Efficient In Vitro and In Vivo Anti-Inflammatory Activity of a Diamine-PEGylated Oleanolic Acid Derivative

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti-inflammatory effects of a diamine-PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub-cytotoxic concentrations for anti-inflammatory assays of OADP in RAW 264.7 cells. The inflammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 µg/mL of OADP. Cell-cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS-stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF-α, IL-1β, iNOS, and COX-2; and the blocking of p-IκBα production in LPS-stimulated RAW 264.7 cells. Finally, we have analyzed the anti-inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti-inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes.

Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

Ectodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase Cδ (PKCδ). Downregulation of PKCδ inhibits neuregulin release and reducesex vivoneurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate.

PKCα and PKCδ Activation Regulates Transcriptional Activity and Degradation of Progesterone Receptor in Human Astrocytoma Cells

Progesterone regulates cancer cell proliferation and invasion through its receptors (PR-A and PR-B), whose phosphorylation modifies their transcriptional activity and induce their degradation. We identified by in silico analysis a putative residue (Ser400) in PR that might be phosphorylated by protein kinase C (PKC), a family of enzymes involved in the proliferation and infiltration of astrocytomas, the most frequent and aggressive brain tumors. A grade III human astrocytoma-derived cell line was used to study the role of PKC in PR phosphorylation, transcriptional activity, and degradation. Treatment with PKC activator [tetradecanoyl phorbol acetate (TPA)] increased PR phosphorylation in Ser400 after 5 minutes, which in turn induced PR transcriptional activity and its subsequent degradation by the 26S proteasome 3–5 hours after treatment. Silencing or inhibition of PKCα and PKCδ blocked PR phosphorylation and degradation induced by TPA. Both PR isoforms were associated with PKCα and reached the maximum association after 5 minutes of TPA addition. These data correlated with immunnofluorescence assays in which nuclear colocalization of PKCα with PR increased after TPA treatment. We observed a 2-fold increase in cell proliferation after PKC activation with TPA that was reduced with the PR antagonist, RU486. The PR S400A mutant revealed that this residue is essential for PKC-mediated PR phosphorylation and degradation. Our results show a key participation of PKCα and PKCδ in PR regulation and function.

Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3′-untranslated region (3′-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3′-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3′-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

Muscle Regeneration and Myogenic Differentiation Defects in Mice Lacking TIS7

ABSTRACT The tetradecanoyl phorbol acetate-induced sequence 7 gene (tis7) is regulated during cell fate processes and functions as a transcriptional coregulator. Here, we describe the generation and analysis of mice lacking the tis7 gene. Surprisingly, TIS7 knockout mice show no gross histological abnormalities and are fertile. Disruption of the tis7 gene by homologous recombination delayed muscle regeneration and altered the isometric contractile properties of skeletal muscles after muscle crush damage in TIS7−/− mice. Cultured primary myogenic satellite cells (MSCs) from TIS7−/− mice displayed marked reductions in differentiation potential and fusion index in a strictly cell-autonomous fashion. Loss of TIS7 caused the down-regulation of muscle-specific genes, such as those for MyoD, myogenin, and laminin-α2. Fusion potential in TIS7−/− MSCs could be rescued by TIS7 expression or laminin supplementation. Therefore, TIS7 is not essential for mouse development but plays a novel regulatory role during adult muscle regeneration.

Early Activation of the Kaposi's Sarcoma-Associated Herpesvirus RTA, RAP, and MTA Promoters by the Tetradecanoyl Phorbol Acetate-Induced AP1 Pathway

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma (PEL) cells, but treatment with tetradecanoyl phorbol acetate (TPA) can trigger the full lytic-cycle replication in some of these cells. During lytic-cycle replication, the KSHV-encoded replication and transcription activator (RTA or ORF50), the mRNA transport and accumulation protein (MTA), and the replication-associated protein (RAP) all play crucial roles in expression of downstream viral genes as well as in mediation of viral DNA replication. The cellular CCAAT/enhancer-binding protein alpha (C/EBPα) is induced in TPA-treated PEL cells and contributes to transactivation of the promoters for all of these genes through both direct binding and cooperative interactions with RTA and RAP targeted to upstream C/EBP sites. However, little is known about how RTA expression is triggered initially at the earliest stages after TPA induction when the C/EBPα levels are still limited. Treatment with TPA proved to significantly induce both AP1 DNA-binding activity and levels of activated phosphorylated cJUN in PEL cells and ectopic expression of cJUN-plus-cFOS-induced RTA protein expression in PEL cells. Cotransfected cJUN plus cFOS or TPA treatment transactivated the KSHV RTA, RAP, and MTA promoters in an AP1-binding site-dependent manner in all three promoters. Chromatin immunoprecipitation assays confirmed that cJUN associates with these KSHV target promoters in PEL cells as early as 4 h after TPA treatment. Furthermore, the KSHV RTA and RAP proteins both interact with cJUN or both cJUN and cFOS in vitro or by coimmunoprecipitation from induced PEL cells and enhance cJUN-plus-cFOS-mediated transactivation of these viral promoters. Both increased phosphorylated cJUN and AP1 DNA-binding activity was detected as early as 1 h after TPA treatment in PEL cells, suggesting that AP1 activity may be crucial for very early activation of the RAP, MTA, and RTA promoters during the KSHV lytic cycle. Finally, expression of RTA alone increased cJUN protein levels severalfold in DG75 cells but did not induce cJUN phosphorylation. Therefore, we suggest that the initiating effects of TPA via the AP1 pathway in PEL cells need to be amplified by RTA for full lytic-cycle induction.

Suppression of Tetradecanoyl Phorbol Acetate-Induced Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus by K1 Signal Transduction

ABSTRACT The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-κB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.

MSK1 and MSK2 Are Required for the Mitogen- and Stress-Induced Phosphorylation of CREB and ATF1 in Fibroblasts

ABSTRACT Using mouse knockouts for mitogen- and stress-activated protein kinase 1 (MSK1) and MSK2 and a double knockout of both MSK1 and MSK2, we show that these protein kinases are required for the stress-induced phosphorylation of transcription factors CREB and ATF1 in primary embryonic fibroblasts. In contrast mitogen-induced phosphorylation of CREB and ATF1 is greatly reduced but not totally abolished. The mitogen- and stress-induced phosphorylation of CREB at Ser133 has been linked to the transcription of several immediate early genes, including c-fos, junB, and egr1. The knockout of both MSK1 and MSK2 resulted in a 50% reduction in c-fos and junB gene transcription in response to anisomycin or UV-C radiation but only a small reduction in response to tetradecanoyl phorbol acetate or epidermal growth factor in fibroblasts. The transcription of egr1 in response to both mitogenic and stress stimuli, as well as stress-induced apoptosis, was unaffected in the MSK1/MSK2 double knockout.