Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells

Робочий зошит до конспекту лекцій з дисциплін “Основи побудови мікропроцесорних систем керування”, “Мікропроцесорна техніка”, “Програмні засоби систем керування” для студентів спеціальностей АГ – 8.092501 Автоматизоване управління технологічними процесами; АТ,ME – 8.091401 Системи управління й автоматики; СМ – 8.091501 Комп`ютерні системи та мережі

Дані методичні вказівки призначуються для вивчення апаратних та програмних засобів мікропроцесорних систем управління (МПС). У процесі вивчення дисципліни "Мікропроцесорні системи" студенти вивчають структуру, архітектуру, сигнали та системи команд однокристального мікроконтролера К1816 ВЕ51. Розглянуті питання організації паралельного та послідовного вводу- виводу, оганізація мікропроцесорних контролерів МПК). Схемні рішення, які приведені у методичних вказівках можуть бути використані при виконанні курсових і дипломних проектів студентами спеціальностей "Комп’ютеризовані системи управління і автоматики" (АТ) і "Автоматизація технологічних процесів гірничих підприємств" (АГ) та "Комп’ютерні системи та мережі" (СМ)

Software and hardware platform for testing of Automatic Generation Control algorithms

Development and implementation of new Automatic Generation Control (AGC) algorithms requires testing them on a model that adequately simulates primary energetic, information and control processes. In this article an implementation of a test platform based on HRTSim (Hybrid Real Time Simulator) and SCADA CK-2007 (which is widely used by the System Operator of Russia) is proposed. Testing of AGC algorithms on the test platform based on the same SCADA system that is used in operation allows to exclude errors associated with the transfer of AGC algorithms and settings from the test platform to a real power system. A power system including relay protection, automatic control systems and emergency control automatics can be accurately simulated on HRTSim. Besides the information commonly used by conventional AGC systems HRTSim is able to provide a resemblance of Phasor Measurement Unit (PMU) measurements (information about rotor angles, magnitudes and phase angles of currents and voltages etc.). The additional information significantly expands the number of possible AGC algorithms so the test platform is useful in modern AGC system developing. The obtained test results confirm that the proposed system is applicable for the tasks mentioned above

Mononuclear Phagocyte System Depletion Blocks Interstitial Tonicity-Responsive Enhancer Binding Protein/Vascular Endothelial Growth Factor C Expression and Induces Salt-Sensitive Hypertension in Rats

We showed recently that mononuclear phagocyte system (MPS) cells provide a buffering mechanism for salt-sensitive hypertension by driving interstitial lymphangiogenesis, modulating interstitial Na(+) clearance, and increasing endothelial NO synthase protein expression in response to very high dietary salt via a tonicity-responsive enhancer binding protein/vascular endothelial growth factor C regulatory mechanism. We now tested whether isotonic saline and deoxycorticosterone acetate (DOCA)-salt treatment leads to a similar regulatory response in Sprague-Dawley rats. Male rats were fed a low-salt diet and received tap water (low-salt diet LSD), 1.0% saline (high-salt diet HSD), or DOCA+1.0% saline (DOCA-HSD). To test the regulatory role of interstitial MPS cells, we further depleted MPS cells with clodronate liposomes. HSD and DOCA-HSD led to Na(+) accumulation in the skin, MPS-driven tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated hyperplasia of interstitial lymph capillaries, and increased endothelial NO synthase protein expression in skin interstitium. Clodronate liposome MPS cell depletion blocked MPS infiltration in the skin interstitium, resulting in unchanged tonicity-responsive enhance binding protein/vascular endothelial growth factor C levels and absent hyperplasia of the lymph capillary network. Moreover, no increased skin endothelial NO synthase protein expression occurred in either clodronate liposome-treated HSD or DOCA-salt rats. Thus, absence of the MPS-cell regulatory response converted a salt-resistant blood-pressure state to a salt-sensitive state in HSD rats. Furthermore, salt-sensitive hypertension in DOCA-salt rats was aggravated. We conclude that MPS cells act as onsite controllers of interstitial volume and blood pressure homeostasis, providing a local regulatory salt-sensitive tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated mechanism in the skin to maintain normal blood pressure in states of interstitial Na(+) and Cl(-) accumulation. Failure of this physiological extrarenal regulatory mechanism leads to a salt-sensitive blood pressure response

Thrombospondin 1 inhibits inflammatory lymphangiogenesis by CD36 ligation on monocytes

By engaging CD36 on murine macrophages, thrombospondin-1 prevents excessive macrophage VEGF-C production and corneal neovascularization

Intralymphatic CCL21 promotes tissue egress of dendritic cells through afferent lymphatic vessels

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient