Dissociation of the AhR/ARNT complex by TGF-ß/Smad signaling represses CYP1A1 gene expression and inhibits benze[a]pyrene-mediated cytotoxicity.

Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-ß (TGF-ß), how TGF-ß signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-ß signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-ß stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-ß signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-ß abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-ß may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.

PDZK1-interacting protein 1 (PDZK1IP1) traps Smad4 protein and suppresses transforming growth factor-ß (TGF-ß) signaling.

Transforming growth factor (TGF)-ß signaling in humans is stringently regulated to prevent excessive TGF-ß signaling. In tumors, TGF-ß signaling can both negatively and positively regulate tumorigenesis dependent on tumor type, but the reason for these opposite effects is unclear. TGF-ß signaling is mainly mediated via the Smad-dependent pathway, and herein we found that PDZK1-interacting protein 1 (PDZK1IP1) interacts with Smad4. PDZK1IP1 inhibited both the TGF-ß and the bone morphogenetic protein (BMP) pathways without affecting receptor-regulated Smad (R-Smad) phosphorylation. Rather than targeting R-Smad phosphorylation, PDZK1IP1 could interfere with TGF-ß- and BMP-induced R-Smad/Smad4 complex formation. Of note, PDZK1IP1 retained Smad4 in the cytoplasm of TGF-ß-stimulated cells. To pinpoint PDZK1IP1's functional domain, we created several PDZK1IP1 variants and found that its middle region, from Phe40 to Ala49, plays a key role in its Smad4-regulating activity. PDZK1IP1 knockdown enhanced the expression of the TGF-ß target genes Smad7 and prostate transmembrane protein androgen-induced (TMEPAI) upon TGF-ß stimulation. In contrast, PDZK1IP1 overexpression suppressed TGF-ß-induced reporter activities, cell migration, and cell growth inhibition. In a xenograft tumor model in which TGF-ß was previously shown to elicit tumor-promoting effects, PDZK1IP1 gain of function decreased tumor size and increased survival rates. Taken together, these findings indicate that PDZK1IP1 interacts with Smad4 and thereby suppresses the TGF-ß signaling pathway.

TMED10 Protein Interferes with Transforming Growth Factor (TGF)-ß Signaling by Disrupting TGF-ß Receptor Complex Formation.

The intensity and duration of TGF-ß signaling determine the cellular biological response. How this is negatively regulated is not well understood. Here, we identified a novel negative regulator of TGF-ß signaling, transmembrane p24-trafficking protein 10 (TMED10). TMED10 disrupts the complex formation between TGF-ß type I (also termed ALK5) and type II receptors (TßRII). Misexpression studies revealed that TMED10 attenuated TGF-ß-mediated signaling. A 20-amino acid-long region from Thr91 to Glu110 within the extracellular region of TMED10 was found to be crucial for TMED10 interaction with both ALK5 and TßRII. Synthetic peptides corresponding to this region inhibit both TGF-ß-induced Smad2 phosphorylation and Smad-dependent transcriptional reporter activity. In a xenograft cancer model, where previously TGF-ß was shown to elicit tumor-promoting effects, gain-of-function and loss-of-function studies for TMED10 revealed a decrease and increase in the tumor size, respectively. Thus, we determined herein that TMED10 expression levels are the key determinant for efficiency of TGF-ß receptor complex formation and signaling.

Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model.

The transforming growth factor-ß (TGF-ß) family is known to play critical roles in cancer progression. While the dual role of TGF-ß is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo.

TGF- ß Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α -Fetoprotein Promoter.

α-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-ß signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-ß signaling. To further understand how TGF-ß suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-ß. We found that the TGF-ß signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-ß stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-ß signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-ß signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.

C18 ORF1, a novel negative regulator of transforming growth factor-ß signaling.

Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.

Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion.

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of paclitaxel-induced thermal hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after paclitaxel treatment. Behavioral assessment using the tail-flick test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced thermal hyperalgesia after days 7, 14, and 21. Paclitaxel-induced thermal hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Paclitaxel (2 and 4 mg/kg) treatment increased the expression of TRPV1 mRNA and protein in DRG neurons. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment increased TRPV1 protein expression in small and medium DRG neurons 14 days after treatment. Antibody double labeling revealed that isolectin B4-positive small DRG neurons co-expressed TRPV1. TRPV1 immunostaining was up-regulated in paw skin day 14 after paclitaxel treatment. Moreover, in situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small or medium in size. These results suggest that paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain.

Identification of amino acids in the pore region of Kv1.2 potassium channel that regulate its glycosylation and cell surface expression.

The Kv1.4 potassium channel is reported to exhibit higher cell surface expression than the Kv1.1 potassium channel when expressed as a homomer in cell lines. Kv1.4 also shows highly efficient trans-Golgi glycosylation whereas Kv1.1 is not glycosylated. The surface expression and glycosylation of Kv1.2 is intermediate between those of Kv1.1 and Kv1.4. Amino acid determinants controlling the surface expression of Kv1 channels were localized to the highly conserved pore region and both positive and negative determinants of Kv1.1 and Kv1.4 trafficking have been reported. In this study, we analyzed the effect of substituting amino acids in the pore region of Kv1.2 with the corresponding amino acid present in Kv1.1 or Kv1.4 on glycosylation and trafficking of Kv1.2. Mutations in the outer pore region of Kv1.2 of Arg(354) to Pro (corresponding to Kv1.4) and to Ala (corresponding to Kv1.1) enhanced and reduced, respectively, cell surface expression of Kv1.2. Mutations in a different outer pore region of Val(381) to Lys (Kv1.4) and Tyr (Kv1.1) both reduced the cell surface expression. In contrast, mutation in the deep pore region of Ser(371) to Thr (Kv1.4) markedly enhanced cell surface expression. These results suggest that the cell surface expression of Kv1.2 is regulated by specific amino acids in the pore region in a similar manner to Kv1.1 and Kv1.4, and that the cell surface expression of Kv1.2, a channel intermediate between Kv1.1 and Kv1.4, can be attributed to these specific residues.

DNA sequencing and transcriptional analysis of the kasugamycin biosynthetic gene cluster from Streptomyces kasugaensis M338-M1.

Streptomyces kasugaensis M338-M1 produces the aminoglycoside antibiotic kasugamycin (KSM). We previously cloned, sequenced and characterized the KSM acetyltransferase, transporter, and some of the biosynthetic genes from this strain. To identify other potential genes in a chromosome walk experiment, a 6.8-kb EcoRI-PstI region immediately downstream from the KSM transporter genes was sequenced. Five open reading frames (designated as kasN, kasO, kasP, kasQ, kasR) and the 5' region of kasA were found in this region. The genes are apparently co-transcribed as bicistrons, all of which are co-directional except for the kasPQ transcript. Homology analysis of the deduced products of kasN, kasP, kasQ and kasR revealed similarities with known enzymes: KasN, D-amino acid oxidase from Pseudomonas aeruginosa (35% identity); KasP, F420-dependent H4MPT reductase from Streptomyces lavendulae (33% identity); KasQ, UDP-N-acetylglucosamine 2-epimerase from Streptomyces verticillus (45% identity); and KasR, NDP-hexose 3,4-dehydratase from Streptomyces cyanogenus (38% identity); respectively. A gel retardation assay showed that KasT, a putative pathway-specific regulator for this gene cluster, bound to the upstream region of kasN and to the intergenic region of kasQ-kasR, suggesting that the expression of these operons is under the control of the regulator protein.

Destruxin E, a cyclodepsipeptide antibiotic, reduces cyclin D1 levels and inhibits anchorage-independent growth of v-Ki-ras-expressed pMAM-ras-REF cells.

Destruxin E (DE), a cyclodepsipeptide isolated from fermentation broths of Metarhizium sp. MA324, inhibited the growth of v-Ki-ras-expressed pMAM-ras-REF (rasREF) cells in the suspension (anchorage-independent) culture (a) more strongly than that in the substratum-attached (anchorage-dependent) culture (b) or that of v-Ki-ras-unexpressed pMAM-ras-REF (REF) cells in the substratum-attached culture (c); the IC(50) values of DE were 0.07 microM (a), 0.4 microM (b), and 1.2 microM (c). DE arrested G1 phase cell cycle progression of rasREF cells in the substratum-attached culture (b). In rasREF cells treated with DE for 72 h in suspension culture (a), the levels of cyclin D1, cyclin A, p27(Kip1), and hyperphosphorylated Rb were decreased, but the levels of cdk4, cdk6, cdk2, p16(INK4a), and p21(Cip1) were not affected. Among these effects, the decrease in cyclin D1 was prominent. DE decreased the level of cyclin D1 in rasREF cells in the suspension culture (a) at 0.1 microM and in the substratum-attached culture (b) at 1 microM, while the level of cyclin D1 in REF cells in the substratum-attached culture (c) was not decreased at 1 microM. The extent of growth inhibition correlated with the decrease in cyclin D1. The level of cyclin D1 mRNA of rasREF cells in the suspension culture (a) was also decreased by DE. DE decreased cyclin D1 mRNA, resulting in inhibition of anchorage-independent growth of rasREF cells.

Panepophenanthrin, from a mushroom strain, a novel inhibitor of the ubiquitin-activating enzyme.

Screening for inhibitors of the ubiquitin-proteasome pathway, considered to regulate important cellular events and linked to serious diseases as well, led to isolation of a new compound, panepophenanthrin, from the fermented broth of a mushroom strain, Panus rudis Fr. IFO 8994. This is the first inhibitor of the ubiquitin-activating enzyme, which is indispensable for the ubiquitin-proteasome pathway. The structure of panepophenanthrin was determined by NMR and X-ray crystallographic analyses as 1,3a,10-trihydroxy-10c-(3-hydroxy-3-methylbut-1-enyl)-5,5-dimethyl-1,2,3,3a,5,5a,8,9,10,10a,10b,10c-dodecahydro-4-oxa-2,3,8,9-diepoxyacephenanthrylen-7-one.

kasT gene of Streptomyces kasugaensis M338-M1 encodes a DNA-binding protein which binds to intergenic region of kasU-kasJ in the kasugamycin biosynthesis gene cluster.

We previously reported that a 4.2 kb SacI-EcoRI DNA region from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM transporter genes (kasKLM). As an extension of that study, a 3.7 kb Psti-SacI DNA region, located at 1.5 approximately 5.2 kb upstream of kasK, was cloned and sequenced, revealing three complete open reading frames, designated kasT, kasU and kasJ. The kasJ gene encodes a protein (KasJ) with a conserved dinucleotide (FAD)-binding motif Homology search for KasJ showed its similarity to NADH: N-amidino-scyllo-inosamine oxidoreductase (StsB) which is involved in biosynthesis of the streptidine moiety of streptomycin (SM) in S. griseus. The kasT gene encodes a DNA-binding protein (KasT), including a helix-turn-helix motif near the center of the sequence. This protein is similar in structure to a pathway-specific activator protein (StrR) that plays a role in regulating the SM biosynthesis gene cluster of S. griseus. A fusion protein (Trx-KasT) clearly showed DNA binding activity with the intergenic region of kasU-kasJ, suggesting that KasT is a pathway-specific regulator of the KSM biosynthesis gene cluster.