Correction: A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan.

[This corrects the article DOI: 10.1371/journal.pone.0124655.].

Genetic variations in complement factors in patients with congenital thrombotic thrombocytopenic purpura with renal insufficiency.

The congenital form of thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13. Some, but not all, congenital TTP patients manifest renal insufficiency in addition to microangiopathic hemolysis and thrombocytopenia. We included 32 congenital TTP patients in the present study, which was designed to assess whether congenital TTP patients with renal insufficiency have predisposing mutations in complement regulatory genes, as found in many patients with atypical hemolytic uremic syndrome (aHUS). In 13 patients with severe renal insufficiency, six candidate complement or complement regulatory genes were sequenced and 11 missense mutations were identified. One of these missense mutations, C3:p.K155Q mutation, is a rare mutation located in the macroglobulin-like 2 domain of C3, where other mutations predisposing for aHUS cluster. Several of the common missense mutations identified in our study have been reported to increase disease-risk for aHUS, but were not more common in patients with as compared to those without renal insufficiency. Taken together, our results show that the majority of the congenital TTP patients with renal insufficiency studied do not carry rare genetic mutations in complement or complement regulatory genes.

A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

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.

The integrin-linked kinase-PINCH-parvin complex supports integrin αIIbß3 activation.

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 (αIIbα6Bß3) and mutant CHO cells expressing inactive αIIbα6Bß3 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 αIIbα6Bß3 activation. In the parental cells expressing active αIIbα6Bß3, 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 ß-parvin mRNA in the parent cells impaired the αIIbα6Bß3 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 αIIbα6Bß3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bß3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbß3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbß3 activation and the THD-induced αIIbß3 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 αIIbß3-expressing CHO cells further augmented THD-induced αIIbß3 activation, whereas they did not induce αIIbß3 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.

Integrin-linked kinase associated with integrin activation.

Platelet integrin alphaIIbbeta3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for alphaIIbbeta3 activation. However, the whole pathway associated with alphaIIbbeta3 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 alphaIIbalpha6Bbeta3, and then obtained mutant cells expressing inactivated alphaIIbalpha6Bbeta3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrin-linked kinase (ILK) complements defective integrin activation in the mutant cells. ILK mRNAs in the mutant cells contained 2 nonsense mutations, R317X and W383X, in a compound heterozygous state, resulting in a complete loss of ILK expression. Moreover, the mutant cells showed partially impaired activation of endogenous beta1 integrins. Knockdown of ILK in parental cells significantly suppressed the activated state of alphaIIbalpha6Bbeta3. However, ILK overexpression did not rescue the impaired integrin activation in talin knocked-down parental cells, whereas overexpression of talin-F3, a subdomain of the talin head domain, restored the function. Our present data suggest that ILK contributes to inside-out integrin activation.

Differential gene expression in the rat skeletal and heart muscle in glucocorticoid-induced myopathy: analysis by microarray.

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.

Tissue factor pathway inhibitor induces expression of JUNB and GADD45B mRNAs.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates tissue factor-triggered blood coagulation. It has previously been reported that TFPI inhibits the proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that TFPI may act as more than just a mediator of coagulation through changes in gene expression. By using DNA-array techniques and Northern blot analysis, we here revealed that TFPI transiently induced the mRNA expression of JUNB and GADD45B. The inducible effects were not observed in TFPIdeltaC (lacking the C-terminal basic region) or antithrombin (heparin-binding anticoagulant protease inhibitor). Moreover, the TFPI-induced expression of GADD45B was blocked by receptor-associated protein, which masks the ligand-binding domain of very low density lipoprotein receptor (VLDL-R). In conclusion, this is the first report to show an effect of TFPI on mRNA expression, and suggests that TFPI modulates cellular functions by inducing JUNB and GADD45B expression through binding to VLDL-R.

Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts.

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.