We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1␣ and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1␤ and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation.Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. Hepatitis B virus (HBV)1 infection causes acute hepatitis in humans, 10% of cases resulting in chronic hepatitis (1), which is linked to development of hepatocellular carcinoma (HCC) (1); integrated HBV DNA is present in virtually all HBV-mediated liver cancers (2, 3). However, the mechanism of HBV-mediated hepatocarcinogenesis remains elusive.HBV encodes a 16.5-kDa protein termed the X antigen (pX) (4). pX is expressed during viral infection (5-7), is required for the viral life cycle (7), and is highly conserved in all oncogenic mammalian hepadnaviruses (8, 9). In contrast, the avian hepatitis virus, which lacks oncogenic potential, is devoid of an X open reading frame (10). pX promotes liver tumor formation in transgenic mice expressing high levels of pX (11), potentiates c-Myc-induced hepatocarcinogenesis in c-myc/pX bitransgenics (12), and acts as a tumor promoter in hepatocarcinogenesis (13). While the published data clearly implicate pX in HCC, the direct oncogenic effect of pX in hepatocyte transformation has not been demonstrated. Herein, we provide evidence demonstrating the causal link of pX expression to the transformation of immortalized hepatocytes and describe a conditional pX expression system that is amenable to the study of the mechanism of pX-mediated transformation.pX is a multifunctional protein, with reported activities affecting transcription (14), cell growth (15, 16), and apoptotic cell death (17, 18). Although pX does not directly bind doublestranded DNA, pX acts as a promiscuous transactivator (reviewed in Ref. 14), via interaction with several components of the transcriptional apparatus (19 -23). In addition, specific pXresponsive cis-acting elements have been identified, e.g. NF-B (24 -28), AP-1 (29 -34), AP-2 (29), and CRE sites (35-37). Transcriptional activation of the AP-1 and NF-B sites by pX is via cytoplasmic signaling pathways, including the protein kinase C pathway (31, 32) a...

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14 -/-mice. We observed that MRP-14 and MRP-8/ MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14 -/-mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14 -/-mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y 12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1-and PAR4-mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y 12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y 12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.

Key Points In Bdkrb2−/− mice, compensatory Mas and AT2R overexpression elevates NO and PGI2 to prolong bleeding times and delay arterial thrombosis. This NO and PGI2 elevation attenuates platelet integrin-dependent spreading and GPVI responses without altering thrombin or ADP activation.

Cells of the vertebrate neural crest (crest cells) differentiate in vitro to melanocytes and sympathoadrenal (SA) progenitor cells. We have shown previously, using primary J. quail neural crest cultures, the combinatorial effect of bone morphogenetic protein-2 (BMP-2) and cAMP signaling on SA cell development. Herein, we report that in primary J. quail neural crest cultures, BMP-2 and cAMP signaling similarly exert a combinatorial effect on melanocyte development. We demonstrate that BMP-2 treatment of neural crest cells increases melanogenesis by promoting the synthesis of melanin. This increased melanin synthesis by BMP-2 is effected by the selective increase in the transcription of the tyrosinase gene, encoding the rate-limiting enzyme of the melanin biosynthetic pathway. By contrast, BMP-2 exerts no effect on the expression of the tyrosine-related proteins 1 and 2 (Tyrpl and Dct), also involved in the melanin biosynthetic process, or on the expression of microphalmia (Mitf) gene, supporting the fact that BMP-2 does not affect melanocyte differentiation. Employing transient transfection analysis of tyrosinase-reporter constructs in B16 melanoma cells, we demonstrate that the BMP-2 response-element is localized between 900 and 1,100 bp upstream from the tyrosinase transcriptional start site. These studies support a role for BMP-2 in melanogenesis by selectively targeting the expression of the tyrosinase gene involved in melanin biosynthesis.