The Effect And Molecular Mechanism Of Forkhead Transcription Factor O1on High Glucose-Induced Extracellular Matrix Deposition Of Rat Mesangial Cells

BackgroundDiabetes nephropathy (DN) is one of the common microvascular complicationswhich are caused by diabetes mellitus (DM). In the genesis and development of DN,the imbalances between secretion and degradation of ECM proteins which are causedby the dysfunction of mesangial cells (MCs), lead to their redundant deposition inmesangium and glomerular basement membrane (GBM), thereby resulting inmorphological and functional changes in glomerulus and ultimately leadingto glomerulosclerosis. Forkhead transcription factor (FoxO1) is a transcription factorbelonging to Forkhead transcription family, which plays vital roles in oxidative stresssuppression, cell cycle arrest, proliferation regulation, apoptosis induction, proteinsynthesis and other biological activities. The transcriptional activity of FoxO1isnegatively regulated by phosphatidylinositol-3-hydroxy kinase/protein kinase B(PI3K/Akt) pathway. Previous studies have demonstrated that the bioactivity ofFoxO1is decreased in renal cortex tissues of STZ-induced DN rats, accompanied byaccumulation ECM protein including collagen IV and fibronectin (FN), whereasresveratrol may activate FoxO1to eliminate these ECM proteins. There might be apossible connection between the attenuation of FoxO1bioactivity and the secretion of ECM proteins, but the specific mechanisms are still unclear.Previous studies have shown that the excessive deposition of ECM proteins isassociated with the activation of transforming growth factor-β (TGF-β)/Smadpathway in MCs under high glucose conditions. Previous studies have demonstratedthat FoxO1may have interaction with TGF-β pathway. In HepG2cell line, PAI-1（Plasminogen activator inhibitor1）which can inhibit the transformation fromplasminogen to plasmin thus leading to inhibit ECM degradation is elevated byTGF-β1treatment, whereas overexpression of FoxO1may inhibit this effect.Therefore, it is probable that the interaction between FoxO1and TGF-β/Smadpathway may have influence on ECM accumulation. Through lentiviral vector–mediated overexpressing of constitutively active FoxO1in MCs, we investigate theeffect and molecular mechanism of FoxO1on the deposition of ECM protein and theactivation of TGF-β/Smad pathway.ObjectiveTo study the roles and mechanisms of FoxO1on activation of TGF-β/Smadpathway and accumulation of ECM in mesangial cells cultured in high glucosecondition.MethodsMCs cultured in normal glucose (5.6mmol/L) medium were transfected withconstitutively active FoxO1lentiviral vectors (LV-CA-FoxO1) or empty lentiviralvectors (LV-NC-GFP) which were constructed by our research group in previousstudy. MCs were divided into4groups: MCs without transfection cultured in normalglucose（5.6mmol/L）medium or high glucose (25mmol/L) medium were served asnormal control group (group NG) or high glucose group (group HG), while the MCstransfected with LV-CA-FoxO1or LV-NC-GFP and treated in high glucosemedium(25mmol/L) were served as group LV-CA or group LV-NC. After MCs ineach group were cultured in corresponding conditions for72h, the mRNA levels ofFoxO1, Fibronectin (FN), Collagen I (Col I), Plasminogen activator inhibitor 1(PAI-1), TGF-β1, TGF-β type I receptor (TGF-βRI), TGF-β type II receptor(TGF-βRII), Smad3, and Smad7were measured by real-time quantitative PCR. Theexpression levels of protein were assessed by Western blotting, including PAI-1,FoxO1, phosphorylation FoxO1(p-FoxO1), TGF-β1, TGF-βRI, TGF-βRII, Smad3,phosphorylation Smad3(p-Smad3), and Smad7. The expressions and distributions ofTGF-βRI, TGF-βRII, Smad3, and p-Smad3were detected by immunofluorescence.The interactions between CBP (CREB-Binding protein, CBP) and Smad3weredetected by immunoprecipitation.Results1. LV-CA-FoxO1promoted the expression and bioactivity of FoxO1Compared with group NG, p-FoxO1protein level was elevated in group HG(P<0.05) which indicated an attenuation of its bioactivity. The protein levels ofFoxO1and p-FoxO1were both increased (all P<0.05) in group LV-CA, but the ratioof p-FoxO1/FoxO1was decreased which indicated an increase of FoxO1bioactivity.2. Overexpression of FoxO1led to the decrease of ECM accumulationCompared with group NG, the expression levels of FN, Col I and PAI-1weresignificantly increased (all P<0.05) in group HG, while these indexes in group LV-CAwere decreased (all P<0.05) compared with group HG.3. Overexpression of FoxO1inhibited the activation of TGF-β pathwayCompared with group NG, the mRNA and protein levels of TGF-β1, TGF-βRI,and TGF-βRII were elevated in group HG (all P<0.05), whereas the indexes in groupLV-CA were attenuated (all P<0.05) compared with group HG. The results ofimmunofluorescence suggested that the distributions of TGF-βRI and TGF-βRII weredecreased in MCs in group LV-CA.4. Overexpression of FoxO1didn’t alter the expression of Smad3, butsuppressed its phosphorylationSmad3, a kind of receptor-regulated Smad(R-Smad) which promotes thesecretions of ECM proteins and PAI-1, were elevated in both group HG and groupLV-CA(all P<0.05), with no significant difference between these two groups(P>0.05). But p-Smad3, the phosphorylated from of Smad3, were increased in group HG butdecreased in Group LV-CA (all P<0.05).5. Overexpression of FoxO1restrained the transcriptional activity of Smad3The results of immunofluorescence indicated that both Smad3and p-Smad3aggregated in the nucleus in group HG, while the same indexes distributed evenly incytoplasm and nucleus in group LV-CA. Co-IP results demonstrated that theinteraction between Smad3and CBP were enhanced in group HG, whereas reduced ingroup LV-CA. The results mentioned above suggested that the transcriptional activityof Smad3increased in group HG and decreased in group LV-CA.6. Overexpression of FoxO1up-regulated the expression of Smad7Smad7, an inhibitory Smad protein (I-Smad), was decreased in group HG andelevated in group LV-CA(all P<0.05), which indicated an inhibitory effect onTGF-β/Smad pathway.Conclusions1. High glucose induces the inactivation of FoxO1, leading to the increase ofECM secretion and decrease of ECM degradation.2. Overexpression of constitutively active FoxO1can ameliorate deposition ofECM proteins thus alleviating dysfunction of mesangial cells, possibly through themechanisms of blocking the activation and signaling of TGF-β/Smad pathway inMCs.