Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis similar to 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging

Emergence of Circulating Recombinant Form 56_cpx in Pakistani HIV-infected patient

In Pakistan, HIV has recently converted from outbreak to concentrated epidemic and has also bridged into the low-risk population. The HIV epidemic in Pakistan is mainly derived by subtype A. Here we present first case and genetic analysis of a circulating recombinant form 56_cpx in Pakistani HIV-infected patient. Genetic analysis of the sequence indicated that Pakistani 56_cpx sequence had more drug resistance mutation than other 56_cpx sequences available in the database

Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway

Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H2O2 or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable sup eroxide-disinutase/catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide- and H2O2-mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt(2)cAMP and Bt(2)cAMP+hHDL(3)-stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPK alpha isoform, along with relatively low-level expression of p38 MAPK gamma. By contrast, little or no expression was detected for p38 MAPK beta and p38 MAPK delta isofoms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPK alpha or dnP38 MAPK beta construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPK gamma or dnp38 MAPK delta construct. These results indicate that activated p38 NIAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis

Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elementss⃞

The 3′untranslated region (UTR) of human LDL receptor (LDLR) mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover and mediates the stabilization induced by berberine (BBR). However, the identities of the specific RNA binding proteins involved in the regulation of LDLR mRNA stability at the steady state level or upon BBR treatment are unknown. By conducting small interfering RNA library screenings, biotinylated RNA pull-down, mass spectrometry analysis, and functional assays, we now identify heterogeneous nuclear ribonucleoprotein D (hnRNP D), hnRNP I, and KH-type splicing regulatory protein (KSRP) as key modulators of LDLR mRNA stability in liver cells. We show that hnRNP D, I, and KSRP interact with AREs of the LDLR 3′UTR with sequence specificity. Silencing the expression of these proteins increased LDLR mRNA and protein levels. We further demonstrate that BBR-induced mRNA stabilization involves hnRNP I and KSRP, as their cellular depletions abolished the BBR effect and BBR treatment reduced the binding of hnRNP I and KSRP to the LDLR mRNA 3′UTR. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE binding proteins, including hnRNP D, hnRNP I, and KSRP. Our results suggest that interference with the ability of destabilizing ARE binding proteins to interact with LDLR-ARE motifs is likely a mechanism for regulating LDLR expression by compounds such as BBR and perhaps others

Blockage of oncostatin M-induced LDL receptor gene transcription by a dominant-negative mutant of C/EBPβ

OM (oncostatin M) activates the human LDLR [LDL (low-density lipoprotein) receptor] gene transcription in HepG2 cells through the SIRE (sterol-independent regulatory element) of LDLR promoter. The SIRE sequence consists of a C/EBP (CCAAT/enhancer-binding protein)-binding site and a CRE (cAMP-response element). Our previous studies [Zhang, Ahlborn, Li, Kraemer and Liu (2002) J. Lipid Res. 43, 1477–1485; Zhang, Lin, Abidi, Thiel and Liu (2003) J. Biol. Chem. 278, 44246–44254] have demonstrated that OM transiently induces EGR-1 (early growth response gene product 1) expression and EGR-1 activates LDLR transcription primarily through a protein–protein interaction with C/EBPβ, which serves as a co-activator of EGR-1. In the present study, we examined the direct role of C/EBPβ as a transactivator in OM-regulated LDLR gene transcription independent of EGR-1. We show that OM induces C/EBPβ expression with kinetics slower than EGR-1 induction. A significant increase in C/EBPβ protein level is detected by 2 h of OM treatment and remains elevated for 24 h. Chromatin immunoprecipitation assays demonstrate that the amount of C/EBPβ bound to the LDLR SIRE sequence is increased 2.8-fold of control by 2 h of OM treatment, reached the highest level of 8-fold by 4 h, and slowly declined thereafter. To further examine the requirement of C/EBPβ in OM-stimulated LDLR expression, we developed a His-tagged dominant-negative mutant of C/EBPβ (His–C/EBPβ-P4; where P4 is plasmid 4 in our mutation series), consisting of the DNA-binding and leucine zipper domains of C/EBPβ (amino acids 246–345). Expression of His–C/EBPβ-P4 in HepG2 cells significantly diminishes the OM-induced increase in LDLR promoter activity and the elevation of endogenous LDLR mRNA expression. Taken together, these new findings identify C/EBPβ as an OM-induced transactivator in LDLR gene transcription and provide a better understanding of the molecular mechanism underlying the sterol-independent regulation of LDLR expression