In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

Platelet integrin ␣IIb␤3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for ␣IIb␤3 activation. However, the whole pathway associated with ␣IIb␤3 activation remains to be determined. To address this issue, we established a Chinese hamster ovary cell line (parental cells) that expresses constitutively activated chimeric integrin ␣IIb␣6B␤3, and then obtained mutant cells expressing inactivated ␣IIb␣6B␤3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrinlinked kinase ( IntroductionIntegrins are cell -surface transmembrane receptors that serve as heterodimeric complexes composed of an ␣-and a ␤-subunit. 1 Integrins are expressed in various cells and mediate the binding of cells to adhesive proteins. Integrin ␣IIb␤3 is mainly expressed in megakaryocyte/platelet lineage cells and functions as a major receptor for fibrinogen and von Willebrand factor (VWF), enabling crosslinking between platelets in the process of thrombus growth. 2 In patients with Glanzmann thrombasthenia, platelet aggregation is impaired and a bleeding tendency is observed. 3 In addition, a blockade of platelet aggregation using ␣IIb␤3 antagonists beneficially reduced thrombotic events in pathologic states, such as percutaneous coronary intervention or acute coronary syndromes. 4,5 Thus, ␣IIb␤3 plays a critical role in both physiologic and pathologic thrombus formation.Activation of ␣IIb␤3 is regulated by an intracellular process called inside-out signaling, which is triggered by agonist stimulation of G protein-coupled receptors or binding of VWF and collagen to their receptors glycoprotein Ib-V-IX and glycoprotein VI, respectively. 6-8 Activated ␣IIb␤3 shows a shift from a low-to high-affinity state for ligands, fibrinogen, and VWF. The bound ligands accelerate ␣IIb␤3 clustering and promote further conformational changes that initiate intracellular signal pathways through the cytoplasmic domains, termed outside-in signaling. Studies of platelet integrin activation have recently been advanced by RNA interference technology and knockout mice. For example, talin, a major cytoskeletal actin-binding protein, is composed of an N-terminal head domain, and a large C-terminal rod domain is associated with ␤ integrin cytoplasmic domains. The talin head domain contains a FERM (protein 4.1, ezrin, radixin, moesin) domain and plays a critical role in the final common step in integrin activation. [9][10][11] The FERM domain consists of 3 subdomains: F1, F2, and F3. The F3 subdomain binds to the ␤3 cytoplasmic domain with high affinity and results in ␣IIb␤3 activation. 12 Recently, kindlin-3, another FERM domain-containing protein, one of the kindlin family (kindlin-1, -2, and -3), was shown to be an essential factor for platelet integrin activation, 13 and kindlin-2 also proved to function as a coactivator of ␤3 integrins. 14 However, although t...

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.

Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.

Administration of glucocorticoids results in hypertension, cardiac hypertrophy, and general myopathy. The present study analyzed the acute effect of dexamethasone (0.5 mg/100 g for 3 days) or dexamethasone plus insulin-like growth factor-1 (0.35 mg/100 g for 3 days) on differential gene expression in the gastrocnemius muscle and the left ventricular myocardium of rats. Dexamethasone induced atrophy of gastrocnemius muscle. Cathepsin L, and not ubiquitin, was the earliest mediator of skeletal muscle proteolysis induced by dexamethasone. Insulin-like growth factor-1 reversed gastrocnemius muscle mass, and deleted a part of downregulated genes by dexamethasone. On the other hand, dexamethasone administration did not result in cardiac hypertrophy or hypertension. Only prostaglandin D synthase gene was upregulated by dexamethasone in myocardium, and genes related to extracellular matrix and proteinase inhibitor were downregulated. Molecular alteration for hypertrophy might have initiated. Dexamethasone-induced proteolysis and reversal with insulin-like growth factor-1 occurred rapidly in skeletal muscle; but was relatively delayed in the myocardium.