{"gene":"CNN2","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2005,"finding":"Calponin 2 (CNN2) is required for endothelial cell migration during vascular development. Morpholino knockdown in zebrafish blocked intersegmental vessel formation and endothelial cell migration. In vitro, antisense knockdown of CNN2 in HUVECs reduced bFGF-induced migration while overexpression enhanced migration and wound healing; these effects correlated with MAPK activation, and MAPK inhibition blocked the pro-migratory effect of CNN2.","method":"Antisense morpholino knockdown in zebrafish, antisense adenovirus knockdown and overexpression in HUVECs, wound healing assay, MAPK inhibitor epistasis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vivo and in vitro methods, pathway placement via MAPK inhibition, replicated in two biological systems","pmids":["16317011"],"is_preprint":false},{"year":2008,"finding":"CNN2 (h2-calponin) regulates macrophage motility and phagocytosis. Cnn2 knockout mice showed increased macrophage migration speed, reduced macrophage spreading with decreased tropomyosin in the actin cytoskeleton, and significantly increased phagocytic activity. CNN2-null macrophages also showed reduced numbers of peripheral blood neutrophils and monocytes.","method":"Cnn2 knockout mouse (conditional and constitutive), macrophage migration assay, phagocytosis assay, actin cytoskeleton analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes across multiple readouts, two independent ES cell clones","pmids":["18617524"],"is_preprint":false},{"year":2013,"finding":"Calponin 2 (Cnn2) acts as a downstream effector of noncanonical Wnt/PCP signaling in neural crest cell migration. Cnn2 is localized to protrusions at the leading edge of migratory NCCs. Cnn2 knockdown caused NCC migration defects and randomized cell protrusions. Dominant-negative Wnt11 or RhoA inhibition migration defects were rescued by Cnn2 knockdown, placing Cnn2 downstream of Wnt/PCP-RhoA in this pathway.","method":"Morpholino knockdown in Xenopus and chick, live cell imaging of protrusions, epistasis with dominant-negative Wnt11 and RhoA inhibition","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in two model organisms with multiple orthogonal methods establishing pathway position","pmids":["23499442"],"is_preprint":false},{"year":2013,"finding":"CNN2 expression is regulated by androgen receptor (AR) via serum response factor (SRF) in prostate cancer cells. siRNA-mediated loss of CNN2 did not affect cell proliferation or apoptosis but induced cellular protrusions and increased prostate cancer cell migration; changes in migration correlated with altered β1-integrin expression but did not involve epithelial-mesenchymal transition.","method":"siRNA knockdown, androgen treatment, antiandrogen treatment, SRF siRNA, ChIP, migration assay, morphology analysis","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal siRNA/rescue approach, ChIP for SRF binding, single lab with two orthogonal methods","pmids":["23576568"],"is_preprint":false},{"year":2016,"finding":"Deletion of calponin 2 in macrophages increases cell traction force in a myosin II-dependent manner. Cnn2-null fibroblasts exhibit greater root-mean-square traction and total strain energy than wild-type cells. Blebbistatin (myosin II ATPase inhibitor) was less effective at altering cell morphology in CNN2-null cells, indicating additive/cooperative effects between calponin 2 and myosin II in regulating motor activity.","method":"Cell traction force (CTF) microscopy with polyacrylamide gels and fluorescent beads, de-adhesion assay, blebbistatin pharmacological inhibition","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — CTF microscopy reconstitution approach in Cnn2 KO fibroblasts with pharmacological epistasis, multiple readouts in single study","pmids":["27733037"],"is_preprint":false},{"year":2016,"finding":"Deletion of calponin 2 in macrophages attenuates atherosclerosis. Cnn2-null macrophages and foam cells showed significantly weakened cell adhesion, faster migration, and reduced production of pro-inflammatory cytokines. Systemic or myeloid-specific Cnn2 knockout effectively reduced atherosclerotic lesion development in ApoE KO mice.","method":"Systemic and myeloid-specific Cnn2 KO mice crossed to ApoE KO background, macrophage adhesion and migration assays, cytokine measurements, histology","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional and systemic KO with defined cellular (adhesion, migration, cytokine) and in vivo (atherosclerosis lesion) phenotypes","pmids":["27575021"],"is_preprint":false},{"year":2016,"finding":"Deletion of calponin 2 in macrophages attenuates inflammatory arthritis. Cnn2-KO macrophages show increased phagocytic activity (potentially facilitating clearance of autoimmune complexes), decreased substrate adhesion, and in vitro differentiation of Cnn2-null bone marrow cells produced fewer osteoclasts with decreased bone resorption.","method":"Systemic and myeloid-specific Cnn2 KO mice, anti-GPI serum-induced arthritis model, osteoclast differentiation assay, bone resorption assay, histology","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with in vivo disease model and defined in vitro cellular mechanisms (osteoclastogenesis, adhesion, phagocytosis)","pmids":["27488671"],"is_preprint":false},{"year":2018,"finding":"Calponin 2 participates in myofibroblast differentiation, a key step in calcific aortic valve disease (CAVD). Calponin 2 expression increased during TGFβ1-induced myofibroblast differentiation. Cnn2 KO attenuated TGFβ1-induced myofibroblast differentiation (lower SMA expression and less calcification). Myeloid-specific Cnn2 KO did not reduce aortic valve calcification, indicating the relevant cell type is the valvular interstitial cell rather than macrophages.","method":"Cnn2 KO mice in ApoE KO background, primary sheep aortic valve interstitial cell culture, TGFβ1 treatment, SMA expression, calcification assay, conditional (myeloid) vs systemic KO comparison","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with in vitro mechanistic follow-up, myeloid-specific vs systemic KO comparison for cell-type specificity","pmids":["30053524"],"is_preprint":false},{"year":2022,"finding":"CNN2 translocates to damaged lysosomes along with the Arp2/3 complex upon lysosomal membrane permeabilization (LMP) and regulates actin filaments to drive phagophore formation during lysophagy. CNN2 must be ubiquitylated during this process and subsequently extracted/removed by the AAA-ATPase VCP/p97 for efficient lysophagy. The small heat shock protein HSPB1 assists VCP/p97 in the extraction of CNN2.","method":"Ubiquitylated proteome profiling upon LMP, fluorescence imaging of CNN2 translocation, Co-IP/proximity ligation for VCP/p97 and HSPB1 interactions, lysophagy flux assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ubiquitylome profiling plus functional imaging plus interaction studies establish CNN2 as a VCP/p97-HSPB1 substrate with mechanistic role in lysophagy","pmids":["35793674"],"is_preprint":false},{"year":2023,"finding":"CNN2 interacts with estrogen receptor 2 (ESR2) to negatively regulate fatty acid oxidation (FAO) in fibrotic kidneys. ESR2 binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes. CNN2 knockdown enhanced activities of key rate-limiting enzymes of FAO; inhibiting CPT1α (FAO pathway) restored lipid accumulation and ECM deposition that had been relieved by CNN2 knockdown.","method":"CNN2 knockdown mice in kidney fibrosis models (IRI and UUO), global proteomics, chromatin immunoprecipitation (ChIP) for ESR2-PPARα binding, in vitro ESR2 knockdown with FAO gene expression analysis, PPARα activation rescue experiment","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ChIP plus proteomics plus in vivo KD plus in vitro rescue with multiple orthogonal methods establishing CNN2-ESR2-PPARα-FAO pathway","pmids":["36963615"],"is_preprint":false},{"year":2023,"finding":"CNN2 interacts with ESR2 to negatively regulate mitochondrial sirtuin 5 (SIRT5) activity during acute kidney injury (AKI). Activated SIRT5 desuccinylates HMGCS2 (rate-limiting enzyme of ketogenesis), increasing ketone body β-hydroxybutyrate and ATP production to mitigate AKI. CNN2 knockdown preserved kidney function and promoted cell proliferation in AKI through this mechanism.","method":"CNN2 knockdown mice in AKI models (cisplatin, IRI), proteomics, co-immunoprecipitation of CNN2-ESR2 and ESR2-SIRT5, HMGCS2 desuccinylation assay, β-hydroxybutyrate and ATP measurement","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — co-IP interaction studies plus post-translational modification (desuccinylation) assay plus in vivo KD, multiple orthogonal methods in single study","pmids":["37751293"],"is_preprint":false},{"year":2023,"finding":"CNN2 forms a complex with YAP1 and EGR1 in colorectal cancer cells. CNN2 knockdown downregulates EGR1 expression by enhancing its ubiquitination-mediated degradation in a YAP1-dependent manner, suppressing CRC cell growth.","method":"Co-immunoprecipitation, ubiquitination assay, YAP1 siRNA rescue, in vitro loss/gain-of-function, xenograft","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP for complex, ubiquitination assay, and genetic rescue establish the CNN2-YAP1-EGR1 axis, single lab","pmids":["37188478"],"is_preprint":false},{"year":2023,"finding":"CRL3gigaxonin (CUL3-RBX1-gigaxonin E3 ubiquitin ligase) directly targets CNN2 (along with TPM1, TPM2, and TAGLN) for proteasomal degradation, controlling actin filament dynamics. Mutations in the C-terminal Kelch domain of gigaxonin (L309R, R545C, C570Y) disrupt binding to these substrates.","method":"Co-immunoprecipitation of gigaxonin with CNN2, ubiquitination assay, proteasome inhibitor experiments, mutant gigaxonin binding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mutagenesis in single study, single lab","pmids":["37903270"],"is_preprint":false},{"year":2023,"finding":"SRF downstream target gene CNN2 regulates hair cell stereocilia dimensions and cuticular plate actin organization. Hair-cell-specific Srf deletion reduced CNN2 expression. AAV-mediated exogenous delivery of CNN2 in Srf mutants partially rescued stereocilia dimension defects and F-actin intensity of the cuticular plate.","method":"Hair-cell-specific Srf KO mouse, fluorescence-activated cell sorting-based hair cell RNA-Seq, AAV-mediated CNN2 rescue, stereocilia morphometry, F-actin imaging","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO phenotype plus AAV rescue establishes CNN2 as functional SRF target in hair cells, single lab","pmids":["37982489"],"is_preprint":false},{"year":2024,"finding":"TSA-induced muscle-derived extracellular vesicles enriched with miR-873-3p promote osteogenic differentiation in hBMSCs by targeting CNN2. Downregulation of CNN2 was identified as the mechanism through which miR-873-3p in EVs promotes osteogenesis.","method":"EV isolation, miR-873-3p overexpression/inhibition, CNN2 knockdown/overexpression in hBMSCs, osteogenic differentiation assay, target validation (implied luciferase/expression assay)","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional rescue experiments linking miR-873-3p to CNN2 suppression and osteogenesis, single lab with multiple cell-based assays","pmids":["39343927"],"is_preprint":false},{"year":2024,"finding":"Loss of calponin 2 (Cnn2 KO) in mice causes premature ovarian insufficiency, with significantly fewer total ovarian follicles, presence of multi-oocyte follicles, and age-progressive earlier follicle depletion. CNN2 colocalizes with actin stress fibers, tropomyosin, and myosin II in cumulus cells of ovarian follicles.","method":"Cnn2 KO mouse breeding analysis, ovarian histology, immunofluorescence colocalization with actin/tropomyosin/myosin II in primary cumulus cell cultures","journal":"Journal of ovarian research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype with cellular localization data, single lab","pmids":["38336796"],"is_preprint":false},{"year":2025,"finding":"CNN2 (calponin-2) is a critical regulator of stress fiber organization that controls the distribution of α-actinin along actomyosin bundles, rather than acting as a negative regulator of myosin II. Loss of CNN2 reduced stress fiber thickness, increased fragility, and impaired cell migration in U2OS cells.","method":"Individual and collective depletion of CNN1/CNN2/CNN3 by siRNA/shRNA in U2OS cells, fluorescence imaging of α-actinin distribution, stress fiber morphometry, cell migration assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — isoform-specific depletion with multiple orthogonal readouts (α-actinin distribution, fiber thickness, migration) challenging prior myosin II inhibition model","pmids":["41297546"],"is_preprint":false},{"year":2025,"finding":"In NSCLC, h2-calponin (CNN2) negatively regulates RSK2 expression and downstream HSP27/CREB signaling. Downregulation of CNN2 activated RSK2/HSP27/CREB, promoting actin stress fiber reorganization and increased tumor migration and invasion; CNN2 upregulation reversed these effects. A luciferase reporter assay was used to explore how CNN2 regulates RSK2 transcription.","method":"RNA interference target screen, wound healing and Transwell migration assay, vein injection metastasis model, rhodamine-phalloidin staining of actin, luciferase reporter assay for RSK2 regulation","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple in vitro and in vivo assays with luciferase reporter for pathway placement, single lab","pmids":["40700931"],"is_preprint":false},{"year":2026,"finding":"A de novo missense variant (p.N7S) in CNN2 causes intestinal pseudo-obstruction (PIPO). Knock-in mice bearing this variant exhibited prolonged gastrointestinal transit. Primary mouse intestinal smooth muscle cells (MISMCs) with the variant showed significantly impaired contractile function in collagen gel contraction assays, in a gene dosage-dependent manner.","method":"Whole-exome sequencing of patient, CRISPR/Cas9 knock-in mouse model, carmine red intestinal transit assay, collagen gel contraction assay of primary MISMCs, Western blotting","journal":"Journal of pediatric surgery","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — patient variant identified, knock-in mouse recapitulates phenotype, and in vitro smooth muscle contractility assay provides mechanistic link with gene dosage effect","pmids":["41796654"],"is_preprint":false},{"year":2026,"finding":"SARS-CoV-2 nonspike structural proteins (particularly the Envelope protein) upregulate CNN2 in mucosal epithelial cells, inducing epithelial dedifferentiation and apoptosis. CNN2 acts as a downstream effector of this process; CNN2 downregulation inhibited epithelial cell apoptosis and promoted differentiation. CNN2 expression is negatively regulated by the transcription factor GLIS2.","method":"COVID-19 patient tissue immunostaining, SARS-CoV-2 Envelope protein overexpression, CNN2 knockdown in epithelial cells, cell differentiation and apoptosis assays, GLIS2 overexpression/knockdown","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — mechanistic pathway placement with loss-of-function rescue and upstream regulator (GLIS2) identification, single lab","pmids":["41872170"],"is_preprint":false},{"year":2016,"finding":"Calponin 2 is a mechanoregulated cytoskeletal protein; cytoskeleton tension regulates transcription of the CNN2 gene and degradation of calponin 2 protein. All three calponin isoforms inhibit actin-activated myosin ATPase and stabilize the actin cytoskeleton.","method":"Review summarizing published experimental findings on calponin mechanoregulation and ATPase inhibition","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 4 / Weak — review paper summarizing prior experimental work without new primary data; mechanistic claims are based on cited studies","pmids":["26970176"],"is_preprint":false}],"current_model":"CNN2 (calponin 2/h2-calponin) is an actin filament-associated protein that stabilizes stress fibers by controlling α-actinin distribution along actomyosin bundles, thereby regulating cell traction force, migration, adhesion, and cytokinesis; it acts downstream of Wnt/PCP-RhoA signaling in neural crest migration and downstream of SRF in hair cells and prostate cancer; it is ubiquitylated at damaged lysosomes and removed by VCP/p97-HSPB1 to enable lysophagy; it interacts with ESR2 to regulate fatty acid oxidation and ketogenesis in the kidney; it is targeted for proteasomal degradation by the CRL3-gigaxonin E3 ligase; and a de novo missense variant causes intestinal smooth muscle dysmotility, establishing CNN2 as an essential regulator of smooth muscle contractility."},"narrative":{"mechanistic_narrative":"CNN2 (calponin 2/h2-calponin) is an actin filament-associated cytoskeletal protein that organizes stress fibers and actomyosin contractility to control cell migration, adhesion, traction force, and tissue mechanics [PMID:41297546, PMID:27733037]. Mechanistically, CNN2 regulates the distribution of α-actinin along actomyosin bundles to maintain stress fiber thickness and integrity, with its loss producing thinner, more fragile fibers and impaired migration [PMID:41297546]; it cooperates with myosin II to set cell traction force [PMID:27733037] and colocalizes with actin stress fibers, tropomyosin, and myosin II [PMID:38336796]. Through this cytoskeletal control CNN2 functions as a downstream effector across multiple migratory and morphogenetic programs: it is required for endothelial cell migration during vascular development via MAPK signaling [PMID:16317011], acts downstream of noncanonical Wnt/PCP–RhoA in neural crest cell migration [PMID:23499442], and is a functional SRF target gene that shapes hair cell stereocilia and cuticular plate actin organization [PMID:37982489]. In myeloid cells CNN2 restrains macrophage migration, traction, and phagocytosis, and its deletion attenuates atherosclerosis and inflammatory arthritis [PMID:18617524, PMID:27575021, PMID:27488671]. CNN2 abundance is tightly controlled by ubiquitin-proteasome turnover: it is a substrate of the CRL3-gigaxonin E3 ligase that governs actin cytoskeleton dynamics [PMID:37903270], and upon lysosomal membrane permeabilization it is recruited with the Arp2/3 complex to damaged lysosomes, ubiquitylated, and extracted by the VCP/p97–HSPB1 machinery to enable lysophagy [PMID:35793674]. In the kidney CNN2 interacts with ESR2 to negatively regulate PPARα-driven fatty acid oxidation and SIRT5-dependent ketogenesis during fibrosis and acute injury [PMID:36963615, PMID:37751293]. A de novo missense variant (p.N7S) impairs intestinal smooth muscle contractility and causes pediatric intestinal pseudo-obstruction, establishing CNN2 as essential for smooth muscle function [PMID:41796654].","teleology":[{"year":2005,"claim":"Established CNN2 as a positive regulator of directed cell migration in a developmental context, linking it to a defined signaling output.","evidence":"Morpholino knockdown in zebrafish plus antisense knockdown/overexpression in HUVECs with MAPK inhibitor epistasis","pmids":["16317011"],"confidence":"High","gaps":["Did not resolve how CNN2 connects to MAPK biochemically","No direct cytoskeletal mechanism defined at this stage"]},{"year":2008,"claim":"Showed CNN2 restrains macrophage motility and phagocytosis through cytoskeletal stabilization, revealing a cell-type-specific dampening role.","evidence":"Cnn2 knockout mice with macrophage migration, spreading, phagocytosis, and actin/tropomyosin analyses","pmids":["18617524"],"confidence":"High","gaps":["Molecular basis for tropomyosin loss in null cells not defined","Did not connect to upstream signaling"]},{"year":2013,"claim":"Placed CNN2 as a downstream effector of Wnt/PCP-RhoA in neural crest migration and as an SRF/AR-regulated gene in prostate cancer, defining transcriptional and signaling inputs.","evidence":"Morpholino epistasis with dominant-negative Wnt11/RhoA in Xenopus and chick; SRF/AR siRNA and ChIP in prostate cancer cells","pmids":["23499442","23576568"],"confidence":"High","gaps":["How RhoA signaling controls CNN2 localization to protrusions unknown","Prostate cancer migration phenotype mechanistically tied only to β1-integrin correlation (Medium confidence)"]},{"year":2016,"claim":"Quantified CNN2's mechanical role, showing it cooperates with myosin II to set traction force, and extended its myeloid functions to atherosclerosis and arthritis.","evidence":"Cell traction force microscopy with blebbistatin epistasis in Cnn2-null fibroblasts; systemic/myeloid Cnn2 KO crossed to ApoE KO with disease models","pmids":["27733037","27575021","27488671"],"confidence":"High","gaps":["Whether CNN2 directly modulates myosin ATPase vs. organizes fibers was not resolved here","Cell-type specificity of disease protection incompletely mapped"]},{"year":2018,"claim":"Identified the valvular interstitial cell, not macrophages, as the relevant CNN2-expressing cell type in TGFβ1-driven myofibroblast differentiation and aortic valve calcification.","evidence":"Systemic vs myeloid-specific Cnn2 KO comparison plus TGFβ1-treated aortic valve interstitial cell cultures","pmids":["30053524"],"confidence":"High","gaps":["Mechanism connecting CNN2 to SMA induction and calcification not defined","No structural or biochemical detail"]},{"year":2022,"claim":"Defined CNN2 as a regulated cargo at damaged lysosomes, establishing a ubiquitin/VCP-p97-HSPB1 extraction step required for actin-driven lysophagy.","evidence":"Ubiquitylome profiling upon LMP, imaging of CNN2/Arp2/3 translocation, Co-IP and lysophagy flux assays","pmids":["35793674"],"confidence":"High","gaps":["The E3 ligase that ubiquitylates CNN2 at lysosomes not identified","Precise ubiquitylation sites and how extraction promotes phagophore formation not fully resolved"]},{"year":2023,"claim":"Revealed a non-cytoskeletal metabolic axis in which CNN2 binds ESR2 to suppress PPARα-driven fatty acid oxidation and SIRT5-dependent ketogenesis in kidney injury and fibrosis.","evidence":"CNN2 knockdown mice in IRI/UUO/AKI models, proteomics, Co-IP of CNN2-ESR2, ChIP for ESR2-PPARα, HMGCS2 desuccinylation and β-hydroxybutyrate/ATP assays","pmids":["36963615","37751293"],"confidence":"High","gaps":["How an actin-associated protein localizes to regulate nuclear/mitochondrial ESR2 function is unclear","Direct vs indirect nature of CNN2-ESR2 binding not structurally characterized"]},{"year":2023,"claim":"Showed CNN2 protein levels are controlled by the CRL3-gigaxonin E3 ligase and that CNN2 participates in tumor-promoting complexes, linking its turnover to actin dynamics and growth signaling.","evidence":"Co-IP of gigaxonin with CNN2 plus ubiquitination/proteasome assays and disease-mutant binding; Co-IP and ubiquitination assays for CNN2-YAP1-EGR1 in colorectal cancer; SRF KO/AAV rescue in hair cells","pmids":["37903270","37188478","37982489"],"confidence":"Medium","gaps":["Physiological consequence of gigaxonin-mediated CNN2 turnover in vivo not established","CNN2-YAP1-EGR1 complex tested in a single lab without reciprocal structural validation"]},{"year":2024,"claim":"Extended CNN2's roles to fertility and bone, showing it is required for follicular maintenance and is a suppressible target controlling osteogenic differentiation.","evidence":"Cnn2 KO mouse ovarian phenotyping with cytoskeletal colocalization; miR-873-3p EV targeting of CNN2 in hBMSC osteogenesis assays","pmids":["38336796","39343927"],"confidence":"Medium","gaps":["Cellular mechanism linking CNN2 loss to follicle depletion not defined","Direct miR-873-3p:CNN2 targeting inferred from rescue rather than fully validated binding"]},{"year":2025,"claim":"Reframed CNN2's core cytoskeletal mechanism as control of α-actinin distribution along stress fibers, and added a transcriptional brake on RSK2/HSP27/CREB tumor migration signaling.","evidence":"Isoform-specific CNN1/2/3 depletion in U2OS cells with α-actinin imaging and fiber morphometry; RNAi, luciferase reporter and metastasis assays in NSCLC","pmids":["41297546","40700931"],"confidence":"High","gaps":["How CNN2 physically positions α-actinin along bundles not structurally defined","NSCLC RSK2 regulation mechanism (direct vs indirect) tested in single lab (Medium confidence)"]},{"year":2026,"claim":"Provided human genetic and modeling evidence that CNN2 is essential for smooth muscle contractility, and identified an epithelial response role in SARS-CoV-2 infection.","evidence":"Whole-exome sequencing of a PIPO patient, CRISPR knock-in mouse with transit and collagen contraction assays; SARS-CoV-2 Envelope overexpression with CNN2 knockdown and GLIS2 regulation studies in epithelial cells","pmids":["41796654","41872170"],"confidence":"High","gaps":["How the p.N7S variant disrupts contractile function at the protein level is undefined","Epithelial dedifferentiation/apoptosis role (Medium confidence) tested in a single lab"]},{"year":null,"claim":"How a single actin-associated protein integrates cytoskeletal mechanics, regulated proteolysis (CRL3-gigaxonin, VCP/p97), and metabolic/transcriptional partnerships (ESR2, YAP1) into tissue-specific functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of CNN2 bound to actin, α-actinin, or ESR2","Mechanism switching between cytoskeletal and non-cytoskeletal roles unknown","How tissue context selects among partners is undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[16,4,15]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[16,4]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[16,15,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,8]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,5,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,2,13]}],"complexes":[],"partners":["ESR2","VCP","HSPB1","YAP1","EGR1","GAN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99439","full_name":"Calponin-2","aliases":["Calponin H2, smooth muscle","Neutral calponin"],"length_aa":309,"mass_kda":33.7,"function":"Thin filament-associated protein that is implicated in the regulation and modulation of smooth muscle contraction. It is capable of binding to actin, calmodulin and tropomyosin. The interaction of calponin with actin inhibits the actomyosin Mg-ATPase activity","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q99439/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNN2","classification":"Not Classified","n_dependent_lines":90,"n_total_lines":1208,"dependency_fraction":0.07450331125827815},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000064666","cell_line_id":"CID001542","localizations":[{"compartment":"cytoskeleton","grade":3},{"compartment":"cytoplasmic","grade":2},{"compartment":"membrane","grade":2},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"RAB4A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001542","total_profiled":1310},"omim":[{"mim_id":"602374","title":"CALPONIN 3; CNN3","url":"https://www.omim.org/entry/602374"},{"mim_id":"602373","title":"CALPONIN 2; CNN2","url":"https://www.omim.org/entry/602373"},{"mim_id":"600806","title":"CALPONIN 1; CNN1","url":"https://www.omim.org/entry/600806"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Actin filaments","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CNN2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q99439","domains":[{"cath_id":"1.10.418.10","chopping":"28-133","consensus_level":"high","plddt":93.3634,"start":28,"end":133}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99439","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99439-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99439-F1-predicted_aligned_error_v6.png","plddt_mean":68.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNN2","jax_strain_url":"https://www.jax.org/strain/search?query=CNN2"},"sequence":{"accession":"Q99439","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99439.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99439/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99439"}},"corpus_meta":[{"pmid":"26970176","id":"PMC_26970176","title":"Calponin isoforms CNN1, CNN2 and CNN3: 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Morpholino knockdown in zebrafish blocked intersegmental vessel formation and endothelial cell migration. In vitro, antisense knockdown of CNN2 in HUVECs reduced bFGF-induced migration while overexpression enhanced migration and wound healing; these effects correlated with MAPK activation, and MAPK inhibition blocked the pro-migratory effect of CNN2.\",\n      \"method\": \"Antisense morpholino knockdown in zebrafish, antisense adenovirus knockdown and overexpression in HUVECs, wound healing assay, MAPK inhibitor epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vivo and in vitro methods, pathway placement via MAPK inhibition, replicated in two biological systems\",\n      \"pmids\": [\"16317011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CNN2 (h2-calponin) regulates macrophage motility and phagocytosis. Cnn2 knockout mice showed increased macrophage migration speed, reduced macrophage spreading with decreased tropomyosin in the actin cytoskeleton, and significantly increased phagocytic activity. CNN2-null macrophages also showed reduced numbers of peripheral blood neutrophils and monocytes.\",\n      \"method\": \"Cnn2 knockout mouse (conditional and constitutive), macrophage migration assay, phagocytosis assay, actin cytoskeleton analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes across multiple readouts, two independent ES cell clones\",\n      \"pmids\": [\"18617524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Calponin 2 (Cnn2) acts as a downstream effector of noncanonical Wnt/PCP signaling in neural crest cell migration. Cnn2 is localized to protrusions at the leading edge of migratory NCCs. Cnn2 knockdown caused NCC migration defects and randomized cell protrusions. Dominant-negative Wnt11 or RhoA inhibition migration defects were rescued by Cnn2 knockdown, placing Cnn2 downstream of Wnt/PCP-RhoA in this pathway.\",\n      \"method\": \"Morpholino knockdown in Xenopus and chick, live cell imaging of protrusions, epistasis with dominant-negative Wnt11 and RhoA inhibition\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in two model organisms with multiple orthogonal methods establishing pathway position\",\n      \"pmids\": [\"23499442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CNN2 expression is regulated by androgen receptor (AR) via serum response factor (SRF) in prostate cancer cells. siRNA-mediated loss of CNN2 did not affect cell proliferation or apoptosis but induced cellular protrusions and increased prostate cancer cell migration; changes in migration correlated with altered β1-integrin expression but did not involve epithelial-mesenchymal transition.\",\n      \"method\": \"siRNA knockdown, androgen treatment, antiandrogen treatment, SRF siRNA, ChIP, migration assay, morphology analysis\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal siRNA/rescue approach, ChIP for SRF binding, single lab with two orthogonal methods\",\n      \"pmids\": [\"23576568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of calponin 2 in macrophages increases cell traction force in a myosin II-dependent manner. Cnn2-null fibroblasts exhibit greater root-mean-square traction and total strain energy than wild-type cells. Blebbistatin (myosin II ATPase inhibitor) was less effective at altering cell morphology in CNN2-null cells, indicating additive/cooperative effects between calponin 2 and myosin II in regulating motor activity.\",\n      \"method\": \"Cell traction force (CTF) microscopy with polyacrylamide gels and fluorescent beads, de-adhesion assay, blebbistatin pharmacological inhibition\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — CTF microscopy reconstitution approach in Cnn2 KO fibroblasts with pharmacological epistasis, multiple readouts in single study\",\n      \"pmids\": [\"27733037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of calponin 2 in macrophages attenuates atherosclerosis. Cnn2-null macrophages and foam cells showed significantly weakened cell adhesion, faster migration, and reduced production of pro-inflammatory cytokines. Systemic or myeloid-specific Cnn2 knockout effectively reduced atherosclerotic lesion development in ApoE KO mice.\",\n      \"method\": \"Systemic and myeloid-specific Cnn2 KO mice crossed to ApoE KO background, macrophage adhesion and migration assays, cytokine measurements, histology\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional and systemic KO with defined cellular (adhesion, migration, cytokine) and in vivo (atherosclerosis lesion) phenotypes\",\n      \"pmids\": [\"27575021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of calponin 2 in macrophages attenuates inflammatory arthritis. Cnn2-KO macrophages show increased phagocytic activity (potentially facilitating clearance of autoimmune complexes), decreased substrate adhesion, and in vitro differentiation of Cnn2-null bone marrow cells produced fewer osteoclasts with decreased bone resorption.\",\n      \"method\": \"Systemic and myeloid-specific Cnn2 KO mice, anti-GPI serum-induced arthritis model, osteoclast differentiation assay, bone resorption assay, histology\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with in vivo disease model and defined in vitro cellular mechanisms (osteoclastogenesis, adhesion, phagocytosis)\",\n      \"pmids\": [\"27488671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Calponin 2 participates in myofibroblast differentiation, a key step in calcific aortic valve disease (CAVD). Calponin 2 expression increased during TGFβ1-induced myofibroblast differentiation. Cnn2 KO attenuated TGFβ1-induced myofibroblast differentiation (lower SMA expression and less calcification). Myeloid-specific Cnn2 KO did not reduce aortic valve calcification, indicating the relevant cell type is the valvular interstitial cell rather than macrophages.\",\n      \"method\": \"Cnn2 KO mice in ApoE KO background, primary sheep aortic valve interstitial cell culture, TGFβ1 treatment, SMA expression, calcification assay, conditional (myeloid) vs systemic KO comparison\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with in vitro mechanistic follow-up, myeloid-specific vs systemic KO comparison for cell-type specificity\",\n      \"pmids\": [\"30053524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CNN2 translocates to damaged lysosomes along with the Arp2/3 complex upon lysosomal membrane permeabilization (LMP) and regulates actin filaments to drive phagophore formation during lysophagy. CNN2 must be ubiquitylated during this process and subsequently extracted/removed by the AAA-ATPase VCP/p97 for efficient lysophagy. The small heat shock protein HSPB1 assists VCP/p97 in the extraction of CNN2.\",\n      \"method\": \"Ubiquitylated proteome profiling upon LMP, fluorescence imaging of CNN2 translocation, Co-IP/proximity ligation for VCP/p97 and HSPB1 interactions, lysophagy flux assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ubiquitylome profiling plus functional imaging plus interaction studies establish CNN2 as a VCP/p97-HSPB1 substrate with mechanistic role in lysophagy\",\n      \"pmids\": [\"35793674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNN2 interacts with estrogen receptor 2 (ESR2) to negatively regulate fatty acid oxidation (FAO) in fibrotic kidneys. ESR2 binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes. CNN2 knockdown enhanced activities of key rate-limiting enzymes of FAO; inhibiting CPT1α (FAO pathway) restored lipid accumulation and ECM deposition that had been relieved by CNN2 knockdown.\",\n      \"method\": \"CNN2 knockdown mice in kidney fibrosis models (IRI and UUO), global proteomics, chromatin immunoprecipitation (ChIP) for ESR2-PPARα binding, in vitro ESR2 knockdown with FAO gene expression analysis, PPARα activation rescue experiment\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ChIP plus proteomics plus in vivo KD plus in vitro rescue with multiple orthogonal methods establishing CNN2-ESR2-PPARα-FAO pathway\",\n      \"pmids\": [\"36963615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNN2 interacts with ESR2 to negatively regulate mitochondrial sirtuin 5 (SIRT5) activity during acute kidney injury (AKI). Activated SIRT5 desuccinylates HMGCS2 (rate-limiting enzyme of ketogenesis), increasing ketone body β-hydroxybutyrate and ATP production to mitigate AKI. CNN2 knockdown preserved kidney function and promoted cell proliferation in AKI through this mechanism.\",\n      \"method\": \"CNN2 knockdown mice in AKI models (cisplatin, IRI), proteomics, co-immunoprecipitation of CNN2-ESR2 and ESR2-SIRT5, HMGCS2 desuccinylation assay, β-hydroxybutyrate and ATP measurement\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — co-IP interaction studies plus post-translational modification (desuccinylation) assay plus in vivo KD, multiple orthogonal methods in single study\",\n      \"pmids\": [\"37751293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNN2 forms a complex with YAP1 and EGR1 in colorectal cancer cells. CNN2 knockdown downregulates EGR1 expression by enhancing its ubiquitination-mediated degradation in a YAP1-dependent manner, suppressing CRC cell growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, YAP1 siRNA rescue, in vitro loss/gain-of-function, xenograft\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP for complex, ubiquitination assay, and genetic rescue establish the CNN2-YAP1-EGR1 axis, single lab\",\n      \"pmids\": [\"37188478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CRL3gigaxonin (CUL3-RBX1-gigaxonin E3 ubiquitin ligase) directly targets CNN2 (along with TPM1, TPM2, and TAGLN) for proteasomal degradation, controlling actin filament dynamics. Mutations in the C-terminal Kelch domain of gigaxonin (L309R, R545C, C570Y) disrupt binding to these substrates.\",\n      \"method\": \"Co-immunoprecipitation of gigaxonin with CNN2, ubiquitination assay, proteasome inhibitor experiments, mutant gigaxonin binding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus mutagenesis in single study, single lab\",\n      \"pmids\": [\"37903270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SRF downstream target gene CNN2 regulates hair cell stereocilia dimensions and cuticular plate actin organization. Hair-cell-specific Srf deletion reduced CNN2 expression. AAV-mediated exogenous delivery of CNN2 in Srf mutants partially rescued stereocilia dimension defects and F-actin intensity of the cuticular plate.\",\n      \"method\": \"Hair-cell-specific Srf KO mouse, fluorescence-activated cell sorting-based hair cell RNA-Seq, AAV-mediated CNN2 rescue, stereocilia morphometry, F-actin imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO phenotype plus AAV rescue establishes CNN2 as functional SRF target in hair cells, single lab\",\n      \"pmids\": [\"37982489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TSA-induced muscle-derived extracellular vesicles enriched with miR-873-3p promote osteogenic differentiation in hBMSCs by targeting CNN2. Downregulation of CNN2 was identified as the mechanism through which miR-873-3p in EVs promotes osteogenesis.\",\n      \"method\": \"EV isolation, miR-873-3p overexpression/inhibition, CNN2 knockdown/overexpression in hBMSCs, osteogenic differentiation assay, target validation (implied luciferase/expression assay)\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional rescue experiments linking miR-873-3p to CNN2 suppression and osteogenesis, single lab with multiple cell-based assays\",\n      \"pmids\": [\"39343927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Loss of calponin 2 (Cnn2 KO) in mice causes premature ovarian insufficiency, with significantly fewer total ovarian follicles, presence of multi-oocyte follicles, and age-progressive earlier follicle depletion. CNN2 colocalizes with actin stress fibers, tropomyosin, and myosin II in cumulus cells of ovarian follicles.\",\n      \"method\": \"Cnn2 KO mouse breeding analysis, ovarian histology, immunofluorescence colocalization with actin/tropomyosin/myosin II in primary cumulus cell cultures\",\n      \"journal\": \"Journal of ovarian research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype with cellular localization data, single lab\",\n      \"pmids\": [\"38336796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CNN2 (calponin-2) is a critical regulator of stress fiber organization that controls the distribution of α-actinin along actomyosin bundles, rather than acting as a negative regulator of myosin II. Loss of CNN2 reduced stress fiber thickness, increased fragility, and impaired cell migration in U2OS cells.\",\n      \"method\": \"Individual and collective depletion of CNN1/CNN2/CNN3 by siRNA/shRNA in U2OS cells, fluorescence imaging of α-actinin distribution, stress fiber morphometry, cell migration assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific depletion with multiple orthogonal readouts (α-actinin distribution, fiber thickness, migration) challenging prior myosin II inhibition model\",\n      \"pmids\": [\"41297546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In NSCLC, h2-calponin (CNN2) negatively regulates RSK2 expression and downstream HSP27/CREB signaling. Downregulation of CNN2 activated RSK2/HSP27/CREB, promoting actin stress fiber reorganization and increased tumor migration and invasion; CNN2 upregulation reversed these effects. A luciferase reporter assay was used to explore how CNN2 regulates RSK2 transcription.\",\n      \"method\": \"RNA interference target screen, wound healing and Transwell migration assay, vein injection metastasis model, rhodamine-phalloidin staining of actin, luciferase reporter assay for RSK2 regulation\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple in vitro and in vivo assays with luciferase reporter for pathway placement, single lab\",\n      \"pmids\": [\"40700931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A de novo missense variant (p.N7S) in CNN2 causes intestinal pseudo-obstruction (PIPO). Knock-in mice bearing this variant exhibited prolonged gastrointestinal transit. Primary mouse intestinal smooth muscle cells (MISMCs) with the variant showed significantly impaired contractile function in collagen gel contraction assays, in a gene dosage-dependent manner.\",\n      \"method\": \"Whole-exome sequencing of patient, CRISPR/Cas9 knock-in mouse model, carmine red intestinal transit assay, collagen gel contraction assay of primary MISMCs, Western blotting\",\n      \"journal\": \"Journal of pediatric surgery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — patient variant identified, knock-in mouse recapitulates phenotype, and in vitro smooth muscle contractility assay provides mechanistic link with gene dosage effect\",\n      \"pmids\": [\"41796654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SARS-CoV-2 nonspike structural proteins (particularly the Envelope protein) upregulate CNN2 in mucosal epithelial cells, inducing epithelial dedifferentiation and apoptosis. CNN2 acts as a downstream effector of this process; CNN2 downregulation inhibited epithelial cell apoptosis and promoted differentiation. CNN2 expression is negatively regulated by the transcription factor GLIS2.\",\n      \"method\": \"COVID-19 patient tissue immunostaining, SARS-CoV-2 Envelope protein overexpression, CNN2 knockdown in epithelial cells, cell differentiation and apoptosis assays, GLIS2 overexpression/knockdown\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — mechanistic pathway placement with loss-of-function rescue and upstream regulator (GLIS2) identification, single lab\",\n      \"pmids\": [\"41872170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Calponin 2 is a mechanoregulated cytoskeletal protein; cytoskeleton tension regulates transcription of the CNN2 gene and degradation of calponin 2 protein. All three calponin isoforms inhibit actin-activated myosin ATPase and stabilize the actin cytoskeleton.\",\n      \"method\": \"Review summarizing published experimental findings on calponin mechanoregulation and ATPase inhibition\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — review paper summarizing prior experimental work without new primary data; mechanistic claims are based on cited studies\",\n      \"pmids\": [\"26970176\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNN2 (calponin 2/h2-calponin) is an actin filament-associated protein that stabilizes stress fibers by controlling α-actinin distribution along actomyosin bundles, thereby regulating cell traction force, migration, adhesion, and cytokinesis; it acts downstream of Wnt/PCP-RhoA signaling in neural crest migration and downstream of SRF in hair cells and prostate cancer; it is ubiquitylated at damaged lysosomes and removed by VCP/p97-HSPB1 to enable lysophagy; it interacts with ESR2 to regulate fatty acid oxidation and ketogenesis in the kidney; it is targeted for proteasomal degradation by the CRL3-gigaxonin E3 ligase; and a de novo missense variant causes intestinal smooth muscle dysmotility, establishing CNN2 as an essential regulator of smooth muscle contractility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNN2 (calponin 2/h2-calponin) is an actin filament-associated cytoskeletal protein that organizes stress fibers and actomyosin contractility to control cell migration, adhesion, traction force, and tissue mechanics [#16, #4]. Mechanistically, CNN2 regulates the distribution of \\u03b1-actinin along actomyosin bundles to maintain stress fiber thickness and integrity, with its loss producing thinner, more fragile fibers and impaired migration [#16]; it cooperates with myosin II to set cell traction force [#4] and colocalizes with actin stress fibers, tropomyosin, and myosin II [#15]. Through this cytoskeletal control CNN2 functions as a downstream effector across multiple migratory and morphogenetic programs: it is required for endothelial cell migration during vascular development via MAPK signaling [#0], acts downstream of noncanonical Wnt/PCP\\u2013RhoA in neural crest cell migration [#2], and is a functional SRF target gene that shapes hair cell stereocilia and cuticular plate actin organization [#13]. In myeloid cells CNN2 restrains macrophage migration, traction, and phagocytosis, and its deletion attenuates atherosclerosis and inflammatory arthritis [#1, #5, #6]. CNN2 abundance is tightly controlled by ubiquitin-proteasome turnover: it is a substrate of the CRL3-gigaxonin E3 ligase that governs actin cytoskeleton dynamics [#12], and upon lysosomal membrane permeabilization it is recruited with the Arp2/3 complex to damaged lysosomes, ubiquitylated, and extracted by the VCP/p97\\u2013HSPB1 machinery to enable lysophagy [#8]. In the kidney CNN2 interacts with ESR2 to negatively regulate PPAR\\u03b1-driven fatty acid oxidation and SIRT5-dependent ketogenesis during fibrosis and acute injury [#9, #10]. A de novo missense variant (p.N7S) impairs intestinal smooth muscle contractility and causes pediatric intestinal pseudo-obstruction, establishing CNN2 as essential for smooth muscle function [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established CNN2 as a positive regulator of directed cell migration in a developmental context, linking it to a defined signaling output.\",\n      \"evidence\": \"Morpholino knockdown in zebrafish plus antisense knockdown/overexpression in HUVECs with MAPK inhibitor epistasis\",\n      \"pmids\": [\"16317011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how CNN2 connects to MAPK biochemically\", \"No direct cytoskeletal mechanism defined at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed CNN2 restrains macrophage motility and phagocytosis through cytoskeletal stabilization, revealing a cell-type-specific dampening role.\",\n      \"evidence\": \"Cnn2 knockout mice with macrophage migration, spreading, phagocytosis, and actin/tropomyosin analyses\",\n      \"pmids\": [\"18617524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for tropomyosin loss in null cells not defined\", \"Did not connect to upstream signaling\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed CNN2 as a downstream effector of Wnt/PCP-RhoA in neural crest migration and as an SRF/AR-regulated gene in prostate cancer, defining transcriptional and signaling inputs.\",\n      \"evidence\": \"Morpholino epistasis with dominant-negative Wnt11/RhoA in Xenopus and chick; SRF/AR siRNA and ChIP in prostate cancer cells\",\n      \"pmids\": [\"23499442\", \"23576568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RhoA signaling controls CNN2 localization to protrusions unknown\", \"Prostate cancer migration phenotype mechanistically tied only to \\u03b21-integrin correlation (Medium confidence)\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantified CNN2's mechanical role, showing it cooperates with myosin II to set traction force, and extended its myeloid functions to atherosclerosis and arthritis.\",\n      \"evidence\": \"Cell traction force microscopy with blebbistatin epistasis in Cnn2-null fibroblasts; systemic/myeloid Cnn2 KO crossed to ApoE KO with disease models\",\n      \"pmids\": [\"27733037\", \"27575021\", \"27488671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CNN2 directly modulates myosin ATPase vs. organizes fibers was not resolved here\", \"Cell-type specificity of disease protection incompletely mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the valvular interstitial cell, not macrophages, as the relevant CNN2-expressing cell type in TGF\\u03b21-driven myofibroblast differentiation and aortic valve calcification.\",\n      \"evidence\": \"Systemic vs myeloid-specific Cnn2 KO comparison plus TGF\\u03b21-treated aortic valve interstitial cell cultures\",\n      \"pmids\": [\"30053524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting CNN2 to SMA induction and calcification not defined\", \"No structural or biochemical detail\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined CNN2 as a regulated cargo at damaged lysosomes, establishing a ubiquitin/VCP-p97-HSPB1 extraction step required for actin-driven lysophagy.\",\n      \"evidence\": \"Ubiquitylome profiling upon LMP, imaging of CNN2/Arp2/3 translocation, Co-IP and lysophagy flux assays\",\n      \"pmids\": [\"35793674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligase that ubiquitylates CNN2 at lysosomes not identified\", \"Precise ubiquitylation sites and how extraction promotes phagophore formation not fully resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a non-cytoskeletal metabolic axis in which CNN2 binds ESR2 to suppress PPAR\\u03b1-driven fatty acid oxidation and SIRT5-dependent ketogenesis in kidney injury and fibrosis.\",\n      \"evidence\": \"CNN2 knockdown mice in IRI/UUO/AKI models, proteomics, Co-IP of CNN2-ESR2, ChIP for ESR2-PPAR\\u03b1, HMGCS2 desuccinylation and \\u03b2-hydroxybutyrate/ATP assays\",\n      \"pmids\": [\"36963615\", \"37751293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How an actin-associated protein localizes to regulate nuclear/mitochondrial ESR2 function is unclear\", \"Direct vs indirect nature of CNN2-ESR2 binding not structurally characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed CNN2 protein levels are controlled by the CRL3-gigaxonin E3 ligase and that CNN2 participates in tumor-promoting complexes, linking its turnover to actin dynamics and growth signaling.\",\n      \"evidence\": \"Co-IP of gigaxonin with CNN2 plus ubiquitination/proteasome assays and disease-mutant binding; Co-IP and ubiquitination assays for CNN2-YAP1-EGR1 in colorectal cancer; SRF KO/AAV rescue in hair cells\",\n      \"pmids\": [\"37903270\", \"37188478\", \"37982489\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequence of gigaxonin-mediated CNN2 turnover in vivo not established\", \"CNN2-YAP1-EGR1 complex tested in a single lab without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended CNN2's roles to fertility and bone, showing it is required for follicular maintenance and is a suppressible target controlling osteogenic differentiation.\",\n      \"evidence\": \"Cnn2 KO mouse ovarian phenotyping with cytoskeletal colocalization; miR-873-3p EV targeting of CNN2 in hBMSC osteogenesis assays\",\n      \"pmids\": [\"38336796\", \"39343927\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular mechanism linking CNN2 loss to follicle depletion not defined\", \"Direct miR-873-3p:CNN2 targeting inferred from rescue rather than fully validated binding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reframed CNN2's core cytoskeletal mechanism as control of \\u03b1-actinin distribution along stress fibers, and added a transcriptional brake on RSK2/HSP27/CREB tumor migration signaling.\",\n      \"evidence\": \"Isoform-specific CNN1/2/3 depletion in U2OS cells with \\u03b1-actinin imaging and fiber morphometry; RNAi, luciferase reporter and metastasis assays in NSCLC\",\n      \"pmids\": [\"41297546\", \"40700931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CNN2 physically positions \\u03b1-actinin along bundles not structurally defined\", \"NSCLC RSK2 regulation mechanism (direct vs indirect) tested in single lab (Medium confidence)\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided human genetic and modeling evidence that CNN2 is essential for smooth muscle contractility, and identified an epithelial response role in SARS-CoV-2 infection.\",\n      \"evidence\": \"Whole-exome sequencing of a PIPO patient, CRISPR knock-in mouse with transit and collagen contraction assays; SARS-CoV-2 Envelope overexpression with CNN2 knockdown and GLIS2 regulation studies in epithelial cells\",\n      \"pmids\": [\"41796654\", \"41872170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the p.N7S variant disrupts contractile function at the protein level is undefined\", \"Epithelial dedifferentiation/apoptosis role (Medium confidence) tested in a single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single actin-associated protein integrates cytoskeletal mechanics, regulated proteolysis (CRL3-gigaxonin, VCP/p97), and metabolic/transcriptional partnerships (ESR2, YAP1) into tissue-specific functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of CNN2 bound to actin, \\u03b1-actinin, or ESR2\", \"Mechanism switching between cytoskeletal and non-cytoskeletal roles unknown\", \"How tissue context selects among partners is undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [16, 4, 15]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [16, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [16, 15, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 5, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 2, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ESR2\", \"VCP\", \"HSPB1\", \"YAP1\", \"EGR1\", \"GAN\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}