The repeated low doses of UVB protected to a limited extent against the effects of an erythemal UVB dose on cytokine expression and thymine dimer formation, but not on CHS or COX enzymes.

Thymosin ␤4 (T␤4), a 4.9-kDa polypeptide primarily known as a main G-actin-sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that T␤4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. T␤4

Our data demonstrate that increased intracellular expression of thymosin ␤4 (T␤4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced T␤4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of T␤4 (AcSDKPT/4A) , T␤4 (KLKKTET/7A) , and T␤4 (K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type T␤4. Overexpression of T␤4, T␤4 (AcSDKPT/4A) , and T␤4 (K16A) affected intracellular formation of actin filaments. As expected, T␤4 (K16A) uptake by nuclei was impaired. On the other hand, overexpression of T␤4 (KLKKTET/7A) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular T␤4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both T␤4 and T␤4 (AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants T␤4 (KLKKTET/7A) and T␤4 (K16A) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATPdependent DNA helicase II was found to be associated with T␤4. Ku80 and T␤4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and T␤4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for T␤4 and mediates its intracellular activity.2 is the most abundant member of the highly conserved family of acidic polypeptides called ␤-thymosins. Although it is a typical intracellular polypeptide, it plays numerous roles, both intracellularly and extracellularly. Numerous observations indicate that T␤4 is involved in adhesion and spreading of fibroblasts (1, 2), differentiation of endothelial cells (3, 4), directional migration of endothelial cells and keratinocytes (5-8), angiogenesis (3-6, 9, 10), wound healing (6, 7, 11), hair follicle growth (8), and apoptosis (12, 13), and has been described to possess anti-inflammatory properties (11,14). In addition, elevated T␤4 expression has been observed in various malignant cell lines and tumors (15-17) and its levels seem to be associated with increased tumorigenicity and metastatic potential (10, 13, 18 -20). The increased expression of T␤4 correlates with the invasive capability of the cells, the degree of morphologic transformation, and disintegration of actin filaments (21). Recent studies have also correlated increased T␤4 expression with potentiated cell growth (13, 22) but this observation seems not to be universal (2, 10).T␤4 is conside...

In this report we compared the mechanism by which nitric oxide (NO), generated exogenously and endogenously, affects the plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells. For this purpose, we stimulated the endothelial cell line EA.hy 926 with tumour necrosis factor a (TNFa) in the presence of the exogenously NO-releasing donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, or regulators of nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester hydrochloride and substrate l-Arg. Expression of PAI-1 in EA.hy 926 cells was determined by measuring the level of mRNA, using relative quantitative reverse transcriptase PCR, and protein, using ELISA. In addition, we estimated the level of activation of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK1/2), in the cells before and after treatment with TNFa, in the presence or absence of NO donors and inhibitors. In contrast to exogenously released NO that significantly reduced mostly basal PAI-1 expression, endogenously generated NO by NOS potentiated TNFa-induced upregulation of PAI-1 expression. Exogenously and endogenously generated NO causes different effects on activation of the MAPKs ERK1/2 and JNK1/2. Specifically, the SNP-released NO activates only ERK1/2, while endogenously generated NO in a pathway induced by TNFa activates both MAPKs. Thus our data indicate that due to different cellular locations and mechanisms of generation, NO may participate in various signalling pathways leading to opposite effects on PAI-1 expression in endothelial cells.Keywords: plasminogen activator inhibitor type 1 expression; nitric oxide donors; tumor necrosis factor a activation; mitogen-activated protein kinases.Plasminogen-activator inhibitor type 1 (PAI-1) is an essential component of the plasmin/plasminogen activator cascade that regulates both vascular fibrinolysis and extravascular generation of plasmin. Plasmin/plasminogen activator-mediated proteolysis not only solubilizes fibrin, and digests various basement membrane components [1], but also is implicated in the activation of collagenase [2] and transforming growth factor b [3] and in the release of basic fibroblast growth factor complexed with heparan sulfate from endothelial cells [4]. It also regulates the proteolytic activity generated extravascularly after activation of plasminogen accompanying tissue repair. In addition, PAI-1 appears to be involved in controlling migration of endothelial cells [5] and vascular smooth muscle cells [6], a critical process in vascular remodelling and atherogenesis. PAI-1 expression has been demonstrated in various cell types, and multiple factors have been described that play a role in the regulation of PAI-1 synthesis and secretion [7,8]. Increased levels of PAI-1 have been identified in atherosclerotic human arteries [9], in neontima formed after vein graft failure [10], and balloon-injured blood vessels [11]. Elevated PAI-1 synthesis and secre...

The midsegment of the ␤ 3 subunit has been implicated in the ligand and cation binding functions of the ␤ 3 integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, ␤ 3 -(95-373) and ␤ 3 -(95-301), were expressed and found to bind fibrinogen. ␣ IIb ␤ 3 is a typical member of the integrin family of cell adhesion receptors (1), being composed of an ␣ (␣ IIb ) and a ␤ (␤ 3 ) subunit, which associate to form a noncovalent heterodimer. This integrin is the most abundant membrane protein on the platelet surface and serves as a receptor for multiple adhesive proteins including fibrinogen (Fg).1 Two sets of peptides (HHLGGAKQAGDV, corresponding to the sequence at the C terminus of the Fg ␥-chain (2) and RGDX, corresponding to a sequence present in many protein ligands of ␣ IIb ␤ 3 and recognized by many other integrins as well) define the recognition specificity of ␣ IIb ␤ 3 for its macromolecular ligands (reviewed in Ref.3). ␣ V ␤ 3 , which shares the same ␤ 3 subunit as ␣ IIb ␤ 3 , is broadly distributed and binds many but not all of the same ligands as ␣ IIb ␤ 3 , including Fg, von Willebrand factor, and fibronectin (reviewed in Refs. 4 and 5). This integrin also exhibits an RGD recognition specificity but shows a much weaker recognition of Fg ␥-chain peptides (6). Numerous studies have suggested that binding of macromolecular ligands to ␣ IIb ␤ 3 as well as ␣ V ␤ 3 involves multiple contacts in each subunit (7-13). Essential residues for ligand binding to ␣ IIb ␤ 3 reside in two major regions: a midsegment of ␤ 3 , ␤ 3 -(95-400), and the amino-terminal aspect of ␣ IIb , ␣ IIb -(1-334) (11), which contains seven structural repeats (14). The midsegment of the ␤ 3 subunit is highly conserved among integrin ␤ subunits and exhibits some structural and functional features of an I domain. I domains are present in nine integrin ␣ subunits and play major roles in the ligand binding functions of their integrin heterodimers (15)(16)(17)(18)(19)(20). The relationship between the conserved ␤ midsegments and ␣ I domains was proposed based upon similarities in their hydropathy profiles, secondary structural predictions, and mutational analyses (18, 20 -22). A central feature of I domains is a metal ion-dependent adhesion site, a MIDAS motif (15,18,19,23). In MIDAS motifs, three of the five cation coordination sites are provided by a DXSXS sequence and two other coordination sites are provided by oxygenated residues distant in the primary sequence. Mutations of the cation-coordinating residues in a MIDAS motif often cause loss of ligand binding functions of an integrin, and ligand binding sites map in close proximity to MIDAS motifs (18,24,25). The ␤ 3 midsegment does contain a 119 DXSXS sequence, and these residues also have been implicated in the ligand binding functions of ␣ IIb ␤ 3 (24, 26 -28). While there is broad consensus that the midsegment of integrin ␤ subunit contains a functional MIDAS, other structural algorithms have predicted ...