BBA - Molecular Cell Research (v.1803, #11)

MicroRNAs: Synthesis, mechanism, function, and recent clinical trials by Fazli Wahid; Adeeb Shehzad; Taous Khan; You Young Kim (1231-1243).
MicroRNAs (miRNAs) are a class of small, endogenous RNAs of 21–25 nucleotides (nts) in length. They play an important regulatory role in animals and plants by targeting specific mRNAs for degradation or translation repression. Recent scientific advances have revealed the synthesis pathways and the regulatory mechanisms of miRNAs in animals and plants. miRNA-based regulation is implicated in disease etiology and has been studied for treatment. Furthermore, several preclinical and clinical trials have been initiated for miRNA-based therapeutics. In this review, the existing knowledge about miRNAs synthesis, mechanisms for regulation of the genome, and their widespread functions in animals and plants is summarized. The current status of preclinical and clinical trials regarding miRNA therapeutics is also reviewed. The recent findings in miRNA studies, summarized in this review, may add new dimensions to small RNA biology and miRNA therapeutics.
Keywords: miRNA; Small RNAs; miRNA based gene regulations; miRNA therapeutics; mRNA degradation;

Thymosin β4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1α-dependent manner by Jin-Ok Jo; Su-Ryun Kim; Moon-Kyung Bae; Yun-Jeong Kang; Mee Sun Ock; Hynda K. Kleinman; Hee-Jae Cha (1244-1251).
Thymosin β4 has multi-functional roles in cell physiology, but little is known about its mechanism(s) of action. We previously reported that thymosin β4 stimulated angiogenesis through the induction of vascular endothelial growth factor (VEGF). To identify the mechanism of VEGF induction by thymosin β4, we have used a luciferase assay system with VEGF in the 5' promoter region. We also analyzed the effect of thymosin β4 on VEGF mRNA stability and on the expression and stability of hypoxia-inducible factor (HIF)-1α. We found that thymosin β4 induces VEGF expression by an increase in the stability of HIF-1α protein. Analysis of the expression patterns of thymosin β4 and HIF-1α in colon cancer tissue microarray showed that thymosin β4 and HIF-1α co-localized in these biopsies. These data show that thymosin β4 induces the expression of VEGF indirectly by increasing the protein stability of HIF-1α.►We first identify that thymosin beta-4 up-regulates VEGF expression indirectly by increasing the stability of HIF-1alpha protein which is the key protein induce the expression of VEGF. These findings also suggest regulation mechanism of VEGF expression and angiogenesis in nomoxic condition and thymosin beta-4 is the regulatory protein control the VEGF expression in normoxic condition.
Keywords: Thymosin β4; VEGF; HIF-1α; Hypoxia; Angiogenesis; Protein stability;

γ2-Adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits by Tatjana Döring; Katherina Gotthardt; Jens Stieler; Reinhild Prange (1252-1264).
γ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of γ2-adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of γ2-adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon γ2-adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of γ2-adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify γ2-adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation.►γ2-Adaptin acts within the multivesicular body (MVB) pathway. ►γ2-Adaptin interacts with the ESCRT subunits Vps28 and CHMP2A. ►γ2-Adaptin contributes to MVB-to-lysosome maturation.
Keywords: CHMP2A; Lysosome; Multivesicular body; Virus budding; Vps28;

Insulin stimulation of PKCδ triggers its rapid degradation via the ubiquitin-proteasome pathway by Chagit Brand; Miriam Horovitz-Fried; Aya Inbar; Tamar-Brutman-Barazani; Chaya Brodie; Sanford R. Sampson (1265-1275).
Insulin rapidly upregulates protein levels of PKCδ in classical insulin target tissues skeletal muscle and liver. Insulin induces both a rapid increase in de novo synthesis of PKCδ protein. In this study we examined the possibility that insulin may also inhibit degradation of PKCδ. Experiments were performed on L6 skeletal muscle myoblasts or myotubes in culture. Phorbol ester (PMA)- and insulin-induced degradation of PKCδ were abrogated by proteasome inhibition. Both PMA and insulin induced ubiquitination of PKCδ, but not of that PKCα or PKCε and increased proteasome activity within 5 min. We examined the role of tyrosine phosphorylation of PKCδ in targeting PKCδ for degradation by the ubiquitin-proteasome pathway. Transfection of cells with PKCδY311F, which is not phosphorylated, resulted in abolition of insulin-induced ubiquitination of PKCδ and increase in proteasome activity. We conclude that insulin induces degradation of PKCδ via the ubiquitin-proteasome system, and that this effect requires phosphorylation on specific tyrosine residues for targeting PKCδ for degradation by the ubiquitin-proteasome pathway. These studies provide additional evidence for unique effects of insulin on regulation of PKCδ protein levels.► Both phorbol ester (PMA)- and insulin-induced degradation of PKCδ are abrogated by proteasome inhibition. ► Both PMA and insulin induce ubiquitination of PKCδ, but not of that PKCα or PKCε, and increase proteasome activity within 5 min. ► Prevention of phosphorylation of PKCδ on tyrosine 311 abolishes both ubiquitination of PKCδ and the increase in proteasome activity induced by insulin. ► These studies provide additional evidence for unique effects of insulin on regulation of PKCδ protein levels.
Keywords: PKC; Protein degradation; Proteasome; Ubiquitin;

Primary effect of 1α,25(OH)2D3 on IL-10 expression in monocytes is short-term down-regulation by Juha M. Matilainen; Tiia Husso; Sari Toropainen; Sabine Seuter; Mikko P. Turunen; Petra Gynther; Seppo Ylä-Herttuala; Carsten Carlberg; Sami Väisänen (1276-1286).
The biologically most active vitamin D compound, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), influences the status of inflammation by modulating the expression of several cytokine genes. In this study, we have examined the mechanism of transcriptional regulation of interleukin 10 (IL-10) by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative PCR showed that IL-10 mRNA expression was significantly down-regulated (2.8-fold) during the first 8 h of 1α,25(OH)2D3 treatment, while after 48 h it was up-regulated (3-fold). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays showed that the vitamin D receptor (VDR) binds in a cyclical fashion to a promoter region 1500–1700 bp upstream of the IL-10 transcription start site (TSS) containing two conserved VDR binding sites. Targeting of VDR binding sites by enhancer specific duplex RNAs revealed that only the more distal element is functional and chromosome conformation capture analysis suggested that this region loops 1α,25(OH)2D3-dependently to the TSS. Quantitative ChIP and micrococcal nuclease assays also revealed 1α,25(OH)2D3-dependent cyclical epigenetic changes and nucleosome remodeling at this promoter region. In conclusion, in LPS-treated THP-1 cells the primary effect of 1α,25(OH)2D3 on IL-10 expression is down-regulation, which is achieved via a cyclical recruitment of VDR to the promoter.►IL-10 is bi-directionally regulated by 1α,25(OH)2D3 in monocytes. The primary effect is repression and the secondary effect is induction. ►The effects of 1α,25(OH)2D3 are mediated via ER7- and ER8-type elements at distal IL-10 promoter. ►The expression of IL-10 can be affected by targeting the regulative regions by duplex RNAs.
Keywords: Chromatin; IL-10; VDR; Transcription;

Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP by Seaho Kim; Wei Huang; Emilio P. Mottillo; Anjum Sohail; Yoon-Ah Ham; M. Katie Conley-LaComb; Chong Jai Kim; Guri Tzivion; Hyeong-Reh Choi Kim; Shihua Wang; Yong Q. Chen; Rafael Fridman (1287-1297).
Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN +/-) or homozygous (PTEN −/−) for PTEN deletion or harboring a wild-type PTEN (PTEN +/+) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN −/− PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN +/+ and PTEN +/ cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN −/− cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN −/− cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN +/+ cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.►MT1-MMP protein but not mRNA is upregulated in PTEN null mouse prostate cancer cells. ►MT1-MMP is differentially O-glycosylated in PTEN −/− mouse prostate cancer cells. ►Mouse PTEN −/− but not PTEN +/+ cells display increased MT1-MMP stability and activity. ►MT1-MMP up-regulation in mouse PTEN −/− cells is mediated by the PI3K/AKT pathway. ►Rapamycin upregulates MT1-MMP protein expression in PTEN −/− cells via PI3K activity.
Keywords: Matrix metalloproteinases; Prostate cancer; PTEN; Glycosylation; Posttranslational modification;

Palmitoylation of CD36/FAT regulates the rate of its post-transcriptional processing in the endoplasmic reticulum by Rick F. Thorne; Kylie J. Ralston; Charles Edo de Bock; Nizar M. Mhaidat; Xu Dong Zhang; Andrew W. Boyd; Gordon F. Burns (1298-1307).
CD36/FAT is a transmembrane glycoprotein that functions in the cellular uptake of long-chain fatty acids and also as a scavenger receptor. As such it plays an important role in lipid homeostasis and, pathophysiologically, in the progression of type 2 diabetes and atherosclerosis. CD36 expression is tightly regulated at the levels of both transcription and translation. Here we show that its expression and location are also regulated post-translationally, by palmitoylation. Although palmitoylation of CD36 was not required for receptor maturation and cell surface expression, inhibition of palmitoylation either pharmacologically with cerulenin or by mutation of the relevant cysteines delayed processing at the ER and trafficking through the secretory pathway. The absence of palmitoylation also reduced the half life of the CD36 protein. Additionally, the CD36 palmitoylation mutant did not incorporate efficiently into lipid rafts, a site known to be required for its function of fatty acid uptake, and this reduced the efficiency of uptake of oxidized low density lipoprotein. These findings provide an added level of sophistication where translocation of CD36 to the plasma membrane may be physiologically regulated by palmitoylation.►Palmitoylation of CD36 occurs in the ER and is not required for cell surface expression but does influences secretory pathway trafficking. ►Absence of palmitoylation causes ER accumulation and decreases protein half-life. ► Palmitoylation is required for CD36 to incorporate into plasma membrane rafts. ► Localisation to plasma membrane rafts affects the efficiency of uptake of oxLDL.
Keywords: Acylation; CD36; Detergent-resistant membranes (DRMs); Endoplasmic reticulum (ER); Lipids rafts; Oxidised low density lipoprotein (oxLDL); Palmitoylation;

Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons by Gabriela Schneider; Krzysztof Nieznanski; Jolanta Jozwiak; Lukasz P. Slomnicki; Maria J. Redowicz; Anna Filipek (1308-1317).
CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1–179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178–229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.
Keywords: CacyBP/SIP; Actin; Tubulin; Cytoskeleton; Adhesion; Migration;