Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes.

BACKGROUND: Mechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation. METHODS: Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated. RESULTS: The mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts' miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation. CONCLUSIONS: In osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein.

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained >90% activity and 80-90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85-90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30-40%) and λmax blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.

Roles and Mechanisms of Human Cathelicidin LL-37 in Cancer.

LL-37, the C-terminal peptide of human cathelicidin antimicrobial peptide (CAMP, hCAP18), reportedly increases resistance to microbial invasion and exerts important physiological functions in chemotaxis, promotion of wound closure, and angiogenesis. Accumulating evidence indicates that LL-37 also plays a significant role in human cancer. LL-37 induces tumorigenic effects in cancers of the ovary, lung, breast, prostate, pancreas, as well as in malignant melanoma and skin squamous cell carcinoma. In contrast, LL-37 displays an anti-cancer effect in colon cancer, gastric cancer, hematologic malignancy and oral squamous cell carcinoma. Mechanistically, LL-37-induced activation of membrane receptors and subsequent signaling pathways lead to alteration of cellular functions. Different membrane receptors on various cancer cells appear to be responsible for the tissue-specific effects of LL-37. Meanwhile, the findings that vitamin D-dependent induction of cathelicidin in human macrophages activates the anti-cancer activity of tumor-associated macrophages (TAMs) and enhances antibody-dependent cellular cytotoxicity (ADCC) support critical roles of vitamin D-dependent induction of cathelicidin in cancer progression. This review describes novel advances involving the roles and mechanisms of human cathelicidin LL-37 in cancer.

Mechanical Strain Regulates Osteoblast Proliferation Through Ca2+-CaMK-CREB Signal Pathway.

Objective To investigate the effects of mechanical strain on Ca2+-calmodulin dependent kinase (CaMK)-cAMP response element binding protein (CREB) signal pathway and proliferation of osteoblasts.Methods Using a four-point bending device, MC3T3-E1 cells were exposed to mechanical tensile strains of 2500 µs and 5000 µs at 0.5 Hz respectively. The intracellular free Ca2+ ([Ca2+]i) concentration and calmodulin activity were assayed by fluorospectrophotometry, CaMK II ß, CREB, and phosphorylated (activated) CREB (p-CREB) were assessed by Western blot, and cells proliferation was assayed with MTT. Pretreatment with verapamil was carried out to block Ca2+ channel, and inhibitor U73122 was used to inhibit phospholipase C (PLC).Results Mechanical strains of 2500 µs and 5000 µs for 1 to 10 minutes both increased [Ca2+]i level of the cells. The 2500 µs strain, a periodicity of 1 h/d for 3 days, activated calmodulin, elevated protein levels of CaMK II ß and p-CREB, and promoted cells proliferation, which were attenuated by pretreatment of verapamil or U73122. The effects of 5000 µs strain on calmodulin, CaMK II ß, p-CREB and proliferation were contrary to 2500 µs strain.Conclusion The mechanical strain regulates osteoblasts proliferation through Ca2+-CaMK-CREB signal pathway via Ca2+ channel and PLC/IP3 transduction cascades.

Mechanical Strain Affects Some Microrna Profiles in Pre-Oeteoblasts.

MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.

Integrin-ß1, not integrin-ß5, mediates osteoblastic differentiation and ECM formation promoted by mechanical tensile strain.

BACKGROUND: Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-ß (ß1, ß5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-ß in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear. RESULTS: After transfection with integrin-ß1 siRNA or integrin-ß5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-ß1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-ß5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation. CONCLUSION: Integrin-ß1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-ß5 is not involved in the osteoblasts response to the tensile strain.

Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line.

S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein ß (C/EBPß). Mutated C/EBPß binding sequences or C/EBPß-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPß-dependent transcriptional activity.

Augmentation of epithelial resistance to invading bacteria by using mRNA transfections.

To protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either the CAMP, S100A8, and S100A9 open reading frames, A8-IRES-A9 (fusion sequence), or A8-nIRES-A9 (fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion by Listeria and Salmonella for up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

S100 proteins in head and neck squamous cell carcinoma (Review).

The most common tumor affecting the head and neck is head and neck squamous cell carcinoma (HNSCC). The characteristics of HNSCC include a rapid onset, a lack of early diagnosis, drug resistance, relapse and systemic adverse effects, leading to inadequate prevention, diagnosis and treatment. Notably, previous research suggests that there is an association between S100 proteins and HNSCC. S100A8, S100A9 and S100A14 interfere with tumor cell proliferation by blocking the cell cycle. The present review discusses this association. S100A4 enhances cancer stem cell properties, and interacts with actin and tropomyosin to promote tumor cell migration. S100A1, S100A8, S100A9, S100A10, S100A14 and S100P are involved in the initiation and progression of HNSCC via Hippo, nuclear factor κB, phosphatidylinositol kinase/protein kinase B/mammalian target of rapamycin and other signaling pathways. In addition, certain long non-coding RNAs and microRNAs are involved in regulating the expression of S100 proteins in HNSCC. Reducing the expression of certain members of the S100 protein family may enhance the chemosensitivity of HNSCC. Collectively, it is suggested that S100 proteins may function as markers and targets for the prevention, diagnosis and treatment of HNSCC.

Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; ∼78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.

Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

Pancreatic cancer (PC) is a devastating solid malignancy with a dismal prognosis. The treatment of metastatic PC is a current challenge for medical oncologists due to a lack of early detection, drug resistance, and relapse. Therefore, potential biomarkers and effective therapeutic targets for PC are urgently required. Ceramide-1-phosphate transfer protein (CPTP) is a member of the glycolipid transfer protein family, which is associated with autophagy and inflammation regulation. The roles and mechanisms of CPTP in PC have not been clarified. In this study, by RT-qPCR and immunohistochemistry analysis, we found that CPTP is highly expressed in PC and is associated with a poor prognosis in PC patients. By using cell counting kit-8, colony formation, transwell and matrigel assays in vitro, as well as xenograft model assays in vivo, we further proved that CPTP enhanced PC cells growth and metastasis. In PC cells, human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling. Sp (specific protein)-1 and Sp3 transcription factors also act as upstream positive regulators of CPTP expression in PC cells. Collectively, these findings suggested that CPTP may function as a pro-tumorigenic gene in PC cells and could be a promising therapeutic target in PC.

Ceramide-1-phosphate and its transfer proteins in eukaryotes.

Ceramide-1-phosphate (C1P) is a bioactive phosphorylated sphingolipid (SL), produced through the direct phosphorylation of ceramide by ceramide kinase. It plays important roles in regulating cell survival, migration, apoptosis and autophagy and is involved in inflammasome assembly/activation, which can stimulate group IVA cytosolic phospholipase A2α and subsequently increase the levels of arachidonic acid and pro-inflammatory cytokines. Human C1P transfer protein (CPTP) can selectively transport C1P from the Golgi apparatus to specific cellular sites through a non-vesicular mechanism. Human CPTP also affects specific SL levels, thus regulating cell SL homeostasis. In addition, human CPTP plays a crucial role in the regulation of autophagy, inflammation and cell death; thus, human CPTP is closely associated with autophagy and inflammation-related diseases such as cardiovascular and neurodegenerative diseases, and cancers. Therefore, illustrating the functions and mechanisms of human CPTP is important for providing the research foundations for targeted therapy. The key human CPTP residues for C1P recognition and binding are highly conserved in eukaryotic orthologs, while the human CPTP homolog in Arabidopsis (accelerated cell death 11) also exhibits selective inter-membrane transfer of phyto-C1P. These results demonstrate that C1P transporters play fundamental roles in SL metabolism in cells. The present review summarized novel findings of C1P and its TPs in eukaryotes.

Human cathelicidin antimicrobial peptide suppresses proliferation, migration and invasion of oral carcinoma HSC-3 cells via a novel mechanism involving caspase-3 mediated apoptosis.

Human cathelicidin antimicrobial peptide and its active product, LL­37 (CAMP/LL­37), exhibit a broad spectrum of antimicrobial effects. An increasing number of studies have shown that human CAMP/LL­37 also serves significant roles in various types of cancer. The primary aims of the present study were to investigate the roles and mechanisms of human CAMP/LL­37 in oral squamous cell carcinoma (OSCC) cells. The results indicated that either LL­37 C­terminal deletion mutants (CDEL) or CAMP stable expression in HSC­3 cells reduced colony formation, proliferation, migration and invasion ability of the cells. Expression analysis demonstrated that either CDEL or CAMP stable expression in HSC­3 cells induced caspase­3 mediated apoptosis via the P53­Bcl­2/BAX signalling pathway, whereas the levels of cell cycle­related proteins, cyclin B1 and PKR­like ER kinase, were significantly upregulated in the CAMP, but not in the CDEL overexpressing cells. Transcriptional profile comparisons revealed that CDEL or CAMP stable expression in HSC­3 cells upregulated expression of genes involved in the IL­17­dependent pathway compared with the control. Taken together, these results suggest that CAMP may act as a tumour suppressor in OSCC cells, and the underlying mechanism involves the induction of caspase­3 mediated apoptosis via the P53­Bcl­2/BAX signalling pathway.

Emerging roles for human glycolipid transfer protein superfamily members in the regulation of autophagy, inflammation, and cell death.

Glycolipid transfer proteins (GLTPs) were first identified over three decades ago as ~24kDa, soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. Upon discovery that GLTPs use a unique, all-α-helical, two-layer 'sandwich' architecture (GLTP-fold) to bind glycosphingolipids (GSLs), a new protein superfamily was born. Structure/function studies have provided exquisite insights defining features responsible for lipid headgroup selectivity and hydrophobic 'pocket' adaptability for accommodating hydrocarbon chains of differing length and unsaturation. In humans, evolutionarily-modified GLTP-folds have been identified with altered sphingolipid specificity, e. g. ceramide-1-phosphate transfer protein (CPTP), phosphatidylinositol 4-phosphate adaptor protein-2 (FAPP2) which harbors a GLTP-domain and GLTPD2. Despite the wealth of structural data (>40 Protein Data Bank deposits), insights into the in vivo functional roles of GLTP superfamily members have emerged slowly. In this review, recent advances are presented and discussed implicating human GLTP superfamily members as important regulators of: i) pro-inflammatory eicosanoid production associated with Group-IV cytoplasmic phospholipase A2; ii) autophagy and inflammasome assembly that drive surveillance cell release of interleukin-1ß and interleukin-18 inflammatory cytokines; iii) cell cycle arrest and necroptosis induction in certain colon cancer cell lines. The effects exerted by GLTP superfamily members appear linked to their ability to regulate sphingolipid homeostasis by acting in either transporter and/or sensor capacities. These timely findings are opening new avenues for future cross-disciplinary, translational medical research involving GLTP-fold proteins in human health and disease. Such avenues include targeted regulation of specific GLTP superfamily members to alter sphingolipid levels as a therapeutic means for combating viral infection, neurodegenerative conditions and circumventing chemo-resistance during cancer treatment.

Expression of S100A8 is induced by interleukin­1α in TR146 epithelial cells through a mechanism involving CCAAT/enhancer binding protein ß.

Calprotectin in mucosal epidermal keratinocytes has an important role in fighting microbial infections. S100A8 belongs to the S100 protein family and is a subunit of calprotectin (heterodimer complex of S100A8/A9). Interleukin­1α (IL­1α) is one of the cytokines produced by oral keratinocytes. The primary aims of the present study were to investigate the effect of IL­1α on the expression of S100A8 and its underlying molecular mechanism in oral epithelial cells. Determining the molecular mechanism of the induced expression of S100A8 by IL­1α aims to improve current understanding of the roles of calprotectin during the infection of mucosal epithelial cells. The expression analysis indicated that IL­1α significantly induced the expression of S100A8 in human TR146 epithelial cells at the mRNA and protein levels, respectively. The reporter assay demonstrated that the upregulatory effect of S100A8 induced by IL­1α was dependent on the S100A8 promoter specific region (­165/­111). The results of electrophoresis mobility shift assay and chromatin immunoprecipitation assay also demonstrated that the CCAAT/enhancer binding protein ß (C/EBPß) binding site (­113/­109) in the S100A8 promoter region was involved into the upregulatory effect on the expression of S100A8 induced by IL­1α. Taken together, these results suggested that the activation of the expression of S100A8 induced by IL­1α in TR146 epithelial cells involves a mechanism by which the binding activity of C/EBPß to the specific site (­113/­109) of the S100A8 promoter is increased.

Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution.

BACKGROUND: Glycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. The present investigation provides the first insights into the organization, transcriptional status, phylogenetic/evolutionary relationships of GLTP genes. RESULTS: In human cells, single-copy GLTP genes were found in chromosomes 11 and 12. The gene at locus 11p15.1 exhibited several features of a potentially active retrogene, including a highly homologous (approximately 94%), full-length coding sequence containing all key amino acid residues involved in glycolipid liganding. To establish the transcriptional activity of each human GLTP gene, in silico EST evaluations, RT-PCR amplifications of GLTP transcript(s), and methylation analyses of regulator CpG islands were performed using various human cells. Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent. Heterologous expression and purification of the GLTP paralogs showed glycolipid intermembrane transfer activity only for 12q24.11 GLTP. Phylogenetic/evolutionary analyses indicated that the 5-exon/4-intron organizational pattern and encoded sequence of 12q24.11 GLTP were highly conserved in therian mammals and other vertebrates. Orthologs of the intronless GLTP gene were observed in primates but not in rodentiates, carnivorates, cetartiodactylates, or didelphimorphiates, consistent with recent evolutionary development. CONCLUSION: The results identify and characterize the gene responsible for GLTP expression in humans and provide the first evidence for the existence of a GLTP pseudogene, while demonstrating the rigorous approach needed to unequivocally distinguish transcriptionally-active retrogenes from silent pseudogenes. The results also rectify errors in the Ensembl database regarding the organizational structure of the actively transcribed GLTP gene in Pan troglodytes and establish the intronless GLTP as a primate-specific, processed pseudogene marker. A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

miR­98­5p promotes osteoblast differentiation in MC3T3­E1 cells by targeting CKIP­1.

Casein kinase 2-interacting protein 1 (CKIP-1) is a negative regulator for bone formation. Previously, using bioinformatics analysis, CKIP­1 has been predicted to serve the role of target gene of miR­98­5p. In the present study, the potential role of miR­98­5p in regulating osteoblast differentiation through CKIP­1 was investigated. Following pre­treatment with microRNA (miR)­98­5p agomir or miR­98­5p antagomir, MC3T3­E1 cells were cultured in an osteoinductive medium. Subsequently, the expression of miR­98­5p, CKIP­1 and levels of osteoblast differentiation markers, including alkaline phosphatase, matrix mineralization, osteocaicin, collagen type I, runt­related transcription factor 2 and osteopontin were assayed. Using a dual­luciferase reporter assay, it was demonstrated that CKIP­1 was the target gene of miR­98­5p. miR­98­5p was upregulated as a result of treatment with miR­98­5p agomir and promoted osteoblast differentiation. Conversely, miR­98­5p antagomir inhibited miR­98­5p expression and osteoblast differentiation. miR­98­5p targeted CKIP­1 by binding to its 3'­untranslated region. Furthermore, miR­98­5p overexpression decreased the protein levels of CKIP­1 and inhibition of miR­98­5p increased the protein levels of CKIP­1. The results of the present study indicated that CKIP­1 was a target gene of miR­98­5p and that miR­98­5p regulated osteoblast differentiation in MC3T3­E1 cells by targeting CKIP­1.

Aberrant high expression level of MORC2 is a common character in multiple cancers.

Microrchidia 2 (MORC2) plays important roles in DNA damage repair and lipogenesis, but the clinical and functional role of MORC2 in cancer remains largely unexplored. In this study, we showed that MORC2 was widely expressed in human tissues while significantly up-regulated in most cancer types using immunohistochemical staining and analysis of messenger RNA expression profile of more than 2000 human tissue samples from 15 different organs (lung, prostate, liver, breast, brain, stomach, colon/rectum, pancreas, ovary, endometrium, skin, nasopharynx, kidney, esophagus, and bladder). We also found that the MORC2 expression level in high-grade cancer tissues was much more elevated and associated with unfavorable pathological characteristics, poor overall survival, and disease-free survival in several kinds of cancers such as non-small cell lung cancer and breast cancer. Gene set enrichment analysis was used to predict the genes modulated by MORC2, and the results showed that dysregulation of MORC2 in tumor may take part in the cell cycle regulation and genomic instability. We observed that MORC2 knockdown would arrest the cell cycle progress, and the genome of tumors with high MORC2 expression contained more point mutations and gene copy number variation, which validates our gene set enrichment analysis results. The results also showed that MORC2 knockdown would significantly inhibit the proliferation, colony forming, migration, and invasion in multiple cancer cell lines. Taken together, these results highlight the importance of MORC2 in tumorigenesis and cancer progression, and it may act as a potential diagnostic marker and therapeutic target for these diseases.