Quorum Sensing Inhibitory Potential and Molecular Docking Studies of Phyllanthus emblica Phytochemicals Against Pseudomonas aeruginosa.

Phyllanthus emblica is a traditional medicinal plant that is endowed with curative properties including anti-bacterial, anti-fungal, anti-viral, and analgesic properties. Bacteria make use of cell-cell signaling system known as quorum sensing (QS) and respond to their own population. In most gram-negative bacteria, the transcriptional regulators belonging to the Lux R protein play a crucial role in the QS mechanism by detecting the presence of signaling molecules known as N-acyl homoserine lactones (AHLs). In this present work, the anti-quorum sensing activity of Phyllanthus emblica was evaluated against Pseudomonas aeruginosa. Anti-quorum sensing efficacy of Phyllanthus emblica was estimated with reference to QS bio-monitoring strain Chromobacterium violaceum. The binding efficacy of the phytochemicals of Phyllanthus emblica against CviR protein from Chromobacterium violaceum and LasR protein from Phyllanthus emblica were studied.

Identification, characterization and expression analysis of the VQ motif-containing gene family in tea plant (Camellia sinensis).

BACKGROUND: VQ motif-containing (VQ) proteins are plant-specific proteins that interact with WRKY transcription factors and play important roles in plant growth, development and stress response. To date, VQ gene families have been identified and characterized in many plant species, including Arabidopsis, rice and grapevine. However, the VQ gene family in tea plant has not been reported, and the biological functions of this family remain unknown. RESULTS: In total, 25 CsVQ genes were identified based on the genome and transcriptome of tea plant, and a comprehensive bioinformatics analysis was performed. The CsVQ proteins all contained the typical conserved motif FxxhVQxhTG, and most proteins were localized in the nucleus. The phylogenetic analysis showed that the VQ proteins were classified into 5 groups (I, III-VI); the evolution of the CsVQ proteins is consistent with the evolutionary process of plants, and close proteins shared similar structures and functions. In addition, the expression analysis revealed that the CsVQ genes play important roles in the process of tea plant growth, development and response to salt and drought stress. Furthermore, a potential regulatory network including the interactions of CsVQ proteins with CsWRKY transcription factors and the regulation of upstream microRNA that is closely related to the above-mentioned processes is proposed. CONCLUSIONS: The results of this study increase our understanding and characterization of CsVQ genes and their encoded proteins in tea plant. This systematic analysis provided comprehensive information for further studies investigating the biological functions of CsVQ proteins in various developmental processes of tea plants.

Scaffold of Selenium Nanovectors and Honey Phytochemicals for Inhibition of Pseudomonas aeruginosa Quorum Sensing and Biofilm Formation.

Honey is an excellent source of polyphenolic compounds that are effective in attenuating quorum sensing (QS), a chemical process of cell-to-cell communication system used by the opportunistic pathogen Pseudomonas aeruginosa to regulate virulence and biofilm formation. However, lower water solubility and inadequate bioavailability remains major concerns of these therapeutic polyphenols. Its therapeutic index can be improved by using nano-carrier systems to target QS signaling potently. In the present study, we fabricated a unique drug delivery system comprising selenium nanoparticles (SeNPs; non-viral vectors) and polyphenols of honey (HP) for enhancement of anti-QS activity of HP against P. aeruginosa PAO1. The developed selenium nano-scaffold showed superior anti-QS activity, anti-biofilm efficacy, and anti-virulence potential in both in-vitro and in-vivo over its individual components, SeNPs and HP. LasR is inhibited by selenium nano-scaffold in-vitro. Using computational molecular docking studies, we have also demonstrated that the anti-virulence activity of selenium nano-scaffold is reliant on molecular binding that occurs between HP and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Our preliminary investigations with selenium-based nano-carriers hold significant promise to improve anti-virulence effectiveness of phytochemicals by enhancing effective intracellular delivery.

Structure based virtual screening for identification of potential quorum sensing inhibitors against LasR master regulator in Pseudomonas aeruginosa.

Inter and intracellular communication in bacteria, which is known as quorum sensing (QS), is mediated by small diffusible signaling molecules known as autoinducers. QS regulates various virulence factors responsible for pathogenesis. Increasing resistance of microorganisms against traditional antibiotics has turned the focus towards the QS as it exerts less selective pressure preventing development of resistance among microorganisms. LasR, a transcription factor that controls QS in Pseudomonas aeruginosa, is an attractive therapeutic target for inhibitors. This study aimed to screen natural compounds as potential inhibitors of LasR. About 2603 compounds from ZINC database were virtually screened against the structure of LasR. Then after qualifying compounds were filtered on the parameters of Lipinski's rule and ADME. Six novel potential QS inhibiting compounds were selected on the basis of binding energy. Structures of LasR-ligand complexes were analysed to have insight of binding between inhibitors and target. It is pertinent to mention here that all the molecules are structurally different from 3-oxo-C12HSL,a native autoinducer of LasR, that play key role in formation of LasR dimer which is an active form of the protein to facilitate QS.

Cranberry-derived proanthocyanidins impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa.

Bacteria have evolved multiple strategies for causing infections that include producing virulence factors, undertaking motility, developing biofilms, and invading host cells. N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) tightly regulates the expression of multiple virulence factors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. Thus, inhibiting QS could lead to health benefits. In this study, we demonstrate an anti-virulence activity of a cranberry extract rich in proanthocyanidins (cerPAC) against P. aeruginosa in the model host Drosophila melanogaster and show this is mediated by QS interference. cerPAC reduced the production of QS-regulated virulence determinants and protected D. melanogaster from fatal infection by P. aeruginosa PA14. Quantification of AHL production using liquid chromatography-mass spectrometry confirmed that cerPAC effectively reduced the level of AHLs produced by the bacteria. Furthermore, monitoring QS signaling gene expression revealed that AHL synthases LasI/RhlI and QS transcriptional regulators LasR/RhlR genes were inhibited and antagonized, respectively, by cerPAC. Molecular docking studies suggest that cranberry-derived proanthocyanidin binds to QS transcriptional regulators, mainly interacting with their ligand binding sites. These findings provide insights into the underlying mechanisms of action of a cerPAC to restrict the virulence of P. aeruginosa and can have implications in the development of alternative approaches to control infections.

Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment.

Diabetes mellitus is characterized by disrupted glucose homeostasis due to loss or dysfunction of insulin-producing beta cells. In this work, we characterize pancreatic islet development and function in zebrafish mutant for pdx1, a gene which in humans is linked to genetic forms of diabetes and is associated with increased susceptibility to Type 2 diabetes. Pdx1 mutant zebrafish have the key diabetic features of reduced beta cells, decreased insulin and elevated glucose. The hyperglycemia responds to pharmacologic anti-diabetic treatment and, as often seen in mammalian diabetes models, beta cells of pdx1 mutants show sensitivity to nutrient overload. This unique genetic model of diabetes provides a new tool for elucidating the mechanisms behind hyperglycemic pathologies and will allow the testing of novel therapeutic interventions in a model organism that is amenable to high-throughput approaches.

Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element.

ERα has a ligand-dependent transactivation function in the ligand binding domain of ERα C terminus (AF-2) and a ligand-independent activation function in the N terminus (AF-1). It is still not fully understood how AF-1 and AF-2 activities are regulated cooperatively by ligands. To evaluate the AF-1 involvement in the estrogenic activities of various compounds, we analyzed these transactivation functions using AF-1-truncated and AF-2-mutated ERα mutants. AF-2 is composed of two domains with flexible and static regions. We used an AF-2 flexible region mutant and an AF-2 static region mutant. Both mutants have been reported as non-E2 responsive due to disruption of E2-mediated coactivator recruitment to the AF-2. The AF-2 mutants were not activated by agonists, but surprisingly antagonists and selective estrogen receptor modulators (SERMs) activated the AF-2 mutants. This antagonist reversal activity was derived from AF-1. Furthermore, we demonstrated that the AF-2 contains an AF-1 suppression function using C-terminal-truncated ERα mutants. From these findings we hypothesized that the mutation of AF-2 disrupted its ability to suppress AF-1, causing the antagonist reversal. To assess the AF-2-mediated AF-1 suppression, we analyzed the transcription activity of physically separated AF-1 and AF-2 using a novel hybrid reporter assay. We observed that the AF-1 activity was not suppressed by the physically separated AF-2. Furthermore, SERMs did not induce the AF-1-mediated activity from the separated mutant AF-2, which differed from the intact protein. These results imply that SERM activity is dependent on a conformational change of the full-length ERα molecule, which allows for AF-1 activation.

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

Isolation and functional characterization of the ShCBF1 gene encoding a CRT/DRE-binding factor from the wild tomato species Solanum habrochaites.

Plant growth and productivity are greatly affected by low ambient temperature. Complex cascades of gene expression in cold stress response are regulated by transcription factors. In this study, a cDNA clone, named ShCBF1, was isolated from Solanum habrochaites seedlings (a wild relative of cultivated tomato). It was classified as one of CBF family members based on multiple sequence alignment. The expression analysis confirmed that ShCBF1 was induced by low temperature, high salinity and drought stress. Experiments of subcellular localization in tobacco leaf cells indicated that it was localized in nucleus. Transient expression assay using onion epidermal cells revealed that the ShCBF1 protein could function similarly to AtCBF1 in activating the expression of reporter genes with a CRT/DRE element in their promoter. Moreover, ectopic overexpression of ShCBF1 in Arabidopsis enhanced freezing and high salinity tolerance of transgenic plants by improving the expression levels of some stress-responsive marker genes. Taken together, our results suggest that ShCBF1 behaves as a typical plant CBF transcription factor and might be involved in plant response to various environmental stresses.

Quorum sensing inhibitors: an overview.

Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.

Characterization of nuclear localization signals in the type III effectors HsvG and HsvB of the gall-forming bacterium Pantoea agglomerans.

HsvG and HsvB, two paralogous type III effectors of the gall-forming bacteria Pantoea agglomerans pv. gypsophilae and P. agglomerans pv. betae, determine host specificity on gypsophila and beet, respectively. They were previously shown to be DNA-binding proteins imported into host and non-host nuclei and might act as transcriptional activators. Sequence analysis of these effectors did not detect canonical nuclear localization signals (NLSs), but two basic amino acid clusters designated putative NLS1 and NLS2 were detected in their N-terminal and C-terminal regions, respectively. pNIA assay for nuclear import in yeast and bombardment of melon leaves with each of the NLSs fused to a 2xYFP reporter indicated that putative NLS1 and NLS2 were functional in transport of HsvG into the nucleus. A yeast two-hybrid assay showed that HsvB, HsvG, putative NLS1, putative NLS2, HsvG converted into HsvB, or HsvB converted into HsvG by exchanging the repeat domain, all interacted with AtKAP-α and importin-α3 of Arabidopsis thaliana. Deletion analysis of the NLS domains in HsvG suggested that putative NLS1 or NLS2 were required for pathogenicity on gypsophila cuttings and presumably for import of HsvG into the nucleus. This study demonstrates the presence of two functional NLSs in the type III effectors HsvG and HsvB.

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.

The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

Modulation of transcription factor function by O-GlcNAc modification.

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytoplasmic proteins is important for many cellular processes, and the number of proteins that contain this modification is steadily increasing. This modification is dynamic and reversible, and in some cases competes for phosphorylation of the same residues. O-GlcNAc modification of proteins is regulated by cell cycle, nutrient metabolism, and other extracellular signals. Compared to protein phosphorylation, which is mediated by a large number of kinases, O-GlcNAc modification is catalyzed only by one enzyme called O-linked N-acetylglucosaminyl transferase or OGT. Removal of O-GlcNAc from proteins is catalyzed by the enzyme beta-N-acetylglucosaminidase (O-GlcNAcase or OGA). Altered O-linked GlcNAc modification levels contribute to the establishment of many diseases, such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Many transcription factors have been shown to be modified by O-linked GlcNAc modification, which can influence their transcriptional activity, DNA binding, localization, stability, and interaction with other co-factors. This review focuses on modulation of transcription factor function by O-linked GlcNAc modification.

Calcineurin-responsive zinc finger transcription factor CRZ1 of Botrytis cinerea is required for growth, development, and full virulence on bean plants.

Recently, we showed that the alpha subunit BCG1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin in the gray mold fungus Botrytis cinerea. To identify the transcription factor acting downstream of BCG1 and calcineurin, we cloned the gene encoding the B. cinerea homologue of CRZ1 ("CRaZy," calcineurin-responsive zinc finger transcription factor), the mediator of calcineurin function in yeast. BcCRZ1 is able to partially complement the corresponding Saccharomyces cerevisiae mutant, and the subcellular localization of the green fluorescent protein-BcCRZ1 fusion product in yeast cells depends on the calcium level and calcineurin activity. Bccrz1 deletion mutants are not able to grow on minimal media and grow slowly on media containing plant extracts. Hyphal morphology, conidiation, and sclerotium formation are impaired. The cell wall and membrane integrity, stress response (extreme pH, H(2)O(2), Ca(2+), Li(+)), and ability of the hyphae to penetrate the intact plant surface are affected in the mutants. However, BcCRZ1 is almost dispensable for the conidium-derived infection of bean plants. The addition of Mg(2+) restores the growth rate, conidiation, and penetration and improves the cell wall integrity but has no impact on sclerotium formation or hypersensitivity to Ca(2+) and H(2)O(2). The expression of a set of recently identified BCG1- and calcineurin-dependent genes is also affected in DeltaBccrz1 mutants, confirming that this transcription factor acts downstream of calcineurin in B. cinerea. Since the Bccrz1 mutants still respond to calcineurin inhibitors, we conclude that BcCRZ1 is not the only target of calcineurin.

Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of the ERD1 gene in Arabidopsis.

The ZFHD recognition sequence (ZFHDRS) and NAC recognition sequence (NACRS) play an important role in the dehydration-inducible expression of the Arabidopsisthaliana EARLY RESPONSIVETO DEHYDRATION STRESS 1 (ERD1) gene. Using the yeast one-hybrid system, we isolated a cDNA encoding the ZFHD1 transcriptional activator that specifically binds to the 62 bp promoter region of ERD1, which contains the ZFHDRS. Both in vitro and in vivo analyses confirmed specific binding of the ZFHD1 to ZFHDRS, and the expression of ZFHD1 was induced by drought, high salinity and abscisic acid. The DNA-binding and activation domains of ZFHD1 were localized on the C-terminal homeodomain and N-terminal zinc finger domain, respectively. Microarray analysis of transgenic plants over-expressing ZFHD1 revealed that several stress-inducible genes were upregulated in the transgenic plants. Transgenic plants exhibited a smaller morphological phenotype and had a significant improvement of drought stress tolerance. Using the yeast two-hybrid system, we detected an interaction between ZFHD1 and NACRS-binding NAC proteins. Moreover, co-over-expression of the ZFHD1 and NAC genes restored the morphological phenotype of the transgenic plants to a near wild-type state and enhanced expression of ERD1 in both Arabidopsis T87 protoplasts and transgenic Arabidopsis plants.

The zinc finger-only protein Zfp260 is a novel cardiac regulator and a nuclear effector of alpha1-adrenergic signaling.

alpha1-Adrenergic receptors mediate several biological effects of catecholamines, including the regulation of myocyte growth and contractility and transcriptional regulation of the atrial natriuretic factor (ANF) gene whose promoter contains an alpha1-adrenergic response element. The nuclear pathways and effectors that link receptor activation to genetic changes remain poorly understood. Here, we describe the isolation by the yeast one-hybrid system of a cardiac cDNA encoding a novel nuclear zinc finger protein, Zfp260, belonging to the Krüppel family of transcriptional regulators. Zfp260 is highly expressed in the embryonic heart but is downregulated during postnatal development. Functional studies indicate that Zfp260 is a transcriptional activator of ANF and a cofactor for GATA-4, a key cardiac regulator. Knockdown of Zfp260 in cardiac cells decreases endogenous ANF gene expression and abrogates its response to alpha1-adrenergic stimulation. Interestingly, Zfp260 transcripts are induced by alpha1-adrenergic agonists and are elevated in genetic models of hypertension and cardiac hypertrophy. The data identify Zfp260 as a novel transcriptional regulator in normal and pathological heart development and a nuclear effector of alpha1-adrenergic signaling.

Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius.

The transmembrane receptor Patched (Ptc) mediates the action of the diffusing factor Sonic hedgehog (Shh), which is implicated in establishing morphogenetic gradients during embryonic development. Whereas alteration of Ptc function is associated with developmental abnormalities and brain tumors, its functional activity and roles in the adult brain have yet to be elucidated. Here we describe the complementary pattern of Shh and Ptc expression in the rat dorsal vagal motor nucleus and the ventrolateral nucleus tractus solitarius (vNTS), respectively. Those two interconnected structures regulate the cardiorespiratory function during hypoxia. Bath application of a subnanomolar concentration of aminoterminal Shh protein (ShhN) to a slice preparation of the vNTS induces a rapid decrease in neuronal firing followed by a bursting activity that propagates in the neuronal network. Intracellular current injections show that bursts result from an action on the neuronal membrane electro-responsiveness. Both inhibiting and bursting effects are blocked by the monoclonal Shh antibody 5E1 and may require the Ptc binding site of ShhN. Thus, ShhN acting on specific neuronal sites controls electrophysiological properties of differentiated neurons of the vNTS. We speculate on a retrocontrol of cardiorespiratory signals in the vNTS, by Shh generated in dorsal vagal motoneurons.

Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo.

NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where multiple antioxidant/detoxification pathways are up-regulated in unison. Although small molecule inducers of Nrf2 activity have been reported to protect neurons in vitro, whether similar pathways can be accessed in vivo is not known. We have investigated whether in vivo toxicity of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2 activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP hypersensitivity that became apparent with time and increasing dose, causing motor deficits and striatal lesions on a more rapid time scale than identically treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase activity, the target of 3-NP, was inhibited to the same extent in all genotypes by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 overexpression significantly reduced lesion size compared with green fluorescent protein overexpressing controls. In cultured astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant response element-dependent gene expression providing a potential mechanism for the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We conclude that Nrf2 may underlie a feedback system limiting oxidative load during chronic metabolic stress.

Transcriptional Co-activators CREB-binding protein/p300 increase chondrocyte Cd-rap gene expression by multiple mechanisms including sequestration of the repressor CCAAT/enhancer-binding protein.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a small secreted matrix protein expressed in developing and adult cartilage and by chondrocytes in culture. We have previously shown that the expression of Cd-rap, like many other cartilage matrix proteins, is repressed by interleukin 1beta and that the transcription factor CCAAT/enhancer-binding protein (C/EBP) beta plays an important role in the interleukin 1beta-induced repression (Okazaki, K., Li, J., Yu, H., Fukui, N., and Sandell, L. J. (2002) J. Biol. Chem. 277, 31526-31533). The co-activators CREB-binding protein (CBP) and p300 are transcriptional co-regulators that participate in the activities of many different transcription factors including C/EBP. Here we show that CBP/p300 can reverse the inhibitory effect of C/EBP and moreover can stimulate expression of Cd-rap. The mechanism of this effect is shown to involve a unique synergy whereby CBP/p300 stimulate Cd-rap gene expression by at least two mechanisms. First, binding of CBP/p300 to C/EBPbeta leads to sequestration of C/EBP eliminating DNA binding and subsequent repression; second, binding of CBP/p300 to the transcriptional activator Sox9 increases Sox9 DNA binding to the Cd-rap promoter leading to further stimulation of gene transcription. This is an example of a complementary transcriptional network whereby two very different mechanisms act together to confer a functional increase in transcription. This new paradigm is likely generally applicable to cartilage genes as Col2a1 cartilage collagen gene responds similarly.