Guck T. Ooi

Role of the Suppressor of Cytokine Signaling-3 in Mediating the Inhibitory Effects of Interleukin-1β on the Growth Hormone-dependent Transcription of the Acid-labile Subunit Gene in Liver Cells

During catabolic diseases such as sepsis, inflammation, and infection, a state of growth hormone (GH) resistance develops in liver. This has been attributed in part to increased production of the proinflammatory cytokine interleukin-1β (IL-1β). To determine how IL-1β induces GH resistance, we studied the acid-labile subunit (ALS) gene whose hepatic transcription is increased by GH via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. IL-1β reduced the ability of GH to stimulate ALS mRNA in rat primary hepatocytes and ALS promoter activity in H4-II-E rat hepatoma cells. This inhibition was dependent on ALSGAS1, an element resembling a γ-interferon activated sequence that mediates the transcriptional effects of GH. Inhibition by IL-1β was also associated with a reduction of GH-dependent binding of STAT5 to this element after chronic (8 and 24 h), but not after acute treatment (15 min). Because these results indicated that the inhibition by IL-1β was indirect, expression of the recently discovered suppressors of cytokine action (SOCS) was examined in liver cells. IL-1β did not alter the expression of SOCS1, SOCS2, and CIS, indicating that they are not involved. In contrast, IL-1β increased SOCS3 mRNA by 8-fold after 24 h of treatment, whereas GH had no effect. Forced expression of SOCS3 was just as effective as IL-1β in reducing the GH induction of ALS promoter activity in H4-II-E rat hepatoma cells. Similar results were observed in primary rat hepatocytes. We conclude that the induction of SOCS3 by IL-1β contributes to the development of GH resistance in liver, and represents a mechanism by which cytokines such as IL-1β cross-talk with cytokines using the JAK-STAT pathway.

Inhibin binding sites and proteins in pituitary, gonadal, adrenal and bone cells

Activin signals via complexes of type I (50-55 kDa) and II (70-75 kDa) activin receptors, but the mechanism of inhibin action is unclear. Proposed models range from an anti-activin action at the type II activin receptor to independent actions involving putative inhibin receptors. Two membrane-embedded proteoglycans, betaglycan and p120, have recently been implicated in inhibin binding, but neither appears to be a signalling receptor. The present studies on primary cultures of rat pituitary and adrenal cells, and several murine and human cell lines were undertaken to characterise inhibin binding to its physiological targets. High affinity binding of inhibin to the primary cultures and several of the cell lines, like that previously described for ovine pituitary cells, was saturable and reversible. Scatchard analysis revealed two classes of binding sites (K(d) of 40-400 and 500-5000 pM, respectively). Affinity labelling identified [125I]inhibin binding proteins with apparent molecular weights of 41, 74, 114 and >170 kDa in all cell types that displayed high affinity, high capacity binding of inhibin. Additional labelling of a 124 kDa species was evident in gonadal TM3 and TM4 cell lines. In several cases, activin (> or =20 nM) competed poorly or not at all for binding to these proteins. The 74, 114 and >170 kDa inhibin binding proteins in TM3 and TM4 cells were immunoprecipitated by an anti-betaglycan antiserum. These three proteins correspond in size to the activin receptor type II and the core protein and glycosylated forms of betaglycan, respectively, that have been proposed to mediate anti-activin actions of inhibin, but the identity of the 74 kDa species is yet to be confirmed. Studies of [125I]inhibin binding kinetics and competition for affinity labelling of individual binding proteins in several cell lines suggest these three species and the 41 and 124 kDa proteins form a high affinity inhibin binding complex. In summary, common patterns of inhibin binding and affinity labelling were observed in inhibin target cells. Novel inhibin binding proteins of around 41 and 124 kDa were implicated in the high affinity binding of inhibin to cells from several sources.

Cloning and Characterization of a Functional Promoter of the Rat pp120 Gene, Encoding a Substrate of the Insulin Receptor Tyrosine Kinase

Cloning of the 5′-flanking region of the rat pp120 gene has indicated that it is a housekeeping gene: it lacks a functional TATA box and contains several Sp1 binding sites and multiple transcription initiation sites at nucleotides −101, −71, −41, and −27 spread over a GC-rich area. A fragment between nucleotides −21 and −1609 exhibited promoter activity when ligated in a sense orientation into a promoterless luciferase reporter plasmid and transiently transfected into rat H4-II-E hepatoma cells. 5′ progressive deletion and block substitution analyses revealed that the three proximal Sp1 boxes (boxes 3, 5, and 6) are required for basal transcription of the pp120 gene. Promoter activity was stimulated 2-3-fold in response to insulin, dexamethasone, insulin plus dexamethasone, and cAMP. Although unaltered by phorbol esters alone, promoter activity was stimulated 4-5-fold in response to phorbol esters plus cAMP. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and −2, and liver-specific factors were detected. The role of these sites in tissue-specific expression of pp120 remains to be investigated.

Enzyme assay for 5α-reductase Type 2 activity in the presence of 5α-reductase Type 1 activity in rat testis

Abstract The relative abundance and physiological role of 5α-reductase (5αR) isoforms in rat testis, in particular 5α-reductase Type 2 (5αR2) are poorly understood. Investigation of 5αR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5α-reductase Type 1 (5αR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5αR assays measured the conversion of 3 [H]-testosterone to 5α-reduced metabolites (dihydrotestosterone+3α-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5αR1 activity at pH 5.0, the amount of 5αR1 activity at pH 5.0 was determined by measuring recombinant rat 5αR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5αR1 was determined to be 12.4±1.4% (mean±S.D., n =14). The 5αR2 assay was validated by determining recombinant rat 5αR2 activity in the presence of recombinant rat 5αR1 activity in COS cells. A 99.3±14.7% recovery of 5αR2 activity was obtained when comparing 5αR2 activity recovered versus activity added. 5αR1 and 5αR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50–100-fold) over this age range, with 5αR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5αR2 activity in the presence of high concentrations of 5αR1 was developed and is applicable in the measurement of 5αR2 activity in rat testis.

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, whilst the remaining three (MW 37 K, 34K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands. These data describe, for the first time, the structural nature of the IGF inhibitor protein and raise important questions regarding the relationship of the inhibitor to the primary IGF-binding subunit of the native high MW IGF carrier protein of serum.

Time course proteomic profiling of human myocardial infarction plasma samples: an approach to new biomarker discovery.

BACKGROUND The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. METHODS Ten individuals with recent acute ischemic-type chest pain (<12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0. Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). RESULTS Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p<0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks than those without previous history of AMI (76, p<0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. CONCLUSIONS Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

Post-transcriptional regulation of insulin-like growth factor binding protein-2 mRNA in diabetic rat liver

IGFBP-1 and IGFBP-2 mRNAs are increased in the livers of streptozotocin-diabetic rats. A corresponding increase is observed in transcription of the IGFBP-1 but not the IGFBP-2 gene, indicating that the increase in steady-state levels of IGFBP-2 mRNA is a post-transcriptional effect. IGFBP-1 and IGFBP-2 mRNAs also differ in the rapidity of their response to insulin treatment: hepatic IGFBP-1 mRNA is normalized within 1 h, IGFBP-2 mRNA decreases more slowly. These differences suggest that IGFBP-2 may provide more chronic adaptation to metabolic change than IGFBP-1.

Prostaglandin E and F receptor expression and myometrial sensitivity at labor onset in the sheep

Abstract Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE2 and PGF2α acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1–4) expression. Thus, the relative expression of FP and EP1–4 may determine the responsiveness to PGE2 and PGF2α. The aims of this study were to characterize the expression of EP1–4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE2 and PGF2α. This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.

Early Transcriptional Responses of HepG2-A16 Liver Cells to Infection by Plasmodium falciparum Sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.

Regulation and role of the acid-labile subunit of the 150-kilodalton insulin-like growth factor complex in the mouse

Abstract After birth, the acid-labile subunit (ALS) associates in the circulation with insulin-like growth factor (IGF)-I or -II and with IGF binding protein-3 (IGFBP-3) to form a 150-kilodalton complex. This association leads to the retention of IGFs in the vascular system and promotes their endocrine actions. ALS is synthesized almost exclusively in liver, and both hepatic ALS mRNA and circulating levels are increased by growth hormone (GH). Three major areas of study were pursued to better understand the regulation of ALS synthesis and its role in the circulating IGF system. First, the mouse ALS gene was isolated and shown to be organized into two exons and a single intron on chromosome 17. Second, using transient transfection studies in the rat H4-II-E hepatoma cell line and primary rat hepatocytes, the region of the mouse promoter that is responsive to GH was mapped to a nine-base pair cis-element resembling a γ-interferon-activated sequence. The activation of the mouse ALS gene by GH is mediated by the binding of STAT5 isoforms to this sequence. Finally, an ALS knockout model was created by inactivating the ALS gene in mouse embryonic stem cells. Mice that are homozygous for the mutation grow at a slower rate after birth. This growth depression is associated with large decreases in the plasma concentrations of both IGF-I and IGFBP-3, indicating the critical role of ALS in the regulation of circulating levels of these proteins. Studies of this model will lead to a better understanding of the circulating IGF system.