Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.     

Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.     

Circatidal activity rhythm in the mangrove cricket Apteronemobius asahinai

Mangrove forests are influenced by tidal flooding and ebbing for a period of approximately 12.4 hours (tidal cycle). Mangrove crickets (Apteronemobius asahinai) forage on mangrove forest floors only during low tide. Under constant darkness, most crickets showed a clear bimodal daily pattern in their locomotor activity for at least 24 days; the active phases of approximately 10 hours alternated with inactive phases of approximately 2 hours, which coincided with the time of high tide in the field. The free-running period was 12.56+/-0.13 hours (mean+/-s.d. n=11). This endogenous rhythm was not entrained by the subsequent 24 hours light-dark cycle, although it was suppressed in the photophase; the active phase in the scotophase continued from the active phase in the previous constant darkness, with no phase shift. The endogenous rhythm was assumed to be a circatidal rhythm. On the other hand, the activity under constant darkness subsequent to a light-dark cycle was more intense in the active phase continuing from the scotophase than from the photophase of the preceding light-dark cycle; this indicates the presence of circadian components. These results suggest that two clock systems are involved in controlling locomotor activity in mangrove crickets.     

TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway

Type I interferons (IFN-alpha/beta) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-alpha/beta production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-alpha/beta induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-kappaB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-beta induction in this process, since normal IFN-alpha/beta production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-beta promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-beta, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-beta by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-kappaB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.     

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.     

Quality control of photosystem II: impact of light and heat stresses

Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.     

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13)

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G(alpha13), showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G(alpha13) enhanced the migration of control HeLa cells, the G(alpha13)-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G(alpha13).     

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.