Effects of over-expressed forkhead transcription factor O1 on high glucose induced extracellular matrix secretion in rat mesangial cells

Abstract

To explore the role and molecular mechanism of forkhead transcription factor O1 (FoxO1) for extracellular matrix (ECM) protein accumulation in rat mesangial cells (MCs) cultured under high-glucose conditions. The MCs were transfected with either lentiviral vectors containing the sequences of constitutively active FoxO1 (LV-CA-FoxO1) or empty vectors (LV-NC-GFP). The MCs were divided into 4 groups of normal glucose (5.6 mmol/L) (NG), high glucose (25 mmol/L) (HG), high glucose plus LV-CA-FoxO1 (LV-CA) and high-glucose plus empty lentiviral vectors (LV-NC). After 72 h treatment, the mRNA levels of MCs were measured through real-time polymerase chain reaction (PCR) in each group, including FoxO1, fibronectin (FN), collagen I(Col I), collagen IV (Col IV), transforming growth factor-β1 (TGF-β1), TGF-β type Iand type II receptor (TGF-βRI/II). The protein levels of FoxO1, phosphorylation FoxO1 (p-FoxO1), TGF-β1 and TGF-βRI/II were assessed by Western blot. And the distributions of TGF-βRI in MCs were detected by immunofluorescence. Expression and bioactivity changes of FoxO1: Compared with NG group, there was no significant difference in either mRNA or protein levels of FoxO1 in HG group (P > 0.05) whereas the protein levels of p-FoxO1 markedly increased (P < 0.05). The levels of FoxO1 mRNA and protein increased in LV-CA group versus HG group (mRNA: 17.36 ± 1.13 vs 1.01 ± 0.12, protein: 4.38 ± 0.09 vs 0.93 ± 0.10, both P < 0.05). And there was a significant decrease of p-FoxO1/FoxO1 ratio (P < 0.05). Thus it suggested an enhancement in FoxO1 expression and transcriptional activity. Changes of ECM protein expression. The mRNA expression levels of FN, Col Iand Col IV in HG group were elevated compared with NG group (all P < 0.05) while the expressions of these indices in LV-CA group became attenuated compared with HG group (all P < 0.05). Changes of TGF-β pathway activation: the expressions of TGF-β1 and TGF-βRI/IIboth increased in HG group (all P < 0.05), but decreased in LV-CA group (all P < 0.05). The results of immunofluorescence suggested that the staining of TGF-βRIdecreased in MCs in LV-CA group versus HG group. All indices of LV-NC group had no statistical difference with those of HG group (all P > 0.05). FoxO1 plays a pivotal role in down-regulating the activation of TGF-β pathway and thereby inhibiting the secretion of ECM.