Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2).

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.

Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP.

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

Expression and cell transformation activity of dynactin-associated protein isoforms.

Overexpression of human dynactin-associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single-pass transmembrane protein with a carboxy-terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino-terminal residues (NH2 -MVA in dynAPa and NH2 -MEYQLL in dynAPc). DynAPb has the same amino-terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy-terminal region, including the transmembrane domain. Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki-1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy-terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U-bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.

Naturally Occurring Mutations in HIV-1 CRF01_AE Capsid Affect Viral Sensitivity to Restriction Factors.

TRIM5α and MxB are known as restriction factors that inhibit the early step of intracellular HIV-1 replication cycle. Both factors are believed to interact with the incoming virus core to suppress HIV-1 infection. The extreme diversity of HIV-1 is thought to be a consequence of its propensity to mutate to escape immune responses and host restriction factors. We recently determined the capsid sequences for 144 HIV-1 CRF01_AE viruses obtained in Thailand from 2005 to 2011. In this study, we further analyzed the amino acid variations among the capsid sequences of 204 HIV-1 CRF01_AE obtained in Thailand and China, including 84 of the aforementioned 144 viruses, to detect mutations permitting escape from restriction by host factors. We found a characteristic combination of E79D, V83T, and H87Q in sequences from Chinese viruses and subsequently showed that this combination conferred partial resistance to MxB. Interestingly, this combination conferred resistance to human TRIM5α as well. The H87Q mutation alone conferred resistance to MxB in the CRF01_AE background, but not in subtype B virus. In contrast, the H87Q mutation alone conferred resistance to human TRIM5α in both the CFR01_AE and subtype B backgrounds. BLAST analysis revealed the presence of the E79D, V83T, and H87Q combination in CRF01_AE viruses isolated not only in China but also in many other countries. Although the mechanistic details as well as precise role of MxB antiviral activity in infected individuals remain to be clarified, our data suggest an interaction between MxB and the HIV-1 capsid in vivo.

Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction.

Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell-cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.