[Risk factors for surgical site infection after emergency abdominal surgery: a multicenter cross-sectional study in China].

Objective: Surgical site infection (SSI) is the most common infectious complication after emergency abdominal surgery (EAS). To a large extent, most SSI can be prevented, but there are few relevant studies in China. This study mainly investigated the current situation of SSI occurrence after EAS in China, and further explored risk factors for SSI occurrence. Methods: Multi-center cross-sectional study was conducted. Clinical data of patients undergoing EAS in 33 hospitals across China between May 1, 2019 and June 7, 2019 were prospectively collected, including perioperative data and microbial culture results from infected incisions. The primary outcome was the incidence of SSI after EAS, while the secondary outcomes were postoperative hospital stay, ICU occupancy rate, length of ICU stay, hospitalization cost, and mortality within postoperative 30 days. Univariate and multivariate logistic regression models were used to analyze the risk factors of SSI after EAS. Results: A total of 660 EAS patients aged (47.9±18.3) years were enrolled in this study, including 56.5% of males (373/660). Forty-nine (7.4%) patients developed postoperative SSI. The main pathogen of SSI was Escherichia coli [culture positive rate was 32.7% (16/49)]. As compared to patients without SSI, those with SSI were more likely to be older (median 56 years vs. 46 years, U=19 973.5, P<0.001), male [71.4% (35/49) vs. 56.1% (343/611), χ(2)=4.334, P=0.037] and diabetes [14.3% (7/49) vs. 5.1% (31/611), χ(2)=5.498, P=0.015]; with-lower preoperative hemoglobin (median: 122.0 g/L vs. 143.5 g/L, U=11 471.5, P=0.006) and albumin (median: 35.5 g/L vs. 40.8 g/L, U=9452.0, P<0.001), with higher blood glucose (median: 6.9 mmol/L vs. 6.0 mmol/L, U=17 754.5, P<0.001); with intestinal obstruction [32.7% (16/49) vs. 9.2% (56/611), χ(2)=25.749, P<0.001], with ASA score 3-4 [42.9% (21/49) vs. 13.9% (85/611), χ(2)=25.563, P<0.001] and with high surgical risk [49.0% (24/49) vs. 7.0% (43/611), χ(2)=105.301, P<0.001]. The main operative procedure resulting in SSI was laparotomy [81.6%(40/49) vs. 35.7%(218/611), χ(2)=40.232, P<0.001]. Patients with SSI experienced significantly longer operation time (median: 150 minutes vs. 75 minutes, U=25 183.5, P<0.001). In terms of clinical outcome, higher ICU occupancy rate [51.0% (25/49) vs. 19.5% (119/611), χ(2)=26.461, P<0.001], more hospitalization costs (median: 44 000 yuan vs. 15 000 yuan, U=24 660.0, P<0.001), longer postoperative hospital stay (median: 10 days vs. 5 days, U=23 100.0, P<0.001) and longer ICU occupancy time (median: 0 days vs. 0 days, U=19 541.5, P<0.001) were found in the SSI group. Multivariate logistic regression analysis showed that the elderly (OR=3.253, 95% CI: 1.178-8.985, P=0.023), colorectal surgery (OR=9.156, 95% CI: 3.655-22.937, P<0.001) and longer operation time (OR=15.912, 95% CI:6.858-36.916, P<0.001) were independent risk factors of SSI, while the laparoscopic surgery (OR=0.288, 95% CI: 0.119-0.694, P=0.006) was an independent protective factor for SSI. Conclusions: For patients undergoing EAS, attention should be paid to middle-aged and elderly patients and those of colorectal surgery. Laparoscopic surgery should be adopted when feasible and the operation time should be minimized, so as to reduce the incidence of SSI and to reduce the burden on patients and medical institutions.

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7.

The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.

Patterning small-molecule biocapture surfaces: microcontact insertion printing vs. photolithography.

Chemical patterns prepared by self-assembly, combined with soft lithography or photolithography, are directly compared. Pattern fidelity can be controlled in both cases but patterning at the low densities necessary for small-molecule probe capture of large biomolecule targets is better accomplished using microcontact insertion printing (µCIP). Surfaces patterned by µCIP are used to capture biomolecule binding partners for the small molecules dopamine and biotin.

Postural responses during falling with rapid reach-and-grasp balance reaction in patients with motor complete paraplegia.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: To investigate the kinematic, kinetic and electromyographic (EMG) aspects of postural control during falling with rapid reach-and-grasp balance reaction in thoracic cord-injured individuals wearing knee-ankle-foot orthoses (KAFOs). SETTING: Institutional Motion Analysis Laboratory. METHODS: Seven T7-T12 cord-injured subjects with complete motor loss (ASIA classes A and B) participated in this study. Subjects with KAFOs first stood steady with a modified walker and then released their hold on the walker to maintain self-supported standing until falling with grasping. The center of pressure (COP), center of mass (COM) and joint angles were measured together with EMG of the triceps (TRI), T4 paraspinal and abdominal muscles. RESULTS: After release of the walker, there was a rapid increase of COM-COP distance (that is, from 13.32+/-11.79 to 54.29+/-24.56 mm), with COM in front of COP during a forward fall, which was associated with the increases of T4 muscle activities. After the reach-and-grasp reaction, COM moved behind COP, which was associated with the increase of ankle dorsiflexion and the TRI and abdominal muscle activities. CONCLUSION: The increase of upper back extensor muscle activity might not be enough to correct postural instability during unsupported stance in thoracic spinal cord injury with complete motor loss. The rapid reach-and-grasp reaction is an alternative compensatory mechanism to prevent falling to the ground.

E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer.

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.

Activation of the human PAX6 gene through the exon 1 enhancer by transcription factors SEF and Sp1.

PAX6 is a transcription factor that plays a major role in ocular morphogenesis. PAX6 is expressed in the eye, central nervous system and pancreas. Two alternative promoters, P0 and P1, which are differentially regulated during development, drive PAX6 transcription. We identified a 57 bp cis-regulatory element in exon 1 of the human PAX6 gene exon 1 enhancer (EIE). EIE enhances P1-driven PAX6 expression. Three regions in E1E (E1E-1, E1E-2 and E1E-3) have sequence similarities with binding sites of transcription factors ARP-1, Isl-1 and SEF, respectively. As shown by electrophoretic mobility shift assays, E1E-3, but not E1E-1 or E1E-2, bound to proteins in nuclear extracts of human glioma cells and transcription factor SEF bound to E1E-3. As shown by transient transfection experiments, deletion or site-specific mutations in E1E-3 dramatically decreased P1 promoter activity. Mutations in E1E-2, however, did not affect function of the P1 promoter. Co-transfection of SEF and PAX6 promoter-reporter constructs showed that SEF up-regulates PAX6 gene expression through the P1 promoter. Two Sp1 sites in the E1E region were also shown to be important by transient co-transfection assays. Data from immunoprecipitation and transient transfection assays demonstrated that SEF and Sp1 interacted in vitro and may act together in vivo to regulate PAX6 expression.

Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI.

We have recently identified a maternally imprinted tumor suppressor gene, ARHI (aplysia ras homolog I), the expression of which is lost in ovarian and breast cancers. We have now characterized the genomic structure of the gene including its promoter and the methylation status of its upstream CpG islands. The ARHI gene spans approximately 8 kb containing two exons and one intron. Exon 1 contains 81 non-translated nucleotides, connected to exon 2 with a 3.2-kb intron. The entire protein-coding region is located within exon 2 and encodes a 229-residue small GTP-binding protein belonging to the Ras superfamily. Genomic structure analysis has identified three potential CpG islands. Two of them (CpG island I and II) are located within the promoter and adjacent exon 1 of the ARHI gene. Aberrant methylation of these CpG islands has been detected in breast cancer cells but not in normal epithelial cells, supporting the possibility that appropriate methylation status of the CpG islands in the promoter region may play a role in the downregulation of ARHI gene expression. A TATA box is found 27 bp upstream of the transcription start site associated with several putative transcription factor binding sites. Transient transfection with nested deletion constructs of the 2-kb ARHI promoter regions fused to a luciferase reporter indicated a 121-bp sequence upstream of the transcription initiation site is required for basal promoter activity. Interestingly, this is the region where lower promoter activity has been observed in cancer cells than in normal cells.

Transcription factor AP-2 functions as a repressor that contributes to the liver-specific expression of serum amyloid A1 gene.

We previously identified transcription factor AP-2 as the nuclear factor that interacts with the tissue-specific repressor element in the rat serum amyloid A1 (SAA1) promoter. In this report, we provide evidence for a second AP-2-binding site and show that both AP-2 sites participate in mediating the transcription repression of SAA1 promoter. This proximal AP-2 site overlaps with the NFkappaB-binding site known to be essential for SAA1 promoter activity. Protein binding competition experiments demonstrated that AP-2 and NFkappaB binding to these overlapping sites were mutually exclusive. Furthermore, the addition of AP-2 easily displaced prebound NFkappaB, whereas NFkappaB could not displace AP-2. These results thus suggest that one mechanism by which AP-2 negatively regulates SAA1 promoter activity may be by antagonizing the function of NFkappaB. Consistent with a repression function, transient expression of AP-2 in HepG2 cells inhibited conditioned medium-induced SAA1 promoter activation. This inhibition was dependent on functional AP-2-binding sites, since mutation of AP-2-binding sites abolished inhibitory effects of AP-2 in HepG2 cells as well as resulted in derepression of the SAA1 promoter in HeLa cells. In addition to SAA1, we found that several other liver gene promoters also contain putative AP-2-binding sites. Some of these sequences could specifically inhibit AP-2.DNA complex formation, and for the human complement C3 promoter, overexpression of AP-2 also could repress its cytokine-mediated activation. Finally, stable expression of AP-2 in hepatoma cells significantly reduced the expression of endogenous SAA, albumin, and alpha-fetoprotein genes. Taken together, our results suggest that AP-2 may function as a transcription repressor to inhibit the expression of not only SAA1 gene but also other liver genes in nonhepatic cells.

Functional analysis of a minimal mouse serum amyloid A3 promoter in transgenic mice.

We report the generation of transgenic mice harboring the SAA3/LacZ transgene and analysis of its expression patterns in vivo following LPS-induced inflammation. Our results show that a 210-bp fragment of the mouse SAA3 promoter when placed in front of the LacZ gene was sufficient to confer basal and inflammation-induced reporter gene expression. Consistent with endogenous SAA3 expression, the basal level of LacZ expression was high in the lung and liver of newborn and 1-week-old transgenic mice. Its expression however decreased with increasing age and at 3-weeks ofage, LacZ expression was very low in the lung and was essentially undetectable in the liver. When SAA3/LacZ transgenic mice were injected with lipopolisaccharide to induce inflammation, beta-gal activities were increased approximately 6- and 16-fold in the lung and liver, respectively. Histological examination of lung and liver tissues stained with X-gal revealed that the LacZ transgene was expressed primarily in the macrophages. Thus, this minimal SAA3 promoter fragment contains the necessary regulatory sequences for its expression and cytokine responsiveness in macrophages albeit is insufficient to confer expression in hepatocytes.

NFkappa B interacts with serum amyloid A3 enhancer factor to synergistically activate mouse serum amyloid A3 gene transcription.

We had previously identified a distal regulatory element (DRE) in the mouse serum amyloid A3 (SAA3) promoter that functions as a cytokine-inducible transcription enhancer. Within this DRE, three functional elements interact with CCAAT/enhancer-binding protein (C/EBP) and SAA3 enhancer factor (SEF) transcription factors. In this study, we show that cotransfection of the SEF expression plasmid with an SAA3/luciferase reporter resulted in 3-5-fold activation of the SAA3 promoter. When SEF-transfected cells were further stimulated with conditioned medium or interleukin-1, SAA3 promoter activity was dramatically increased, suggesting that SEF may cooperate functionally with other interleukin-1-inducible transcription factors to synergistically up-regulate SAA3 gene transcription. Indeed, cotransfection of SEF and NFkappaBp65 expression DNAs resulted in synergistic activation of the SAA3 promoter. Intriguingly, no consensus NFkappaB-binding site was found in the SAA3 promoter region; rather a putative NFkappaB-binding sequence with 3-base pair mismatches was identified in the DRE. When this sequence was used in an electrophoretic mobility shift assay, it interacted with NFkappaBp50, albeit with binding affinities that were several hundredfold lower than that with the consensus NFkappaB probe. Functional cooperation between SEF and NFkappaB was further strengthened by the finding that SEF and NFkappaB formed stable cytokine-inducible protein-protein complexes. Finally, despite its weak binding, mutation of this NFkappaB-binding site nevertheless dramatically reduced both NFkappaBp65- and cytokine-mediated induction of SAA3 promoter. Therefore, the molecular basis for the functional synergy between SEF and NFkappaB may, in part, be the ability of SEF to recruit NFkappaB through physical interactions that lead to enhancement or stabilization of NFkappaB binding to the SAA3 promoter element.

Phosphatidylinositol 3-kinase as a mediator of TNF-induced NF-kappa B activation.

The activation of transcription factor NF-kappa B by TNF involves the stimulation of a novel signaling cascade. In this paper we show that phosphatidylinositol 3-kinase (PI 3-kinase) may play a pivotal role in TNF-mediated activation of NF-kappa B-dependent genes. Consistent with its involvement in TNF signaling, PI 3-kinase activities in HepG2 and U937 cells can be stimulated by TNF in a rapid but transient manner through a mechanism that may involve its association with the insulin receptor substrate-1. A dominant-negative mutant of the p85 regulatory subunit of PI 3-kinase, which is a potent inhibitor of PI 3-kinase signaling, effectively blocked the TNF-induced expression of an NF-kappa B-dependent reporter gene. Although PI 3-kinase may be required for NF-kappa B activation, overexpression of its p110 catalytic subunit alone was unable to induce an NF-kappa B/chloramphenicol acetyltransferase (CAT) reporter gene. However, when TNF was added to p110-overexpressing cells, there was a synergistic activation of the NF-kappa B/CAT reporter, suggesting that other TNF-inducible signals may cooperate with PI 3-kinase to activate NF-kappa B. Consistent with its role in NF-kappa B activation, inhibition of PI 3-kinase activity by wortmannin or LY294002 greatly potentiated TNF-induced apoptosis. This TNF/wortmannin-induced apoptosis was markedly prevented in cells overexpressing Rel A. Taken together, our results indicate that a PI 3-kinase-regulated step in TNF-signaling is critical for the expression of NF-kappa B-dependent genes.

Purification and identification of a tissue-specific repressor involved in serum amyloid A1 gene expression.

We have previously demonstrated that the 5'-flanking regions from the rat serum amyloid A1 (SAA1) promoter are necessary and sufficient to confer specific cytokine-induced expression in cultured hepatoma cells. Deletion analysis identified a tissue-specific repressor (TSR) regulatory element, located between bp -289 and -256, that functioned as a silencer, contributing to the transcription repression on SAA1 promoter in nonliver cells. When this 34-base pair TSR-binding element was used as a probe in electrophoretic mobility shift assays, an intense DNA-protein complex was detected in nuclear extracts from HeLa and several other nonliver tissues. This TSR binding activity, however, was undetectable in extracts from liver or liver-derived cells. The distribution of TSR binding activity is therefore consistent with its regulatory role in repressing SAA1 expression in a tissue-specific manner. In this study, we purified TSR protein from HeLa nuclear extracts and showed that it has a molecular mass of approximately 50 kDa. Surprisingly, protein sequencing and antibody supershift experiments identified TSR as transcription factor AP-2. Subsequent functional analysis showed that forced expression of AP-2 in HepG2 cells could indeed inhibit conditioned medium-induced SAA1 promoter activation. Moreover, expression of a dominant-negative mutant of AP-2 in HeLa cells or mutation of the AP-2-binding site led to derepression of the SAA1 promoter, presumably by neutralizing the inhibitory effects of the endogenous wild-type AP-2. Our results therefore demonstrate a novel function for AP-2 in the transcriptional repression of SAA1 promoter. Together with its tissue distribution, AP-2 may contribute to SAA1's highly liver-specific expression pattern by restricting its expression in nonliver cells.

An upstream repressor element that contributes to hepatocyte-specific expression of the rat serum amyloid A1 gene.

Serum amyloid A (SAA) is a major acute-phase protein whose expression can be dramatically induced in response to tissue damage, infection, and inflammation. Its expression is highly tissue-specific, restricted almost exclusively to liver hepatocytes. We have shown that a 320-bp fragment of the rat SAA1 promoter could confer liver-cell-specific expression on a reporter gene when transfected into cultured cells. Here we report the identification of a 29-bp regulatory element that possesses inhibitory activities on SAA1 promoter in HeLa cells but has no such effects in liver cells. Moreover, this regulatory element has properties of a transcriptional repressor; in that, it can function with a heterologous promoter and is independent of orientation and distance from the transcription initiation site. Protein binding studies showed that this regulatory element can form specific protein-DNA complexes with nuclear proteins from several nonliver cell lines (HeLa, 10T(1/2), and C2) and placenta. However, the same DNA-protein complex was not detected in extracts from liver or liver-derived cell lines (HepG2 and Hep3B). Taken together, our results demonstrate the presence of a DNA-binding protein, termed tissue-specific repressor, found only in nonhepatocytes which may function to repress SAA1 gene expression by interacting with a repressor element. Thus, liver-specific expression of the SAA1 gene is apparently regulated by both positive and negative regulatory elements and their interacting transcription factors to ensure that it is expressed only in suitable cell types.

Purification and characterization of the serum amyloid A3 enhancer factor.

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the alpha(2)-macroglobulin and Aalpha-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes.

Synergistic induction of mouse serum amyloid A3 promoter by the inflammatory mediators IL-1 and IL-6.

Serum amyloid A (SAA), one of the major acute-phase proteins, increases several hundredfold in concentration in plasma following acute inflammation, primarily as a result of a 200-fold increase in its transcription rate. We have previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene could confer conditioned medium-induced expression in cultured cells. The induction is mediated through a 42-bp distal response element (DRE) consisting of three functional regulatory elements. In this study, we show that interleukin-1 (IL-1) is the major cytokine in the conditioned medium responsible for SAA3 induction, and the induction by IL-1 can be effectively blocked by H-7, a protein kinase C inhibitor. Although IL-6 alone had no effect on SAA3 promoter activity, the addition of IL-6 and IL-1 resulted in dramatic synergistic activation of the reporter gene. We further show that the DRE is both necessary and sufficient to confer synergistic induction by IL-1 and IL-6. Moreover, individual mutation of the three regulatory elements within DRE either abolished or drastically reduced the synergistic induction. Our results indicate that synergistic activation of SAA3 promoter by IL-1 and IL-6 is achieved through integration of signals triggered by these two cytokines onto the DRE and that all three functionally distinct regulatory elements in the DRE are required to effectively and fully activate SAA3 gene transcription.

Phosphatidylinositol 3-kinase in interleukin 1 signaling. Physical interaction with the interleukin 1 receptor and requirement in NFkappaB and AP-1 activation.

The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFkappaB and activator protein-1 (AP-1) are poorly defined. We present evidence here that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI 3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibited IL-1-induced activation of both NFkappaB and AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFkappaB-dependent gene expression. In this regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate NFkappaB. Our results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFkappaB and AP-1.

Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression.

A highly conserved CCAAT/enhancer-binding protein (C/EBP)-binding site centered around -134 relative to the transcription start site in the rat beta-casein gene promoter is capable of interacting specifically with recombinant and mammary gland C/EBP proteins. Western blot analysis indicates that C/EBP levels change dramatically throughout mammary gland development. C/EBP alpha expression is barely detectable in mammary glands from virgin and pregnant animals but is expressed at high levels during lactation and at lower levels during involution. The expression of three C/EBP beta isoforms [the liver-enriched activating proteins (LAPs); and the liver-enriched inhibiting protein (LIP)] is elevated throughout pregnancy, with LIP expression increasing more than 100-fold. Thus, during pregnancy, a low LAP/LIP ratio (< 5) is maintained. C/EBP beta expression decreases at parturition, with LIP diminishing to levels observed in the virgin gland. Therefore, during lactation a more than 100-fold increase in the LAP/LIP ratio is observed. Treatment of the HC11 mammary epithelial cell line with hydrocortisone results in a 10- to 20-fold inhibition of LIP expression, with only minor changes in LAP levels. Therefore, glucocorticoids may impinge upon beta-casein gene expression by altering the ratio of the inhibitory to the activating isoforms of C/EBP beta. Several previously defined casein gene promoter regions capable of conferring hormone and extracellular matrix inducibility to reporter genes in mammary cells are suggested to be composite response elements, containing putative binding sites for the same set of hormonally and developmentally regulated factors: C/EBP, MGF/Stat5, and the glucocorticoid receptor.

YY1 represses rat serum amyloid A1 gene transcription and is antagonized by NF-kappa B during acute-phase response.

Serum amyloid A (SAA), one of the major acute-phase proteins, increases several hundredfold in concentration in plasma following acute inflammation, primarily as a result of a 200-fold increase in its transcriptional rate. Functional analysis of the rat SAA1 promoter has identified a 65-bp cytokine response unit (CRU; positions -135 to -71) that could confer cytokine responsiveness on a heterologous promoter. Within this CRU, two cis-regulatory elements, corresponding to NF-kappa B- and C/EBP-binding sites, were found to be functionally important and exerted synergistic effects on induced SAA1 expression. In this report, we show that a third transcription factor interacts with the CRU through a region located between the NF-kappa B- and C/EBP-binding sites. On the basis of its gel mobility shift patterns, ubiquitous binding activity, sequence specificity of DNA binding, zinc-dependent binding activity, and gel mobility supershift by specific antibodies, we concluded that this factor is identical to YY1. Methylation interference studies revealed that YY1 binding sequences overlapped with those of NF-kappa B, and gel mobility studies showed that NF-kappa binding to the CRU was effectively inhibited by YY1. Consistent with its presumed antagonistic role to NF-kappa B, YY1 exerted a negative effect on SAA1 expression, whereas disruption of its binding in the promoter elevated basal and cytokine-induced activities. Furthermore, overexpression of YY1 trans-repressed SAA1 promoter activity. Thus, our results demonstrate that SAA1 expression is tightly regulated by an on-off switch of activators and repressors, presumably to ensure that it is expressed only under appropriate physiological conditions.