Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

Obesity is a prevalent global health challenge linked to type 2 diabetes, cardiovascular disease, cancers, and increased disability-adjusted life years. It arises from energy imbalance and involves adipose tissue expansion through adipocyte hypertrophy and hyperplasia, with adipogenesis (differentiation of preadipocytes into mature adipocytes) playing a central role. Adipogenesis is orchestrated by transcription factors including C/EBPβ/δ, PPARγ, and C/EBPα, and is promoted by BMP4 signaling through SMAD proteins, particularly SMAD4, to induce PPARγ and terminal differentiation. Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein with anti-inflammatory functions; it is elevated in adipose tissue in obesity and recombinant ANXA1 reduces body weight in mice. However, most prior work focused on anti-inflammatory effects and not adipogenesis, and the mechanism in adipose progenitors remains unclear. PDLIM7 is a PDZ-LIM protein implicated in NF-κB regulation and Hippo signaling, and MYCBP2 is an E3 ubiquitin ligase important in neural development. Their roles in obesity were unknown. This study investigates whether ANXA1 regulates adipogenesis and obesity by modulating SMAD4 via interactions with PDLIM7 and MYCBP2, and evaluates whether ANXA1 or its peptide Ac2-26 can serve as therapeutic modulators to prevent obesity and metabolic disorders.

Human subjects: Subcutaneous adipose tissue (SAT) samples from lean (n=5) and obese (n=5) individuals undergoing scar surgery were collected under approved protocols with informed consent for RNA/protein analyses. Public datasets of monozygotic twins discordant for BMI (E-MEXP-1425, GSE152991) and a cohort of 45 individuals were analyzed for ANXA1 expression. Animals and in vivo studies: Male C57BL/6 wild-type (WT), Anxa1 knockout (Anxa1−/−), adipose-specific Anxa1 knockout (Anxa1fl/fl crossed with Adipoq-Cre; Anxa1ΔKO), and Anxa1 transgenic (Anxa1Tg/Δ79) mice were used. Mice were fed high-fat diet (HFD; D12492) for 10–18 weeks beginning at 8 weeks of age. Metabolic phenotyping included body weight tracking, indirect calorimetry (VO2, VCO2, energy expenditure) in metabolic chambers, glucose tolerance tests (GTT), insulin tolerance tests (ITT), plasma lipids (TC, TG, HDL-C, LDL-C), NEFA, and glucose assays. Body composition was assessed by NMR/EchoMRI. Tissues (SAT, visceral adipose tissue [VAT], brown adipose tissue [BAT], liver) were analyzed by western blot, qPCR, histology (H&E), and hepatic triglyceride content. Interventions: Ac2-26 peptide (0, 0.5, 1.0, 2.0 mg/kg i.p. every other day for 10 weeks) was administered to db/db mice on HFD; PPARγ antagonist GW9662 (1 mg/kg i.p. every other day for 16 weeks) was given to HFD-fed Anxa1ΔKO mice. Cell isolation and culture: Stromal vascular fractions (SVFs) from mouse WAT were isolated by collagenase digestion and cultured in DMEM/F12 with 10% FBS. Adipogenic induction used insulin, dexamethasone, IBMX, and indomethacin. Differentiation was evaluated by Oil Red O staining and expression of adipogenic markers (Pparg, Fabp4, Adipoq, Cebpa, Cidec, Plin1). 3T3-L1 references were used for context. Pharmacological modulation employed GW9662 and rosiglitazone. Molecular assays: Protein and mRNA levels were measured by western blot and qRT-PCR. Flow cytometry and immunofluorescence assessed ANXA1 and CD105 in SVFs; localization of ANXA1 and interactions of FITC-Ac2-26 with PDLIM7 were evaluated by ICC. Co-immunoprecipitation (co-IP) and mass spectrometry (IP-MS; OMIX006798) identified ANXA1- and PDLIM7-interacting proteins. Ubiquitination assays assessed total, K48- and K63-linked polyubiquitination of SMAD4; proteasome inhibition used MG132 (10 μM, 6 h). ChIP-PCR/qPCR tested SMAD4 binding to the Pparg promoter. Genetic perturbations: siRNA-mediated knockdown of ANXA1, PDLIM7, MYCBP2, SMAD4; adenoviral-mediated silencing (Ad-SMAD4, Ad-MYCBP2) and lentiviral overexpression (LV-SMAD4, LV-PDLIM7) in SVFs (MOI≈400). Peptide mapping used ANXA1 and PDLIM7 domain fragments (pfam segments) and a MYCBP2 RING-FLAG construct to define interaction interfaces. Predicted structures from UniProt/AlphaFold were used for interaction simulations in PyMOL. Statistics: Data are mean ± SEM; tests included Student’s t test, one-way ANOVA with Dunn’s post hoc, Mann–Whitney U, Fisher’s exact/chi-square as appropriate. p<0.05 considered significant.

- ANXA1 expression is elevated in SAT of individuals with obesity and in HFD-fed mouse SAT (protein and mRNA), with no significant change in VAT or BAT. Public twin datasets (n=133 and n=49 pairs) show higher ANXA1 mRNA in higher-BMI co-twins; a cohort of 45 subjects shows elevated SAT ANXA1 in obese vs lean. - Whole-body Anxa1 knockout (KO) mice on HFD exhibit significantly greater weight gain than WT, reduced VO2/VCO2/energy expenditure, glucose intolerance, insulin resistance, and elevated plasma TC, TG, NEFA, HDL-C, LDL-C. Increased fat mass/lean mass ratio and increased subcutaneous, perirenal, and gonadal fat weights and hepatic TG indicate worsened metabolic disorder. - Adipose-specific Anxa1ΔKO mice on HFD show increased body weight, reduced VO2/VCO2/EE, greater fat depot weights, higher fat mass ratio, enlarged adipocytes, elevated plasma TC/TG/LDL-C and insulin, and impaired GTT/ITT compared to controls, establishing ANXA1’s protective role in adipose tissue. - Anxa1 transgenic (overexpression) mice resist HFD-induced obesity: lower body weight gain, higher VO2/VCO2/EE, reduced fat depot weights and adipocyte size, lower plasma TG and insulin, and improved GTT/ITT versus WT. - In SVFs, ANXA1 expression declines during adipogenic differentiation. Overexpression of ANXA1 reduces adipocyte differentiation and adipogenic gene expression; ANXA1 KO or knockdown increases PPARγ protein and adipogenic markers (Pparg, Fabp4, Adipoq, Cebpa, Cidec, Plin1) and promotes adipogenesis. - ANXA1 regulates SMAD4 post-transcriptionally: ANXA1 deficiency increases SMAD4 protein without changing Smad4 mRNA; MG132 rescues differences, indicating proteasomal degradation. ANXA1 promotes SMAD4 ubiquitination specifically via K48-linked chains; K63-ubiquitination not altered. SMAD4 knockdown reduces PPARγ and adipogenesis; SMAD4 overexpression increases them. ChIP shows SMAD4 binds the Pparg promoter; ANXA1 overexpression reduces SMAD4 binding; ANXA1 loss enhances it. - Mechanism: ANXA1 binds PDLIM7 (validated by co-IP/co-IF; domain mapping ANXA1-pfam1 with PDLIM7-pfam4). PDLIM7 stabilizes SMAD4 by inhibiting its K48-linked ubiquitination; PDLIM7 knockdown decreases SMAD4 and PPARγ, reducing adipogenesis; PDLIM7 overexpression increases SMAD4 and adipogenesis. IP-MS identified MYCBP2 as a strong PDLIM7 interactor; MYCBP2 directly binds SMAD4 and mediates its K48-linked ubiquitination and degradation. MYCBP2 knockdown increases SMAD4 and PPARγ and adipogenesis. - Competitive interactions: High ANXA1 enhances ANXA1–PDLIM7 binding, weakens PDLIM7–MYCBP2 interaction, exposing MYCBP2-binding site for SMAD4 and increasing SMAD4 ubiquitination/degradation, thereby lowering SMAD4 protein, reducing PPARγ transcription and adipogenesis. Perturbation experiments (siRNA combinations and MYCBP2 RING domain competition) support this pathway in SVFs, mature adipocytes, and H5V endothelial cells. - Therapeutic peptide: ANXA1-derived Ac2-26 (0.1 mg/mL) reduces SMAD4 protein, downregulates adipogenic genes, and inhibits SVF differentiation in vitro; FITC-Ac2-26 co-localizes with PDLIM7. In db/db mice on HFD, Ac2-26 (2 mg/kg i.p. every other day for 10 weeks) lowers blood glucose, TC, and TG versus vehicle; body weight and GTT/ITT not significantly changed. PPARγ antagonist GW9662 improves obesity and glucose/insulin tolerance in HFD-fed Anxa1ΔKO mice, consistent with ANXA1 acting upstream of PPARγ. - Overall, ANXA1 restrains adipogenesis and protects against HFD-induced obesity by promoting MYCBP2-mediated K48-linked ubiquitination and proteasomal degradation of SMAD4 via competitive binding to PDLIM7, ultimately suppressing PPARγ transcription.

This study addresses how ANXA1 influences adipose tissue biology beyond its known anti-inflammatory roles. The data reveal that ANXA1 acts within adipose stromal vascular fractions to suppress the adipogenic program by reducing SMAD4 protein levels through enhanced K48-linked ubiquitination and proteasomal degradation. Mechanistically, ANXA1 competes with MYCBP2 for binding to PDLIM7, decreasing PDLIM7–MYCBP2 association and facilitating MYCBP2 accessibility to SMAD4. Lower SMAD4 diminishes its binding to the Pparg promoter, reducing PPARγ transcription and adipocyte differentiation. In vivo, loss of ANXA1 (global or adipose-specific) exacerbates HFD-induced obesity and metabolic dysfunction, whereas ANXA1 overexpression protects by increasing energy expenditure and limiting fat mass expansion. The ANXA1 peptide Ac2-26 mimics the molecular and anti-adipogenic effects and improves circulating metabolic parameters in diabetic-obese mice, suggesting translational potential. These findings link ANXA1–PDLIM7–MYCBP2–SMAD4 protein–protein interactions to adipogenesis control, providing a mechanistic framework for targeting adipocyte formation to combat obesity.

ANXA1 is a critical negative regulator of adipogenesis and obesity. It binds PDLIM7 to modulate MYCBP2-mediated K48-linked ubiquitination and degradation of SMAD4, thereby reducing PPARγ transcription and limiting adipocyte differentiation. Genetic loss of ANXA1 aggravates HFD-induced obesity and insulin resistance, while ANXA1 overexpression confers protection. The ANXA1-derived peptide Ac2-26 reproduces key anti-adipogenic and metabolic benefits, highlighting a potential therapeutic avenue. Future research should refine targeting of the ANXA1–PDLIM7–MYCBP2–SMAD4 axis, evaluate long-term efficacy and safety of Ac2-26 or small molecules that modulate these PPIs, explore tissue- and cell-type specificity (including vesicle-mediated delivery), and delineate additional ubiquitination patterns on SMAD4 and related pathway components.

- The paradox of elevated ANXA1 levels in obese SAT likely reflects cell composition changes and compensatory responses, but causality and cell-type contributions require deeper single-cell and lineage-tracing analyses. - While K48-linked ubiquitination of SMAD4 is implicated, other ubiquitin linkages or ubiquitination sites on SMAD4 may contribute; comprehensive mapping is needed. - Some affiliations of molecular effects beyond SVFs were examined in mature adipocytes and H5V cells, but broader tissue-specificity and in vivo cell autonomy need further validation. - Ac2-26 improved circulating glucose and lipids in db/db mice without significant effects on body weight or GTT/ITT within the tested regimen; dose, duration, and pharmacokinetics/target engagement require optimization. - The study primarily uses mouse models; human functional validation in primary human adipose progenitors and clinical translation remain to be established.