Myeloid-associated Differentiation Marker Research Articles

MYADM binds human parechovirus 1 and is essential for viral entry

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

Identification and Validation of MYADM as a Novel Prognostic Marker Related to EMT in ESCC.

Background: Esophageal squamous cell carcinoma (ESCC), one of the most aggressive gastrointestinal malignancies, remains an enormous challenge in terms of medical treatment and prognostic improvement. Based on the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases in R language, the myeloid-associated differentiation marker (MYADM) was confirmed using bioinformatics analysis and experimental verification. MYADM is upregulated in multiple cancer types; however, the oncogenic mechanism by which MYADM promotes ESCC remains largely unknown. Methods: In the present study, we used weighted gene coexpression network analysis to filter four hub genes (AKAP12, ITGA1, JAM2, and MYADM) in GSE45670 and GSE23400 that are related to the malignant progression of ESCC. Transcription factors and target miRNAs of the hub genes were predicted using the TarBase and JASPRAR databases, respectively, and a regulatory network was established. MYADM was selected based on the analysis of expression differences and prognostic value in ESCC. Next, we confirmed the level of MYADM in ESCC samples using immunohistochemistry of the tissue microarray. The molecular mechanisms of MYADM were further elucidated by experimental analyses, including Transwell assays, wound healing assays, and CCK8. Results: The correlation between MYADM levels and the clinical data of patients with ESCC was confirmed, including tumor differentiation, the node and metastasis stage, T stage, lymphatic metastasis, and postoperative distant metastasis. MYADM was significantly upregulated in ESCC and positively correlated with overall survival. MYADM induced cell proliferation, migration, invasion, and wound healing via the epithelial to mesenchymal transition (EMT) pathway in multiple experiments. Moreover, our results supported the hypothesis that MYADM promotes EMT during paclitaxel resistance. Conclusion: MYADM is closely correlated with ESCC progression, metastasis, and paclitaxel resistance and could be regarded as a novel biomarker and therapeutic target for ESCC patients.

Myeloid-associated differentiation marker is associated with type 2 asthma and is upregulated by human rhinovirus infection.

Human rhinoviruses are known to predispose infants to asthma development during childhood and are often associated with exacerbations in asthma patients. MYADM epithelial expression has been shown to associate with asthma severity. The goal of this study was to determine if MYADM expression patterns were altered in asthma and/or rhinovirus infection and if increased MYADM expression is associated with increased asthma-associated factors. Utilizing H1HeLa cells and differentiated primary human airway epithelial cells (AECs), we measured the expression of MYADM and inflammatory genes by qRT-PCR in the presence or absence of RV-1B infection or poly I:C treatment and with siRNA knockdown of MYADM. Expression of MYADM in the asthmatic lung was determined in the ovalbumin (ova)-challenged murine model. MYADM expression was upregulated in the lungs from ova-treated mice and in particular on the subsurface vesicle membrane in airway epithelial cells. Upon infection with RV-1B, human AECs grown at an air-liquid interface had increased the MYADM expression predominantly detected in ciliated cells. We found that the presence of MYADM was required for expression of several inflammatory genes both in a resting state and after RV-1B or poly I:C treatments. Our studies show that in a mouse model of asthma and during RV-1B infection of primary human AECs, increased MYADM expression is observed. In the mouse model of asthma, MYADM expression was predominantly on the luminal side of airway epithelial cells. Additionally, MYADM expression was strongly associated with increases in inflammatory genes, which may contribute to more severe asthma and RV-linked asthma exacerbations.

Myeloid-associated differentiation marker is an essential host factor for human parechovirus PeV-A3 entry

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

A Comprehensive Bioinformatic Analysis for Identification of Myeloid-Associated Differentiation Marker as a Potential Negative Prognostic Biomarker in Non-Small-Cell Lung Cancer

Objectives: This study aimed to identify a molecular marker associated with the prognosis of non-small-cell lung cancer (NSCLC). Materials and Methods: The RNA sequencing data and clinical information of NSCLC patients were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). The weighted gene co-expression network analysis (WGCNA) was used to identify the co-expression gene modules and differentially expressed genes (DEGs) by comparing gene expression between NSCLC tumor tissues and normal tissues. Subsequently, the functional enrichment analysis of the DEGs was performed. Kaplan-Meier survival analysis and the GEPIA2 online tool were performed to investigate the relationship between the expression of these genes of interest and the survival of NSCLC patients, and to validate one most survival-relevent hub gene, as well as validated the hub gene using independent datasets from the GEO database. Further analysis was carried out to characterize the relationship between the hub gene and tumor immune cell infiltration, tumor mutation burden (TMB), microsatellite instability (MSI), and other known biomarkers of lung cancer. The related genes were screened by analyzing the protein-protein interaction (PPI) network and the survival model was constructed. GEPIA2 was applied in the potential analysis of pan-cancer biomarker of hub gene. Results: 57 hub genes were found to be involved in intercellular connectivity from the 779 identified differentially co-expressed genes. Myeloid-associated differentiation marker (MYADM) was strongly associated with overall survival (OS) and disease-free survival (DFS) of NSCLC patients, and high MYADM expression was associated with poor prognosis. Thus, MYADM was identified as a risk factor. Additionally, MYADM was validated as a survival risk factor in NSCLC patients in two independent datasets. Further analysis showed that MYADM was nagetively associated with TMB, and was positively correlated with macrophages, neutrophils, and dendritic cells, suggesting its role in regulating tumor immunity. The MYADM expression differed across many types of cancer and had the potential to serve as a pan-cancer marker. Conclusion: MYADM is an independent prognostic factor for NSCLC patients, which can predict the progression of cancer and play a role in the tumor immune cell infiltration in NSCLC.

Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity

Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.

The miR-182/Myadm axis regulates hypoxia-induced pulmonary hypertension by balancing the BMP- and TGF-β-signalling pathways in an SMC/EC-crosstalk-associated manner.

We recently identified oncologic miR-182 as a new regulator of pulmonary artery hypertension (PAH) that targets myeloid-associated differentiation marker (Myadm), which is expressed in bone marrow stem cells and multipotent progenitors. Both miR-182 and Myadm are expressed in the cardiopulmonary system and correlated with the balance between the bone morphogenetic protein (BMP) and the transforming growth factor (TGF)-β signalling pathways, which are disturbed in PAH. We hypothesize that miR-182/Myadm are involved in BMP-TGF-β-signalling way in PAH. Hypoxia triggered pathological progression in cardiopulmonary PAH in vivo and in vitro; these changes were accompanied by strongly dowregulated BMP/SMAD1/5/8 expression and enhanced TGF-β/SMAD2/3 signalling pathway, favouring SMAD4/SMAD2 transcript formation and inhibiting the PAH negative regulator Id1 expression. miR-182 gain-of-function significantly inhibited the pathological progression in hypoxia-induced PAH (HPH) in vivo and in vitro, with a restoration of the balance in BMP-TGF-β signalling pathway. This recovery was abrogated by overexpression of Myadm. Conversely, loss-of-function of miR-182 increased the pathological progression of HPH followed by severe disturbance of BMP and TGF-β signal transduction and reduced Id1 expression, which was restored by Myadm knockdown. We also showed that the miR-182/Myadm relate BMP-TGF-β pathway is associated with NOS3/NO/cGMP via the crosstalk between endothelial cells and smooth muscle cells. Our findings further support the therapeutic significance of miR-182/Myadm in PAH via the balance of BMP- and TGF-β-associated mechanisms.

Proteomic profiling of biomarkers by MALDI-TOF mass spectrometry for the diagnosis of tracheobronchial stenosis after tracheobronchial tuberculosis.

Tracheobronchial tuberculosis (TB) leads to airway stenosis, irreversible airway damage and even death. The present study aimed to identify biomarkers for the diagnosis of tracheobronchial stenosis (TBS) secondary to tracheobronchial TB. A cohort was recruited, including patients with TBS after tracheobronchial TB, TBS after tracheal intubation or tracheotomy (TIT) and no stenosis of early-stage lung cancer,. Proteomic profiling was performed to gain insight into the mechanisms of the pathological processes. Differentially expressed proteins in the serum and bronchial alveolar lavage fluid (BALF) from patients were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Subsequently, ELISA was performed to validate the changes of protein levels in an additional cohort. MALDI-TOF MS revealed that 8 peptides in the serum, including myeloid-associated differentiation marker, keratin type I cytoskeletal 18, fibrinogen α-chain, angiotensinogen (AGT), apolipoprotein A-I (APOAI), clusterin and two uncharacterized peptides, and nine peptides in BALF, including argininosuccinate lyase, APOAI, AGT and five uncharacterized peptides, were differentially expressed (molecular-weight range, 1,000-10,000 Da) in the TB group compared with the TIT group. The ELISA results indicated that the changes in the protein levels had a similar trend as those identified by proteomic profiling. In conclusion, the present study identified proteins that may serve as potential biomarkers and provide novel insight into the molecular mechanisms underlying TBS after tracheobronchial TB.

MiR-182-3p/Myadm contribute to pulmonary artery hypertension vascular remodeling via a KLF4/p21-dependent mechanism.

Rationale: There is a continued need for investigating the roles of microRNAs and their targets on the pathogenesis of pulmonary arterial hypertension (PAH) vascular remodeling. We recently identified the association of myeloid miR-182-3p and its new target, Myeloid-Associated Differentiation Marker (Myadm), with vascular remodeling. Here, we aimed to determine the role of miR-182-3p/Myadm on PAH vascular remodeling and the underlying molecular mechanism.Methods: The miR-182-3p/Myadm expression profiles were detected in PAH patients and experimental rodent models. Loss-of-function and gain-of-function studies using gene knock-in or gene knock-out and the combinations of the proteomic technology and genome-wide ChIP-Seq were employed to determine the downstream targets of miR-182-3p/Myadm in response to monocrotaline (MCT)-induced PAH.Results: The miR-182-3p/Myadm expression was altered in PAH patients and experimental rodent models. Both miR-182-3p inhibitor and overexpression of Myadm augmented the pathological progression in rats in response to MCT-induced PAH. In contrast, miR-182-3p mimic and Myadm gene knockout attenuated the changes in the hemodynamics and structure of the cardio-pulmonary system in MCT-induced PAH in rats. Myadm mediated the proliferation of pulmonary artery smooth muscle cells (PASMCs) by altering the cell cycle kinase inhibitor (p21/Cip1) expression through the transcription factor Krüppel-like factor 4 (KLF4) translocation into the cytoplasm.Conclusion: Our findings indicate the prognostic and therapeutic significance of miR-182-3p in PAH and provide a new regulatory model of the myeloid-derived miR-182-3p/Myadm/KLF4/p21 axis in PAH vascular remodeling.

Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome†.

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P≤0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P<0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P<0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.

Oncological miR-182-3p, a Novel Smooth Muscle Cell Phenotype Modulator, Evidences From Model Rats and Patients.

Vascular smooth muscle cell (VSMC) phenotype change is a hallmark of vascular remodeling, which contributes to atherosclerotic diseases and can be regulated via microRNA-dependent mechanisms. We recently identified that asymmetrical dimethylarginine positively correlates to vascular remodeling-based diseases. We hypothesized that asymmetrical dimethylarginine induces smooth muscle cell (SMC) phenotypic change via a microRNA-dependent mechanism. Microarray analysis enabled the identification of downregulation of miR-182-3p in asymmetrical dimethylarginine-treated human aortic artery SMCs. The myeloid-associated differentiation marker (MYADM) was identified as the downstream target of miR-182-3p and implicated to contribute to miR-182-3p knockdown-mediated SMC phenotype change, which was evidenced by the increased proliferation and migration and reduced expression levels of phenotype-related genes in human aortic artery SMCs through the ERK/MAP (extracellular signal-regulated kinase/mitogen-activated protein) kinase-dependent mechanism. When inhibiting MYADM in the presence of miR-182-3p inhibitor or overexpressing MYADM in the presence of pre-miR-182-3p, human aortic artery SMCs were reversed to the differentiation phenotype. In vivo, adeno-miR-182-3p markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression, whereas adeno-miR-182-3p inhibitor significantly promoted neointimal formation. Atherosclerotic lesions from patients with high asymmetrical dimethylarginine plasma levels exhibited decreased miR-182-3p expression levels and elevated MYADM expression levels. miR-182-3p is a novel SMC phenotypic modulator by targeting MYADM.

MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. Membrane rafts are ordered assemblies of cholesterol, glycolipids, and proteins that modulate proinflammatory cell signaling and barrier function. In epithelial cells, the MAL family members MAL, MAL2, and myeloid-associated differentiation marker (MYADM) regulate the function and dynamics of ordered membrane domains. We analyzed the expression of these three proteins in human endothelial cells and found that only MYADM is expressed. MYADM was confined in ordered domains at the plasma membrane, where it partially colocalized with filamentous actin and cell-cell junctions. Small interfering RNA (siRNA)-mediated MYADM knockdown increased permeability, ICAM-1 expression, and leukocyte adhesion, all of which are features of an inflammatory response. Barrier function decrease in MYADM-silenced cells was dependent on ICAM-1 expression. Membrane domains and the underlying actin cytoskeleton can regulate each other and are connected by ezrin, radixin, and moesin (ERM) proteins. In endothelial cells, MYADM knockdown induced ERM activation. Triple-ERM knockdown partially inhibited ICAM-1 increase induced by MYADM siRNA. Importantly, ERM knockdown also reduced ICAM-1 expression in response to the proinflammatory cytokine tumor necrosis factor-α. MYADM therefore regulates the connection between the plasma membrane and the cortical cytoskeleton and so can control the endothelial inflammatory response.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.

Identification of diagnostic and therapeutic targets of pancreatic cancer using proteomic approaches

Pancreatic cancer remains one of the most lethal of all the solid malignancies. Using a quantitative proteomic strategy, we have carried out profiling studies to identify novel biomarkers and therapeutic targets in pancreatic cancer. Phosphotyrosine profiling revealed aberrant activation of epidermal growth factor receptor (EGFR) in a subset of pancreatic cancers. This correlated with phospho‐EGFR staining and response to the EGFR inhibitor, erlotinib, in mice carrying pancreatic cancer xenografts demonstrating the utility of activated EGFR as a biomarker that could be used to guide therapeutic decisions. Proteomic profiling of membrane fractions identified several proteins that are differentially expressed in pancreatic cancers. Immunohistochemical validation of one of the candidate markers, myeloid associated differentiation marker (MYADM), confirmed overexpression of this molecule in greater than 80% of pancreatic adenocarcinomas. Our studies emphasize the utility of advanced proteomic approaches in identifying novel biomarkers and therapeutic targets.This work was supported by Sol Goldman Trust for Pancreatic Cancer Research.

A Quantitative Proteomic Approach for Identification of Potential Biomarkers in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. In this study, our objective was to identify differentially regulated proteins in HCC through a quantitative proteomic approach using iTRAQ. More than 600 proteins were quantitated of which 59 proteins were overexpressed and 92 proteins were underexpressed in HCC as compared to adjacent normal tissue. Several differentially expressed proteins were not implicated previously in HCC. A subset of these proteins (six each from upregulated and downregulated groups) was further validated using immunoblotting and immunohistochemical labeling. Some of the overexpressed proteins with no previous description in the context of HCC include fibroleukin, interferon induced 56 kDa protein, milk fat globule-EGF factor 8, and myeloid-associated differentiation marker. Interestingly, all the enzymes of urea metabolic pathway were dramatically downregulated. Immunohistochemical labeling confirmed differential expression of fibroleukin, myeloid associated differentiation marker and ornithine carbamoyl transferase in majority of HCC samples analyzed. Our results demonstrate quantitative proteomics as a robust discovery tool for the identification of differentially regulated proteins in cancers.

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4 +, CD8 +, CD19 +cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34 +stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.

Characterization of the Mouse Myeloid-associated Differentiation Marker (Myadm) Gene: Promoter Analysis and Protein Localization

Hematopoietic differentiation is a complex process involving many genes inducing functional changes and characteristics of different cell lineages. To understand this process, it is important to identify genes involved in lineage commitment and maturation of hematopoietic progenitor cells. Recently we isolated the novel gene MYADM which is strongly up-regulated as multipotent progenitor cells differentiate towards myeloid cells. Because it is not expressed in lymphocytes, understanding the transcriptional control of MYADM could further explain differences in gene expression between myeloid and lymphoid cells. To identify regulatory elements controlling its restricted expression, we have analyzed the 5'-flanking region of the MYADM gene. The proximal promoter was found to lack both TATA and CCAAT boxes, but contained several potential binding sites for both ubiquitous and myeloid-specific transcription factors. Maximal promoter activity was contained within 800 bp from the tentative transcription initiation site, which was reduced as portions of the 5'-end were deleted, and completely abolished when the transcription initiation site was deleted. This promoter sequence had higher activity in myeloid cells compared to B cells, and activity was enhanced during myeloid differentiation, suggesting that we have identified the MYADM core promoter. Computer predictions had suggested MYADM to encode a protein with multiple transmembrane domains. By immunofluorescence and confocal microscopy we demonstrate that the protein is localized to the nuclear envelope and to intracytoplasmic membranes, indicating that MYADM constitutes an integral membrane protein.

Cloning of human myeloid-associated differentiation marker (MYADM) gene whose expression was up-regulated in NB4 cells induced by all-trans retinoic acid.

A full-length cDNA of 3192 bp isolated from human bone marrow cDNA library was predicted an ORF encoding 298 amino acids. The deduced protein, containing seven putative transmembrane segments and sharing 75.8% amino acid identity with mouse Myadm protein, was named as human MYADM. The results of Northern blot analysis showed that MYADM was ubiquitously expressed in 15 of 16 adult tissues tested, except thymus. To determine whether the novel human gene was involved in hematopoietic differentiation process as mouse Myadm did, we examined the mRNA expressive abundance of this gene between normal bone marrow cells and peripheral blood leukocytes, and detected the expression change in NB4 cells induced by all-trans retinoic acid at different induce time by the semi-quantitative RT-PCR. The results showed that the expression of the novel gene was not only significantly higher in peripheral blood leukocytes than in bone marrow cells, but also significantly up-regulated when the NB4 cells(derived from a patient with acute promyelocytic leukemia) were induced by all-trans retinoic acid (ATRA) for 48hr. It is suggested that human MYADM was also associated with the differentiation of hematopoietic cells or acute promyelocytic leukemia cells. In addition, MYADM was mapped to human chromosome 19q 13.33-q 13.4 by Radiation Hybrid mapping, and it consists of 3 exons and 2 introns and spans a 7.1-Kb genomic region.

Isolation of MYADM, a novel hematopoietic-associated marker gene expressed in multipotent progenitor cells and up-regulated during myeloid differentiation

A large number of hematopoietic cytokines and their receptors as well as transcription factors have been shown to be involved in maturation of blood cells. However, many of the genes important for the differentiation of multipotent stem cells to specific cellular lineages are still unknown. To identify novel genes involved in lineage selection of myeloid cells, we have applied differential display analysis during commitment toward granulocytes and macrophages of an IL-3-dependent multipotent progenitor cell line, FDCP-mix. One regulated cDNA represented a novel gene with restricted expression pattern within the hematopoietic system and was strongly up-regulated when FDCP-mix cells differentiated in GM-CSF, G-CSF, and M-CSF. The expression appears to be differentiation stage-specific in myeloid cells and is absent in B and T lymphocytes. Thus we found expression in normal mouse bone marrow enriched for stem cells and multipotent progenitors (c-kit+Sca-1+Lin- cells). When these cells were induced to differentiate toward myeloid cells, MYADM was up-regulated. In contrast, during conditions known to favor the development of B cell progenitors, the gene was down-regulated. The gene, termed MYADM for myeloid-associated differentiation marker gene, shows 100% identity to expressed sequence tags from early mouse embryonic development as well as from the mouse lung and from activated mouse macrophages. The predicted 32-kDa MYADM protein contains multiple hydrophobic putative transmembrane segments and has several potential consensus sites for phosphorylation. In view of its expression pattern, MYADM could serve as a new marker gene for hematopoietic differentiation. Although the function is unknown, antisense oligonucleotides were able to inhibit colony formation of c-kit+ Lin- bone marrow cells, suggesting an important role for MYADM in myeloid differentiation.