{"gene":"DLK2","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2011,"finding":"DLK2 interacts with itself (homodimerizes), with DLK1, and with the same extracellular region of NOTCH1 receptor as DLK1; this interaction results in inhibition of basal NOTCH1 signaling in preadipocytes and mouse embryo fibroblasts, functioning as a non-canonical inhibitory ligand.","method":"Co-immunoprecipitation, overexpression experiments, NOTCH signaling reporter assays in preadipocytes and MEFs","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional reporter assays in multiple cell lines, single lab with two orthogonal methods","pmids":["21419176"],"is_preprint":false},{"year":2011,"finding":"Overexpression of DLK2 inhibits NOTCH signaling, but this inhibition is reversed by co-overexpression of DLK1, and conversely DLK1-mediated NOTCH inhibition is reversed by DLK2 overexpression, demonstrating mutual modulation of each other's NOTCH-inhibitory activities.","method":"NOTCH signaling reporter assays with co-overexpression of DLK1 and DLK2 in preadipocytes","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional reporter assays with genetic perturbation, single lab, two conditions tested","pmids":["21419176"],"is_preprint":false},{"year":2006,"finding":"DLK2 (EGFL9) affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite manner to DLK1; enforced changes in expression of one gene affect expression levels of the other, suggesting coordinated regulation.","method":"Overexpression and knockdown in 3T3-L1 and C3H10T1/2 cell lines with adipogenesis assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with defined cellular phenotype, single lab, multiple cell lines","pmids":["17320102"],"is_preprint":false},{"year":2012,"finding":"KLF4 directly binds to the DLK2 promoter and activates its transcription in response to IBMX during early adipogenesis; KLF4 overexpression increases DLK2 expression and KLF4 knockdown reduces Dlk2 promoter activity.","method":"Chromatin immunoprecipitation (ChIP), promoter reporter assays, KLF4 overexpression and knockdown in 3T3-L1 and C3H10T1/2 cells","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ChIP binding plus functional reporter and expression assays, single lab with multiple orthogonal methods","pmids":["22306741"],"is_preprint":false},{"year":2011,"finding":"Sp1 directly binds to a proximal GC-box element in the DLK2 promoter, activates its transcription, and regulates basal DLK2 expression levels.","method":"5' RACE mapping of transcription start site, in silico promoter analysis, ChIP, promoter reporter assays in mouse cell lines","journal":"BMC molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ChIP binding confirmed with reporter assays, single lab, two orthogonal methods","pmids":["22185379"],"is_preprint":false},{"year":2014,"finding":"DLK2 inhibits NOTCH1 signaling in SK-MEL-2 metastatic melanoma cells and thereby modulates their proliferation; high levels of NOTCH inhibition by DLK proteins decrease melanoma cell proliferation, whereas lower levels increase it, both in vitro and in vivo.","method":"NOTCH reporter assays, overexpression/knockdown of DLK2 in SK-MEL-2 cells, proliferation assays in vitro and xenograft in vivo","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional reporter plus in vitro and in vivo proliferation assays, single lab with multiple methods","pmids":["25093684"],"is_preprint":false},{"year":2023,"finding":"DLK2 interacts with synapse-associated protein 1 (Syap1); this interaction activates Akt phosphorylation at Ser473 and downstream ERK1/2 and p38 signaling, promoting osteoclast formation. DLK2 deletion in osteoclasts inhibits osteoclastogenesis in vitro and produces a high-bone-mass phenotype in vivo, including in ovariectomized mice.","method":"Co-immunoprecipitation, Dlk2 conditional knockout in osteoclasts, phosphorylation assays (western blot), in vitro osteoclast differentiation assays, in vivo bone phenotype analysis","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, KO with defined cellular and in vivo phenotype, signaling pathway validation with multiple orthogonal methods in single study","pmids":["37669921"],"is_preprint":false},{"year":2024,"finding":"DLK2 overexpression in mesenchymal C3H10T1/2 cells promotes osteogenic differentiation, coinciding with increased ERK1/2 MAPK phosphorylation; this is consistent with DLK2 acting as an inhibitor of NOTCH signaling that thereby promotes osteogenesis, in contrast to DLK1 which inhibits osteogenesis.","method":"Overexpression and knockdown of DLK1/DLK2 in C3H10T1/2 cells, osteogenesis assays, western blot for ERK1/2 and p38 phosphorylation, DAPT (NOTCH inhibitor) treatment","journal":"Biological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain/loss-of-function with defined phenotype and signaling readouts, single lab with multiple methods","pmids":["39473022"],"is_preprint":false},{"year":2019,"finding":"EGFL9/DLK2 binds cMET at the cell membrane and within mitochondria, activating cMET-mediated downstream signaling; EGFL9 also interacts with COX assembly factor COA3, regulates cytochrome c oxidase (COX) activity, and promotes a Warburg-like metabolic phenotype in triple-negative breast cancer cells.","method":"Co-immunoprecipitation, co-localization (confocal microscopy), cMET signaling assays, COX activity assay, metabolic assays, knockdown and overexpression in breast cancer cell lines, in vivo metastasis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, co-localization, enzymatic activity assay, in vitro and in vivo functional validation with multiple orthogonal methods in single study","pmids":["31695034"],"is_preprint":false},{"year":2017,"finding":"Dlk2 deletion in mice increases anxiety- and depressive-like behaviors and alters gene expression of NOTCH pathway transcription factors (Hes1, Hes5, Hey1) in brain regions including the paraventricular nucleus, hippocampus, and amygdala; Dlk2-/- mice also show altered GABA-A receptor subunit (Gabra2, Gabrg2) expression and fail to respond to the anxiolytic alprazolam.","method":"Dlk2 knockout mouse behavioral assays, gene expression analysis in specific brain regions, pharmacological challenge with alprazolam","journal":"Psychoneuroendocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined behavioral and molecular phenotype, single lab, multiple brain region analyses","pmids":["28863347"],"is_preprint":false},{"year":2025,"finding":"EGFL9/DLK2 promotes hepatocellular carcinoma cell proliferation, invasion, and metastasis through activation of the EGFR/PI3K/AKT signaling pathway and amino acid metabolic reprogramming.","method":"EGFL9 knockdown and overexpression in HCC cell lines, MTT, apoptosis, wound healing, Transwell, xenograft, transcriptomics, proteomics, metabolomics, western blot, inhibitor rescue experiments","journal":"Discover oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omic plus functional validation with pathway inhibitor rescue, single lab with multiple orthogonal methods","pmids":["41003932"],"is_preprint":false}],"current_model":"DLK2 is a membrane-bound EGF-like protein that functions as a non-canonical inhibitory ligand of NOTCH1 receptor (interacting with the same extracellular domain region as canonical DSL ligands), modulates adipogenesis and osteogenesis through NOTCH signaling and MAPK pathways, promotes osteoclastogenesis via a direct interaction with Syap1 that activates Akt(Ser473)/ERK1/2/p38 signaling, and in cancer contexts (breast, hepatocellular carcinoma) binds cMET and COX assembly factor COA3 to activate PI3K/AKT signaling and rewire mitochondrial metabolism; its transcription is regulated directly by Sp1 basally and by KLF4 in response to adipogenic stimuli."},"narrative":{"mechanistic_narrative":"DLK2 (EGFL9) is a membrane-bound EGF-like protein that acts as a non-canonical inhibitory ligand of NOTCH1, binding the same NOTCH1 extracellular region as the canonical ligand DLK1 and suppressing basal NOTCH1 signaling in preadipocytes and fibroblasts [PMID:21419176]. DLK2 homodimerizes and physically associates with DLK1, and the two proteins mutually antagonize each other's NOTCH-inhibitory activity, providing a reciprocal switch that tunes signaling output [PMID:21419176]. Through this NOTCH-modulating activity DLK2 controls mesenchymal cell fate decisions, acting opposite to DLK1 to influence adipogenesis [PMID:17320102] and to promote osteogenic differentiation with concomitant ERK1/2 MAPK activation [PMID:39473022]. Its expression is set transcriptionally: Sp1 binds a proximal GC-box to drive basal transcription [PMID:22185379], while KLF4 binds the promoter to induce DLK2 during early adipogenesis [PMID:22306741]. Beyond NOTCH, DLK2 engages distinct partners to activate kinase cascades — it binds Syap1 to drive Akt(Ser473)/ERK1/2/p38 signaling and promote osteoclastogenesis, with osteoclast-specific deletion producing a high-bone-mass phenotype [PMID:37669921], and in cancer it binds cMET and the COX assembly factor COA3 to activate downstream signaling and reprogram mitochondrial metabolism toward a Warburg-like state in triple-negative breast cancer [PMID:31695034] and drives hepatocellular carcinoma progression via EGFR/PI3K/AKT signaling and amino acid metabolic rewiring [PMID:41003932]. A Dlk2 knockout mouse additionally links the gene to NOTCH-target and GABA-A receptor expression in the brain and to anxiety- and depressive-like behavior [PMID:28863347].","teleology":[{"year":2006,"claim":"Established that DLK2 is a functional regulator of mesenchymal cell fate, acting in opposition to its paralog DLK1 during adipogenesis and revealing the two genes are coordinately co-regulated.","evidence":"Overexpression and knockdown in 3T3-L1 and C3H10T1/2 cell lines with adipogenesis assays","pmids":["17320102"],"confidence":"Medium","gaps":["Did not define the molecular mechanism linking DLK2 to adipogenic outcome","Cross-regulation of DLK1/DLK2 expression not mechanistically explained"]},{"year":2011,"claim":"Defined the molecular basis of DLK2 action by showing it homodimerizes, binds DLK1, and engages the same NOTCH1 extracellular region as DLK1 to act as a non-canonical inhibitory NOTCH ligand, with the two paralogs reciprocally antagonizing each other.","evidence":"Reciprocal Co-IP and NOTCH reporter assays with DLK1/DLK2 co-overexpression in preadipocytes and MEFs","pmids":["21419176"],"confidence":"Medium","gaps":["Structural details of the DLK2-NOTCH1 interface not resolved","Whether inhibition is competitive with canonical DSL ligands not directly tested"]},{"year":2011,"claim":"Identified Sp1 as the transcription factor setting basal DLK2 expression, mapping its action to a proximal GC-box promoter element.","evidence":"5' RACE, ChIP, and promoter reporter assays in mouse cell lines","pmids":["22185379"],"confidence":"Medium","gaps":["Does not address stimulus-dependent or tissue-specific regulation"]},{"year":2012,"claim":"Connected DLK2 expression to adipogenic signaling by showing KLF4 directly binds the promoter and induces DLK2 in response to IBMX during early adipogenesis.","evidence":"ChIP, promoter reporter assays, KLF4 overexpression and knockdown in 3T3-L1 and C3H10T1/2 cells","pmids":["22306741"],"confidence":"Medium","gaps":["Functional consequence of KLF4-driven DLK2 induction on adipocyte differentiation not directly traced"]},{"year":2014,"claim":"Extended DLK2's NOTCH-inhibitory function to cancer, showing it modulates melanoma proliferation in a dose-dependent manner in vitro and in vivo.","evidence":"NOTCH reporter assays, DLK2 overexpression/knockdown in SK-MEL-2 cells, proliferation and xenograft assays","pmids":["25093684"],"confidence":"Medium","gaps":["Biphasic dependence on NOTCH inhibition level not mechanistically explained","Downstream effectors of the proliferative response not identified"]},{"year":2017,"claim":"Linked DLK2 to neural function in vivo, showing knockout alters NOTCH-target and GABA-A receptor expression in brain and produces anxiety/depressive behaviors with loss of alprazolam response.","evidence":"Dlk2 knockout mouse behavioral assays, regional gene expression analysis, pharmacological challenge","pmids":["28863347"],"confidence":"Medium","gaps":["Causal chain from DLK2 loss to GABA-A subunit changes not established","Cell-autonomous vs systemic origin of phenotype unresolved"]},{"year":2019,"claim":"Revealed a NOTCH-independent arm of DLK2 function in cancer: binding cMET and the COX assembly factor COA3 to activate signaling and drive Warburg-like metabolic reprogramming.","evidence":"Reciprocal Co-IP, confocal co-localization, COX activity and metabolic assays, in vivo metastasis assays in breast cancer cells","pmids":["31695034"],"confidence":"High","gaps":["How a single EGF-like protein localizes to both membrane and mitochondria not fully explained","Stoichiometry of the cMET/COA3 interactions unresolved"]},{"year":2023,"claim":"Identified Syap1 as a direct DLK2 partner that links the protein to Akt(Ser473)/ERK1/2/p38 activation and osteoclastogenesis, with genetic deletion establishing a causal high-bone-mass phenotype.","evidence":"Reciprocal Co-IP, osteoclast-specific conditional knockout, phospho-western blots, in vitro differentiation and in vivo bone phenotyping including ovariectomized mice","pmids":["37669921"],"confidence":"High","gaps":["Mechanism by which DLK2-Syap1 binding triggers Akt phosphorylation not defined","Relationship to DLK2's NOTCH-inhibitory role in the same lineage not reconciled"]},{"year":2024,"claim":"Showed DLK2 promotes osteogenic differentiation of mesenchymal cells via ERK1/2 MAPK and NOTCH inhibition, acting opposite to DLK1.","evidence":"DLK1/DLK2 gain/loss-of-function in C3H10T1/2 cells, osteogenesis assays, phospho-westerns, DAPT NOTCH inhibitor treatment","pmids":["39473022"],"confidence":"Medium","gaps":["Direct demonstration that NOTCH inhibition mediates the osteogenic effect remains correlative"]},{"year":2025,"claim":"Extended the cancer pro-tumor arm to hepatocellular carcinoma, implicating EGFR/PI3K/AKT signaling and amino acid metabolic reprogramming in DLK2-driven proliferation and metastasis.","evidence":"EGFL9 knockdown/overexpression in HCC lines, functional assays, xenografts, multi-omics, inhibitor rescue","pmids":["41003932"],"confidence":"Medium","gaps":["Direct receptor partner driving EGFR/PI3K/AKT activation in HCC not identified","Whether metabolic rewiring is cause or consequence of growth not resolved"]},{"year":null,"claim":"How DLK2's distinct molecular modes — NOTCH1 inhibition versus direct activation of cMET/EGFR/Syap1-driven kinase and metabolic programs — are integrated within a single cell, and the structural basis for its dual membrane/mitochondrial localization, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of any DLK2 interaction interface","Mechanism switching DLK2 between NOTCH ligand and kinase-activating partner unknown","Mitochondrial targeting mechanism uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,6,8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,6,7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,10]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8,10]}],"complexes":[],"partners":["DLK1","NOTCH1","SYAP1","MET","COA3","DLK2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6UY11","full_name":"Protein delta homolog 2","aliases":["Epidermal growth factor-like protein 9","EGF-like protein 9"],"length_aa":383,"mass_kda":40.5,"function":"Regulates adipogenesis","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q6UY11/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DLK2","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DLK2","total_profiled":1310},"omim":[{"mim_id":"621120","title":"DELTA-LIKE NONCANONICAL NOTCH LIGAND 2; DLK2","url":"https://www.omim.org/entry/621120"},{"mim_id":"600276","title":"NOTCH RECEPTOR 3; NOTCH3","url":"https://www.omim.org/entry/600276"},{"mim_id":"600275","title":"NOTCH RECEPTOR 2; NOTCH2","url":"https://www.omim.org/entry/600275"},{"mim_id":"301161","title":"SYNAPSE-ASSOCIATED PROTEIN 1; SYAP1","url":"https://www.omim.org/entry/301161"},{"mim_id":"190198","title":"NOTCH RECEPTOR 1; NOTCH1","url":"https://www.omim.org/entry/190198"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"esophagus","ntpm":11.7},{"tissue":"prostate","ntpm":12.4},{"tissue":"skin 1","ntpm":12.3}],"url":"https://www.proteinatlas.org/search/DLK2"},"hgnc":{"alias_symbol":["MGC2487"],"prev_symbol":["EGFL9"]},"alphafold":{"accession":"Q6UY11","domains":[{"cath_id":"2.10.25.10","chopping":"28-93","consensus_level":"medium","plddt":88.7402,"start":28,"end":93},{"cath_id":"2.10.25.10","chopping":"94-132","consensus_level":"medium","plddt":86.3449,"start":94,"end":132},{"cath_id":"2.10.25.10","chopping":"138-175","consensus_level":"medium","plddt":91.6124,"start":138,"end":175},{"cath_id":"2.10.25.10","chopping":"176-213","consensus_level":"medium","plddt":96.7613,"start":176,"end":213},{"cath_id":"2.10.25.10","chopping":"218-253","consensus_level":"medium","plddt":93.4053,"start":218,"end":253}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UY11","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UY11-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UY11-F1-predicted_aligned_error_v6.png","plddt_mean":73.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DLK2","jax_strain_url":"https://www.jax.org/strain/search?query=DLK2"},"sequence":{"accession":"Q6UY11","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6UY11.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6UY11/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UY11"}},"corpus_meta":[{"pmid":"21419176","id":"PMC_21419176","title":"The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities.","date":"2011","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/21419176","citation_count":86,"is_preprint":false},{"pmid":"17320102","id":"PMC_17320102","title":"The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis.","date":"2006","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17320102","citation_count":48,"is_preprint":false},{"pmid":"31695034","id":"PMC_31695034","title":"EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31695034","citation_count":40,"is_preprint":false},{"pmid":"28970845","id":"PMC_28970845","title":"Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs.","date":"2017","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/28970845","citation_count":38,"is_preprint":false},{"pmid":"25093684","id":"PMC_25093684","title":"The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells.","date":"2014","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/25093684","citation_count":34,"is_preprint":false},{"pmid":"32966595","id":"PMC_32966595","title":"DLK2 regulates arbuscule hyphal branching during arbuscular mycorrhizal symbiosis.","date":"2020","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/32966595","citation_count":25,"is_preprint":false},{"pmid":"28405524","id":"PMC_28405524","title":"Therapeutic efficacy of combined vaccination against tumor pericyte-associated antigens DLK1 and DLK2 in mice.","date":"2017","source":"Oncoimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/28405524","citation_count":22,"is_preprint":false},{"pmid":"22306741","id":"PMC_22306741","title":"DLK2 is a transcriptional target of KLF4 in the early stages of adipogenesis.","date":"2012","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22306741","citation_count":20,"is_preprint":false},{"pmid":"28863347","id":"PMC_28863347","title":"Deletion of Dlk2 increases the vulnerability to anxiety-like behaviors and impairs the anxiolytic action of alprazolam.","date":"2017","source":"Psychoneuroendocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/28863347","citation_count":11,"is_preprint":false},{"pmid":"30888503","id":"PMC_30888503","title":"Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.","date":"2019","source":"Histochemistry and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30888503","citation_count":10,"is_preprint":false},{"pmid":"37669921","id":"PMC_37669921","title":"Dlk2 interacts with Syap1 to activate Akt signaling pathway during osteoclast formation.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/37669921","citation_count":8,"is_preprint":false},{"pmid":"22185379","id":"PMC_22185379","title":"Characterization of a proximal Sp1 response element in the mouse Dlk2 gene promoter.","date":"2011","source":"BMC molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22185379","citation_count":7,"is_preprint":false},{"pmid":"39473022","id":"PMC_39473022","title":"DLK1 and DLK2, two non-canonical ligands of NOTCH receptors, differentially modulate the osteogenic differentiation of mesenchymal C3H10T1/2 cells.","date":"2024","source":"Biological research","url":"https://pubmed.ncbi.nlm.nih.gov/39473022","citation_count":5,"is_preprint":false},{"pmid":"41003932","id":"PMC_41003932","title":"EGFL9 regulates the growth and metastasis of hepatocellular carcinoma through EGFR/PI3K/AKT signaling pathway and amino acid modulation.","date":"2025","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41003932","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9107,"output_tokens":2785,"usd":0.034548,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10027,"output_tokens":3682,"usd":0.071093,"stage2_stop_reason":"end_turn"},"total_usd":0.105641,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"DLK2 interacts with itself (homodimerizes), with DLK1, and with the same extracellular region of NOTCH1 receptor as DLK1; this interaction results in inhibition of basal NOTCH1 signaling in preadipocytes and mouse embryo fibroblasts, functioning as a non-canonical inhibitory ligand.\",\n      \"method\": \"Co-immunoprecipitation, overexpression experiments, NOTCH signaling reporter assays in preadipocytes and MEFs\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional reporter assays in multiple cell lines, single lab with two orthogonal methods\",\n      \"pmids\": [\"21419176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Overexpression of DLK2 inhibits NOTCH signaling, but this inhibition is reversed by co-overexpression of DLK1, and conversely DLK1-mediated NOTCH inhibition is reversed by DLK2 overexpression, demonstrating mutual modulation of each other's NOTCH-inhibitory activities.\",\n      \"method\": \"NOTCH signaling reporter assays with co-overexpression of DLK1 and DLK2 in preadipocytes\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional reporter assays with genetic perturbation, single lab, two conditions tested\",\n      \"pmids\": [\"21419176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DLK2 (EGFL9) affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite manner to DLK1; enforced changes in expression of one gene affect expression levels of the other, suggesting coordinated regulation.\",\n      \"method\": \"Overexpression and knockdown in 3T3-L1 and C3H10T1/2 cell lines with adipogenesis assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with defined cellular phenotype, single lab, multiple cell lines\",\n      \"pmids\": [\"17320102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KLF4 directly binds to the DLK2 promoter and activates its transcription in response to IBMX during early adipogenesis; KLF4 overexpression increases DLK2 expression and KLF4 knockdown reduces Dlk2 promoter activity.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), promoter reporter assays, KLF4 overexpression and knockdown in 3T3-L1 and C3H10T1/2 cells\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP binding plus functional reporter and expression assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22306741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sp1 directly binds to a proximal GC-box element in the DLK2 promoter, activates its transcription, and regulates basal DLK2 expression levels.\",\n      \"method\": \"5' RACE mapping of transcription start site, in silico promoter analysis, ChIP, promoter reporter assays in mouse cell lines\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP binding confirmed with reporter assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"22185379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DLK2 inhibits NOTCH1 signaling in SK-MEL-2 metastatic melanoma cells and thereby modulates their proliferation; high levels of NOTCH inhibition by DLK proteins decrease melanoma cell proliferation, whereas lower levels increase it, both in vitro and in vivo.\",\n      \"method\": \"NOTCH reporter assays, overexpression/knockdown of DLK2 in SK-MEL-2 cells, proliferation assays in vitro and xenograft in vivo\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional reporter plus in vitro and in vivo proliferation assays, single lab with multiple methods\",\n      \"pmids\": [\"25093684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DLK2 interacts with synapse-associated protein 1 (Syap1); this interaction activates Akt phosphorylation at Ser473 and downstream ERK1/2 and p38 signaling, promoting osteoclast formation. DLK2 deletion in osteoclasts inhibits osteoclastogenesis in vitro and produces a high-bone-mass phenotype in vivo, including in ovariectomized mice.\",\n      \"method\": \"Co-immunoprecipitation, Dlk2 conditional knockout in osteoclasts, phosphorylation assays (western blot), in vitro osteoclast differentiation assays, in vivo bone phenotype analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, KO with defined cellular and in vivo phenotype, signaling pathway validation with multiple orthogonal methods in single study\",\n      \"pmids\": [\"37669921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DLK2 overexpression in mesenchymal C3H10T1/2 cells promotes osteogenic differentiation, coinciding with increased ERK1/2 MAPK phosphorylation; this is consistent with DLK2 acting as an inhibitor of NOTCH signaling that thereby promotes osteogenesis, in contrast to DLK1 which inhibits osteogenesis.\",\n      \"method\": \"Overexpression and knockdown of DLK1/DLK2 in C3H10T1/2 cells, osteogenesis assays, western blot for ERK1/2 and p38 phosphorylation, DAPT (NOTCH inhibitor) treatment\",\n      \"journal\": \"Biological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain/loss-of-function with defined phenotype and signaling readouts, single lab with multiple methods\",\n      \"pmids\": [\"39473022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EGFL9/DLK2 binds cMET at the cell membrane and within mitochondria, activating cMET-mediated downstream signaling; EGFL9 also interacts with COX assembly factor COA3, regulates cytochrome c oxidase (COX) activity, and promotes a Warburg-like metabolic phenotype in triple-negative breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, co-localization (confocal microscopy), cMET signaling assays, COX activity assay, metabolic assays, knockdown and overexpression in breast cancer cell lines, in vivo metastasis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, co-localization, enzymatic activity assay, in vitro and in vivo functional validation with multiple orthogonal methods in single study\",\n      \"pmids\": [\"31695034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Dlk2 deletion in mice increases anxiety- and depressive-like behaviors and alters gene expression of NOTCH pathway transcription factors (Hes1, Hes5, Hey1) in brain regions including the paraventricular nucleus, hippocampus, and amygdala; Dlk2-/- mice also show altered GABA-A receptor subunit (Gabra2, Gabrg2) expression and fail to respond to the anxiolytic alprazolam.\",\n      \"method\": \"Dlk2 knockout mouse behavioral assays, gene expression analysis in specific brain regions, pharmacological challenge with alprazolam\",\n      \"journal\": \"Psychoneuroendocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined behavioral and molecular phenotype, single lab, multiple brain region analyses\",\n      \"pmids\": [\"28863347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EGFL9/DLK2 promotes hepatocellular carcinoma cell proliferation, invasion, and metastasis through activation of the EGFR/PI3K/AKT signaling pathway and amino acid metabolic reprogramming.\",\n      \"method\": \"EGFL9 knockdown and overexpression in HCC cell lines, MTT, apoptosis, wound healing, Transwell, xenograft, transcriptomics, proteomics, metabolomics, western blot, inhibitor rescue experiments\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omic plus functional validation with pathway inhibitor rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41003932\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DLK2 is a membrane-bound EGF-like protein that functions as a non-canonical inhibitory ligand of NOTCH1 receptor (interacting with the same extracellular domain region as canonical DSL ligands), modulates adipogenesis and osteogenesis through NOTCH signaling and MAPK pathways, promotes osteoclastogenesis via a direct interaction with Syap1 that activates Akt(Ser473)/ERK1/2/p38 signaling, and in cancer contexts (breast, hepatocellular carcinoma) binds cMET and COX assembly factor COA3 to activate PI3K/AKT signaling and rewire mitochondrial metabolism; its transcription is regulated directly by Sp1 basally and by KLF4 in response to adipogenic stimuli.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DLK2 (EGFL9) is a membrane-bound EGF-like protein that acts as a non-canonical inhibitory ligand of NOTCH1, binding the same NOTCH1 extracellular region as the canonical ligand DLK1 and suppressing basal NOTCH1 signaling in preadipocytes and fibroblasts [#0]. DLK2 homodimerizes and physically associates with DLK1, and the two proteins mutually antagonize each other's NOTCH-inhibitory activity, providing a reciprocal switch that tunes signaling output [#0, #1]. Through this NOTCH-modulating activity DLK2 controls mesenchymal cell fate decisions, acting opposite to DLK1 to influence adipogenesis [#2] and to promote osteogenic differentiation with concomitant ERK1/2 MAPK activation [#7]. Its expression is set transcriptionally: Sp1 binds a proximal GC-box to drive basal transcription [#4], while KLF4 binds the promoter to induce DLK2 during early adipogenesis [#3]. Beyond NOTCH, DLK2 engages distinct partners to activate kinase cascades — it binds Syap1 to drive Akt(Ser473)/ERK1/2/p38 signaling and promote osteoclastogenesis, with osteoclast-specific deletion producing a high-bone-mass phenotype [#6], and in cancer it binds cMET and the COX assembly factor COA3 to activate downstream signaling and reprogram mitochondrial metabolism toward a Warburg-like state in triple-negative breast cancer [#8] and drives hepatocellular carcinoma progression via EGFR/PI3K/AKT signaling and amino acid metabolic rewiring [#10]. A Dlk2 knockout mouse additionally links the gene to NOTCH-target and GABA-A receptor expression in the brain and to anxiety- and depressive-like behavior [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that DLK2 is a functional regulator of mesenchymal cell fate, acting in opposition to its paralog DLK1 during adipogenesis and revealing the two genes are coordinately co-regulated.\",\n      \"evidence\": \"Overexpression and knockdown in 3T3-L1 and C3H10T1/2 cell lines with adipogenesis assays\",\n      \"pmids\": [\"17320102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the molecular mechanism linking DLK2 to adipogenic outcome\", \"Cross-regulation of DLK1/DLK2 expression not mechanistically explained\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the molecular basis of DLK2 action by showing it homodimerizes, binds DLK1, and engages the same NOTCH1 extracellular region as DLK1 to act as a non-canonical inhibitory NOTCH ligand, with the two paralogs reciprocally antagonizing each other.\",\n      \"evidence\": \"Reciprocal Co-IP and NOTCH reporter assays with DLK1/DLK2 co-overexpression in preadipocytes and MEFs\",\n      \"pmids\": [\"21419176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural details of the DLK2-NOTCH1 interface not resolved\", \"Whether inhibition is competitive with canonical DSL ligands not directly tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified Sp1 as the transcription factor setting basal DLK2 expression, mapping its action to a proximal GC-box promoter element.\",\n      \"evidence\": \"5' RACE, ChIP, and promoter reporter assays in mouse cell lines\",\n      \"pmids\": [\"22185379\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address stimulus-dependent or tissue-specific regulation\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected DLK2 expression to adipogenic signaling by showing KLF4 directly binds the promoter and induces DLK2 in response to IBMX during early adipogenesis.\",\n      \"evidence\": \"ChIP, promoter reporter assays, KLF4 overexpression and knockdown in 3T3-L1 and C3H10T1/2 cells\",\n      \"pmids\": [\"22306741\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of KLF4-driven DLK2 induction on adipocyte differentiation not directly traced\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended DLK2's NOTCH-inhibitory function to cancer, showing it modulates melanoma proliferation in a dose-dependent manner in vitro and in vivo.\",\n      \"evidence\": \"NOTCH reporter assays, DLK2 overexpression/knockdown in SK-MEL-2 cells, proliferation and xenograft assays\",\n      \"pmids\": [\"25093684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biphasic dependence on NOTCH inhibition level not mechanistically explained\", \"Downstream effectors of the proliferative response not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked DLK2 to neural function in vivo, showing knockout alters NOTCH-target and GABA-A receptor expression in brain and produces anxiety/depressive behaviors with loss of alprazolam response.\",\n      \"evidence\": \"Dlk2 knockout mouse behavioral assays, regional gene expression analysis, pharmacological challenge\",\n      \"pmids\": [\"28863347\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from DLK2 loss to GABA-A subunit changes not established\", \"Cell-autonomous vs systemic origin of phenotype unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a NOTCH-independent arm of DLK2 function in cancer: binding cMET and the COX assembly factor COA3 to activate signaling and drive Warburg-like metabolic reprogramming.\",\n      \"evidence\": \"Reciprocal Co-IP, confocal co-localization, COX activity and metabolic assays, in vivo metastasis assays in breast cancer cells\",\n      \"pmids\": [\"31695034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single EGF-like protein localizes to both membrane and mitochondria not fully explained\", \"Stoichiometry of the cMET/COA3 interactions unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified Syap1 as a direct DLK2 partner that links the protein to Akt(Ser473)/ERK1/2/p38 activation and osteoclastogenesis, with genetic deletion establishing a causal high-bone-mass phenotype.\",\n      \"evidence\": \"Reciprocal Co-IP, osteoclast-specific conditional knockout, phospho-western blots, in vitro differentiation and in vivo bone phenotyping including ovariectomized mice\",\n      \"pmids\": [\"37669921\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which DLK2-Syap1 binding triggers Akt phosphorylation not defined\", \"Relationship to DLK2's NOTCH-inhibitory role in the same lineage not reconciled\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed DLK2 promotes osteogenic differentiation of mesenchymal cells via ERK1/2 MAPK and NOTCH inhibition, acting opposite to DLK1.\",\n      \"evidence\": \"DLK1/DLK2 gain/loss-of-function in C3H10T1/2 cells, osteogenesis assays, phospho-westerns, DAPT NOTCH inhibitor treatment\",\n      \"pmids\": [\"39473022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration that NOTCH inhibition mediates the osteogenic effect remains correlative\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the cancer pro-tumor arm to hepatocellular carcinoma, implicating EGFR/PI3K/AKT signaling and amino acid metabolic reprogramming in DLK2-driven proliferation and metastasis.\",\n      \"evidence\": \"EGFL9 knockdown/overexpression in HCC lines, functional assays, xenografts, multi-omics, inhibitor rescue\",\n      \"pmids\": [\"41003932\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct receptor partner driving EGFR/PI3K/AKT activation in HCC not identified\", \"Whether metabolic rewiring is cause or consequence of growth not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DLK2's distinct molecular modes — NOTCH1 inhibition versus direct activation of cMET/EGFR/Syap1-driven kinase and metabolic programs — are integrated within a single cell, and the structural basis for its dual membrane/mitochondrial localization, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of any DLK2 interaction interface\", \"Mechanism switching DLK2 between NOTCH ligand and kinase-activating partner unknown\", \"Mitochondrial targeting mechanism uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 6, 7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DLK1\", \"NOTCH1\", \"SYAP1\", \"MET\", \"COA3\", \"DLK2\"]\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":6,"faith_pct":100.0}}