Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

AbstractMonochloramine (NH2Cl) is one of the inflammation-derived oxidants, and has various effects on cell cycle, apoptosis and signal transduction. We studied the effects of NH2Cl on DNA repair response induced by ultraviolet B (UVB) irradiation in normal human diploid fibroblasts, TIG-1. TIG-1 irradiated with 20 mJ/cm2 UVB showed marked increase in thymine dimer, which decreased by about 50% after 24 h. This decrease in thymine dimer was significantly attenuated (P<0.05) by the pretreatment of NH2Cl (200 μM), which indicated DNA repair inhibition. UVB induced p53 phosphorylation at Ser15, Ser20 and Ser37, and p53 accumulation, and NH2Cl also inhibited these changes. Consequently, UVB-induced increase in the downstream effectors of p53, namely p21Cip1 and Gadd45a, were almost completely inhibited by NH2Cl. Immunoprecipitation study indicated that the association of p53 and MDM2, an E3 ubiquitin ligase for p53, did not change substantially by NH2Cl and/or UVB. The phosphorylation of p53 (Ser15 and Ser37) by UVB is catalyzed by ATR (ataxia telangiectasia mutated and Rad3 related kinase), which works as DNA damage sensor, and ATR also phosphorylates checkpoint kinase 1(Chk1) at Ser345. NH2Cl also inhibited the phosphorylation of Chk1 (Ser345). As UVB-induced DNA damage is repaired by nucleotide excision repair (NER) in human cells, these findings indicated that NH2Cl inhibited NER through the inhibition of p53 phosphorylation and accumulation, and NH2Cl probably impaired DNA damage recognition and/or ATR activation. NH2Cl may facilitate carcinogenesis through the inhibition of NER that repairs DNA damages from various carcinogens

Oxidative modification of IκB by monochloramine inhibits tumor necrosis factor α-induced NF-κB activation

AbstractWe have previously reported that monochloramine (NH2Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor α (TNFα)-induced expression of cell adhesion molecules and nuclear factor-κB (NF-κB) activation (Free Radical Research 36 (2002) 845–852). Here, we studied the mechanism how NH2Cl inhibited TNFα-induced NF-κB activation, and compared the effects with taurine chloramine (Tau–NHCl). Pretreatment of Jurkat cells with NH2Cl at 70 μM resulted in suppression of TNFα-induced IκB phosphorylation and degradation, and inhibited NF-κB activation. In addition, a slow-moving IκB band appeared on SDS-PAGE. By contrast, Tau–NHCl for up to 200 μM had no effects. Interestingly, NH2Cl did not inhibit IκB kinase activation by TNFα. Protein phosphatase activity did not show apparent change. When recombinant IκB was oxidized by NH2Cl in vitro and phosphorylated by TNFα-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IκB. In addition, when NF-κB–IκB complex was immunoprecipitated from NH2Cl-treated cells and phosphorylated in vitro by recombinant active IκB kinase, native IκB but not oxidized IκB was phosphorylated. Amino acid analysis of the in vitro oxidized IκB showed methionine oxidation to methionine sulfoxide. Although Tau–NHCl alone had little effects on TNFα-induced NF-κB activation, simultaneous presence of Tau–NHCl and ammonium ion significantly inhibited the NF-κB activation, probably through the conversion of Tau–NHCl to NH2Cl. These results indicated that NH2Cl inhibited TNFα-induced NF-κB activation through the oxidation of IκB, and that NH2Cl is physiologically more relevant than Tau–NHCl in modifying NF-κB-mediated cellular responses

Platelets Strongly Induce Hepatocyte Proliferation with IGF-1 and HGF In Vitro

Background. It is well known that platelets have athrombotic effect. However, platelets play an importantrole not only in hemostasis but also in woundhealing and tissue regeneration. Platelets have beenreported to accumulate in the liver and promote liverregeneration after an extended hepatectomy, but themechanism is unclear. The present study was designedto clarify the mechanism by which plateletshave a direct proliferative effect on hepatocytes invitro.Materials and methods. Hepatocytes obtained frommale BALB/c mice by collagenase digestion and immortalizedhepatocytes (TLR2) were used. To elucidatethe mechanism of the proliferative effect of platelets,DNA synthesis of hepatocytes was measuredunder various conditions and the related cellular signalswere analyzed. Chromatographic analysis wasalso performed to clarify which elements of plateletshave mitogenic activity.Results. DNA synthesis significantly increased in thehepatocytes cultured with platelets (P < 0.001). However,when the platelets and hepatocytes were separated,the platelets did not have a proliferative effect.Whole disrupted platelets, the supernatant fraction,and fresh isolated platelets had a similar proliferativeeffect, while the membrane fraction did not. After theaddition of platelets, both Akt and extracellularsignal-regulated kinases ERK1/2 were activated, butextracellular signal-regulated kinase STAT3 was not activated. Some mitogenic fractions were obtainedfrom the platelet extracts by gel exclusion chromatography;the fractions were rich in hepatocyte growthfactor and IGF-1.Conclusions. Direct contact between platelets andhepatocytes was necessary for the proliferative effect.The direct contact initiated signal transduction involvedin growth factor activation. Hepatocyte growthfactor, vascular endothelial growth factor, and insulin-like growth factor-1, rather than platelet-derivedgrowth factor, mainly contributed to hepatocyteproliferation

Molecular mechanisms of liver regeneration and protection for treatment of liver dysfunction and diseases

Liver regeneration is a necessary process which most liver damage depends on for its recovery. It is achieved by a complex interaction network consisting of liver cells (hepatocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells and stem cells) and extra-hepatic organs (thyroid gland, adrenal gland, pancreas, duodenum and autonomous nervous system). The restoration of liver volume depends on the hepatocyte proliferation which includes initiation/proliferation/termination phases. Hepatocytes are 'primed' mainly by Kupffer cells via cytokines (IL-6 and TNF-alpha) and then 'proliferation' and 'cell growth' of hepatocyte are induced by the stimulations of cytokines and growth factors (HGF and TGF-alpha). Liver regeneration is achieved by cell proliferation and cell growth, where IL-6/STAT3 pathway and PI3-K/PDK1/Akt pathway play pivotal roles, respectively. IL-6/STAT3 pathway regulates hepatocyte proliferation via cyclin D1/p21 and protects against cell death by up-regulating FLIP, Bcl-2, Bcl-xL, Ref1 and MnSOD. PI3-K/PDK1/Akt is known to be responsible for regulation of cell size via its downstream molecules such as mTOR in addition to its survival, anti-apoptotic and anti-oxidative properties. Although the molecular mechanisms of liver regeneration have been actively studied, it is required to elucidate and leverage the mechanisms of liver regeneration for the sufficient treatment of liver diseases

Relevance of FXR-p62/SQSTM1 pathway for survival and protection of mouse hepatocytes and liver, especially with steatosis

Abstract Background Liver injury and regeneration involve complicated processes and are affected by various physio-pathological conditions. Surgically, severe liver injury after surgical resection often leads to fatal liver failure, especially with some underlying pathological conditions such as steatosis. Therefore, protection from the injury of hepatocytes and liver is a serious concern in various clinical settings. Methods We studied the effects of the farnesoid X receptor (FXR) on cell survival and steatosis in mouse hepatocytes (AML12 mouse liver cells) and investigated their molecular mechanisms. We next studied whether or not FXR improves liver injury, regeneration and steatosis in a mouse model of partial hepatectomy (PH) with steatosis. Results An FXR-specific agonist, GW4064, induced expressions of the p62/SQSTM1 gene and protein in AML12 mouse liver cells. Because we previously reported p62/SQSTM1 as a key molecule for antioxidation and cell survival in hepatocytes, we next examined the activation of nuclear factor erythroid 2-related factor-2 (Nrf2) and induction of the antioxidant molecules by GW4064. GW4064 activated Nrf2 and subsequently induced antioxidant molecules (Nrf2, catalase, HO-1, and thioredoxin). We also examined expressions of pro-survival and cell protective molecules associated with p62/SQSTM1. Expectedly, GW4064 induced phosphorylation of Akt, expression of the anti-apoptotic molecules (Bcl-xL and Bcl-2), and reduced harmful hepatic molecules (Fas ligand and Fas). GW4064 promoted hepatocyte survival, which was cancelled by p62/SQSTM1 siRNA. These findings suggest the potential relevance of the FXR-p62/SQSTM1 pathway for the survival and protection of hepatocytes. Furthermore, GW4064 induced the expression of small heterodimer partners (SHP) and suppressed liver X receptor (LXR)-induced steatosis in hepatocytes, expecting the in vivo protective effect of FXR on liver injury especially with steatosis. In the hepatectomy model of db/db mice with fatty liver, pre-treatment by GW4064 significantly reduced post-PH liver injury (serum levels of LDH, AST & ALT and histological study) and improved steatosis. The key molecules, p62/SQSTM1, Nrf2 and SHP were upregulated in fatty liver tissue by GW4064 treatment. Conclusions The present study is the first to demonstrate the relevance of FXR-p62/SQSTM1 and -SHP in the protection against injury of hepatocytes and post-PH liver, especially with steatosis