therapeutically manipulated in human heart failure. Lastly we provide evidence that dysregulation of phosphatase activity contributes to the cellular pathology associated with a previously described inheritable human arrhythmia syndrome, highlighting the importance of the PP2A in cardiovascular physiology and disease. Together, our findings provide new insight into the functional complexity of PP2A expression, activity, and regulation in heart and in human cardiovascular disease and identify potentially new and specific gene and subcellular targets for the treatment of human arrhythmia and heart failure.