A STAT5-Smad3 dyad regulates adipogenic plasticity of visceral adipose mesenchymal stromal cells during chronic inflammation

The study investigates how chronic inflammatory conditions suppress neoadipogenesis from visceral adipose mesenchymal stromal cells (VA-MSCs), a process necessary for healthy adipose tissue expansion and metabolic homeostasis. While terminal adipogenesis has been extensively studied in committed preadipocytes and cell lines, the regulation of multipotent VA-MSC plasticity under physiologically relevant chronic inflammation remains unclear. The authors hypothesize that specific inflammatory cytokines converge on conserved signaling pathways to inhibit early adipogenic commitment in VA-MSCs. Identifying these pathways could reveal druggable targets to restore adipogenesis in metabolic dysfunction and wasting syndromes associated with chronic inflammation.

• Human VA-MSC isolation and characterization: Stromal vascular fraction from intra-abdominal visceral fat of consenting donors was cultured ex vivo with human platelet lysate. MSC identity was confirmed by flow cytometry showing CD105+CD73+CD90+CD45−CD34−CD11b−HLA-DR− phenotype. Cells at passages 2–6 were used.
• Murine VA-MSC model: MSCs isolated from epididymal fat pads (SVF) of adult C57BL/6J (WT) and IFNGR1 knockout (γR1KO) mice; MSC phenotype confirmed by CD105+CD44+CD29+Sca1+CD73−CD45−CD11b−MHCII−.
• Adipogenic induction: For human cells, 14-day induction in DMEM-based cocktail; for mouse cells, 7-day induction in α-MEM-based cocktail; media (and treatments) were refreshed every 48–72 h. Oil Red staining and triglyceride assay quantified lipid accumulation.
• Cytokine/growth factor screen: hVA-MSCs were differentiated with a dose range (1–30 ng/ml) of individual pro-inflammatory cytokines/growth factors; only IFNγ and TGFβ robustly inhibited adipogenesis in a dose-dependent manner.
• Western blots and qRT-PCR: Protein and mRNA levels of adipogenic transcription factors (C/EBPδ, C/EBPα, PPARγ, PPARα) and signaling mediators (JAK2, STAT1/3/5, Smad2/3/4; phosphorylated forms) were assessed at multiple time points (5–14 days). GAPDH was loading control; ΔΔCt normalized to GAPDH for qRT-PCR.
• Immunofluorescence/confocal microscopy: Nuclear localization of pSTAT5, pSmad3, and Smad4 evaluated after chronic IFNγ and/or TGFβ exposure under adipogenic conditions.
• Pharmacological and genetic perturbations: • JAK1/2 inhibitor Ruxolitinib (5 µM), STAT5 inhibitor (STAT5i; 50–200 µM), STAT1 inhibitor Fludarabine (20 µM), STAT3 inhibitor Stattic (5 µM), TGFBR1 inhibitor Galunisertib (20 µM), Smad3 inhibitor SIS3 (5 µM), proteasome inhibitor MG132 (2 µM for final 12 h). • Dicer-substrate siRNA knockdowns: human STAT5B, STAT1, and SMAD3; validation by western blot and functional rescue assays.
• Co-immunoprecipitation (co-IP): Non-denaturing lysates from hVA-MSCs chronically treated with IFNγ under adipogenic conditions; IPs with anti-STAT5, anti-Smad3, anti-Smad4 to test physical interactions; reciprocal IPs performed; effects of Galunisertib probed.
• TGFβ secretion and receptor analysis: ELISA for secreted TGFβ; flow cytometry for cell-surface TGFBR1 under basal, adipogenic, and IFNγ conditions.
• In vivo chronic inflammation model: WT and γR1KO male mice maintained on high-fat diet (45% kcal fat) for ~25 weeks; epididymal and subcutaneous fat analyzed by H&E for adipocyte size distribution (Adiposoft). Oral glucose tolerance tests (oGTT) performed with concurrent plasma insulin measurements; circulating IFNγ measured by ELISA. Flow cytometry assessed MSC fractions in SVF from epididymal fat of HFD-fed mice.
• Statistics: One-way ANOVA with Tukey’s or Dunnett’s tests; two-way ANOVA with Sidak’s multiple comparisons; unpaired t-tests where indicated. Significance denoted by P < 0.05 to P < 0.00005. Biological replicates typically N=3–4 donors or animals per group unless stated.

• Screen identifies IFNγ and TGFβ as potent, dose-dependent inhibitors of human VA-MSC adipogenesis; other cytokines (TNFα, IL-1β, IL-6, IL-15) failed to inhibit in this model (Oil Red staining; triglyceride assays; N≥3; multiple comparisons significant at P < 0.05 to P < 0.00005).
• IFNγ blocks early adipogenic commitment by suppressing C/EBPδ and consequently PPARγ and C/EBPα at both mRNA and protein levels (day 3–10), preventing lipid accumulation (western blots, qRT-PCR; N=3–4; significant reductions).
• Chronic IFNγ exposure transcriptionally upregulates and activates JAK2, STAT1, STAT3, and STAT5 in VA-MSCs, distinct from acute IFNγ signaling which only increases phospho-STATs without changing total levels. Short-term (12 h) IFNγ increased pSTAT1/3/5 without changing total protein; effects blocked by Ruxolitinib.
• STAT5 is the principal mediator of IFNγ anti-adipogenic action: STAT5 inhibitor (STAT5i) specifically decreased pSTAT5 and total STAT5 (dose-dependent) and rescued adipogenesis under chronic IFNγ, restoring PPARγ, C/EBPα, and Perilipin and increasing triglyceride accumulation, comparable to JAK inhibition (Ruxolitinib). Inhibition or knockdown of STAT1 or STAT3 did not rescue adipogenesis.
• TGFβ/Smad3 axis is required for IFNγ-mediated suppression: adipogenesis normally downregulates Smad proteins; chronic IFNγ specifically upregulated pSmad3 and Smad3 (not Smad2) and promoted nuclear pSmad3/Smad4. Galunisertib blocked IFNγ-induced pSmad3 and restored PPARγ, C/EBPα, and adipogenesis (Oil Red, triglycerides; N=3; significant).
• Reciprocal STAT5–Smad3 synergy via Smad4: STAT5 inhibition (STAT5i or STAT5B knockdown) reduced IFNγ-induced pSmad3 and its nuclear localization. Conversely, Galunisertib or Smad3 inhibition (SIS3) reduced pSTAT5, showing mutual dependence. Proteasome inhibition (MG132) increased pSmad3 and pSTATs; STAT5 particularly accumulated under IFNγ, suggesting proteasomal turnover normally limits signaling. STAT5i reversed IFNγ+MG132-induced pSmad3 elevation. Co-IP showed STAT5 binds Smad4 (not Smad3) and Smad3 binds Smad4; STAT5–Smad4 interaction depended on Smad3 activation and was diminished by Galunisertib.
• Conservation in mouse models: In WT mVA-MSCs, IFNγ inhibited adipogenesis and PPARγ; γR1KO mVA-MSCs were resistant to IFNγ. Inhibiting JAK2 (Ruxolitinib), STAT5 (STAT5i), or TGFBR1 (Galunisertib) restored adipogenesis in WT mVA-MSCs under IFNγ.
• In vivo relevance under chronic HFD: After ~25 weeks HFD, WT epididymal fat showed hypertrophy with limited neoadipogenesis, whereas γR1KO showed numerous small adipocytes indicative of enhanced neoadipogenesis; adipocyte size distribution significantly shifted toward smaller adipocytes in γR1KO epididymal fat (N=3/group; multiple t-tests, P < 0.05 to P < 0.00005). Subcutaneous fat showed no significant differences. γR1KO mice displayed improved glucose tolerance (lower oGTT glucose and reduced AUC; N=5/group; two-way ANOVA with Sidak’s, P < 0.05–0.005) with modest, mostly non-significant insulin differences. γR1KO HFD mice had higher MSC fractions in epididymal SVF.

The findings resolve a key question in adipose biology: how chronic inflammatory milieus suppress neoadipogenesis from VA-MSCs. The study demonstrates that IFNγ and TGFβ converge to inhibit early adipogenic commitment by jointly maintaining a STAT5–Smad3 complex via Smad4. This dyad suppresses pro-adipogenic transcriptional cascades (C/EBPδ → PPARγ → C/EBPα) and is sustained by reciprocal stabilization that limits proteasomal degradation, explaining persistent inhibition under chronic inflammation. Importantly, chronic IFNγ signaling in VA-MSCs differs from canonical acute responses, driving transcriptional upregulation and sustained activation of JAK2/STATs, with STAT5 emerging as the dominant effector of anti-adipogenic action. Pharmacologic or genetic disruption of STAT5 or Smad3 restores adipogenesis in human and mouse VA-MSCs and improves adipose regeneration in settings modeling chronic metabolic inflammation. In vivo, loss of IFNγ signaling protects against HFD-induced loss of neoadipogenesis and improves glucose handling, supporting the physiological relevance of the pathway. The STAT5–Smad3 node may represent a broader paradigm for chronic inflammation-induced plasticity control and offers therapeutic leverage to counteract metabolic dysfunction and wasting syndromes.

This work identifies a central, conserved mechanism by which chronic inflammation inhibits adipogenesis from VA-MSCs: IFNγ-activated STAT5 and TGFβ-activated Smad3 form a complex via Smad4 that is protected from proteasomal degradation, thereby suppressing early adipogenic commitment by downregulating C/EBPδ, PPARγ, and C/EBPα. Disrupting this STAT5–Smad3 dyad with JAK/STAT inhibition (Ruxolitinib, STAT5i) or TGFBR1/Smad3 inhibition (Galunisertib, SIS3), or via genetic knockdown (STAT5B, SMAD3), restores adipogenesis under chronic inflammatory stress. Mouse models demonstrate conserved mechanisms and improved metabolic outcomes when IFNγ signaling is abrogated. These findings suggest therapeutic strategies targeting STAT5–Smad3 synergy to re-enable neoadipogenesis in metabolic syndrome, insulin resistance, and wasting conditions, with potential implications in oncology where sustained STAT5/Smad3 activity contributes to malignancy. Future research should define specific STAT5 and Smad3 genomic targets in VA-MSCs, map post-translational modifications governing complex stability, optimize selective inhibitors, and evaluate long-term metabolic and safety outcomes in preclinical models.