High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

Abstract

Aims

This study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.

Main methods

Rat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.

Key findings

Measurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.

Significance

HG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.

Introduction

Cell therapy with bone marrow mesenchymal stem cells (MSCs) remains a viable option for tissue repair and regeneration after injuries including myocardial infarction (Williams and Hare, 2011). While the mechanisms for the beneficial effects of MSCs on cardiac repair are complex and not fully understood, paracrine signaling is believed to be responsible (at least partially) for their beneficial effects including myogenesis, angiogenesis, and collateral development (Mirotsou et al., 2011). A wide spectrum of biologically active growth factors and cytokines including, but not limited to, fibroblast growth factor (FGF), interleukin-1 and 6, transforming growth factor-β (TGF-β), and VEGF are generated from MSCs and released into the culture medium (Mirotsou et al., 2011). It has been shown that cardiovascular risk factors such as hypercholesterolemia and diabetes mellitus (DM) attenuate native collateral development and are associated with incomplete revascularization and decreased re-endothelialization after arterial injury in both human and animals (Ii et al., 2006, Kinnaird et al., 2008, Sodha et al., 2009). The mechanisms for the impaired outcome in the patients with hypercholesterolemia and/or DM have not been well defined. It is possible that these risk factors like DM may impair the function of MSCs involved in cardiovascular repair and regeneration. Indeed, bone marrow-derived progenitor cells from patients with hypercholesterolemia and/or DM exhibit a substantially reduced capacity for neovascularization and decreased paracrine secretion of proangiogenic factors (Tepper et al., 2002, Loomans et al., 2004, Kränkel et al., 2005, Heeschen et al., 2004). Recently, we demonstrated that high glucose (HG) substantially suppressed VEGF expression in MAPCs through inhibition of JAK2/STAT3 signaling (Liu et al., 2008).

TGF-β is the prototypic member of a super family of multifunctional cytokines involved in many cellular functions including cell growth, migration, adhesion, differentiation, and angiogenesis (Iruela-Arispe and Sage, 1993, Schmierer and Hill, 2007). The super family has thirty-three members in mammals including three TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3), activins, and bone morphogenetic proteins (BMPs) (Schmierer and Hill, 2007). Perturbations in TGF-β signaling are associated with cardiovascular diseases such as impaired vascular development and angiogenesis (Grainger, 2007, Goumans et al., 2009). TGF-β1 is produced in various cells including endothelial cells and MSCs (Vinals and Pouyssegur, 2001, Worster et al., 2000). Multipotent adult progenitor cells (MAPCs) are clonally isolated from postnatal human and rodent tissues including bone marrow, muscle and brain (Jiang et al., 2002a, Jiang et al., 2002b). These cells have been well characterized and are able to differentiate into multiple cell lineages including endothelial cells, hepatocytes, cardiomyocytes, and neurons (Jiang et al., 2002a, Jiang et al., 2002b, Ulloa-Montoya et al., 2007, Lu et al., 2010). MAPCs promote angiogenesis and improve cardiac and limb function when injected into the peri-infarct areas in ischemic mice (Pelacho et al., 2007, Aranguren et al., 2008). Interestingly, the increased vascularity and improved cardiac function after myocardial infarction is not due to differentiation and direct contribution of MAPCs to the vascular or cardiomyocyte compartment after cell transplantation. Instead, the beneficial effects are largely contributed by secreting vascular growth factors like VEGF and TGF-β by these cells, resulting in increased angiogenesis and cardioprotection (Pelacho et al., 2007, Agbulut et al., 2006). MSCs have also been shown to enhance wound healing at least partially due to the release of different cytokines and chemokines, such as TGF-β1 and VEGF (Chen et al., 2008). Therefore, optimal expression of these growth factors and cytokines is critical to the optimal outcome of cell therapies especially in the patients with DM that is associated with significant cardiovascular morbidity and mortality (Engelgau et al., 2004).

In the present study, we investigated the effect of HG on the expression of TGF-β1 and the underlying mechanism in rat MAPCs. We observed that detectable level of TGF-β1 was expressed in rat MAPCs with balanced phosphorylation of both ERK1/2 and STAT3. TGF-β1 expression was substantially increased in MAPCs cultured in HG media. HG also significantly enhanced ERK1/2 phosphorylation, and inhibited STAT3 (Tyr705) phosphorylation in MAPCs. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 remarkably suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation, and increased TGF-β1 expression without change in ERK1/2 phosphorylation. These data suggested that HG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.