at 0 min), cell surface levels of TGF- receptors are higher in BFA-treated cells (right) than in the control group (left). The consequent autocrine TGF- signaling in response to glucose led to Akt-TOR pathway activation. Accordingly, preventing MMP-2/MMP-9 or TGF–induced TOR activation inhibited high glucose-induced cell hypertrophy. Introduction Cell size is usually highly controlled and its deregulation has been implicated in obesity, diabetes and cancer. Cell growth is usually defined as increase in cell mass, often associated with increased protein synthesis (Mamane et al., 2006). The best characterized signaling pathway that regulates cell size is usually defined by the sequential activation of phosphatidylinositol 3-kinase (PI3K), Akt, TOR and S6 kinase. Growth factors that take action through tyrosine kinase receptors, such as insulin and IGF-1, activate PI3K, thus enhancing the phosphorylation of Akt. Consequent activation of mTOR results in phosphorylation of S6 kinase and 4ECBP1, leading to enhanced translation (Hay and Sonenberg, 2004; Manning and Cantley, 2007). The contribution of additional signaling pathways that control cell size during homeostasis remains poorly understood. Glucose is an essential nutrient for cells and provides energy for cell growth. After being transported into the cell by glucose transporters, glucose undergoes a metabolic process known as glycolysis, which generates ATP and NADPH as energy source and regulates the activity of TOR, protein synthesis and cell size (Herman and Kahn, 2006). High glucose induces increased protein synthesis and cell size, and promotes cell hypertrophy in various tissues and organs, including muscle, kidney and heart Elevated levels of blood glucose, i.e. hyperglycemia, consequently increase the risk and complications of diseases such as obesity, diabetes and heart disease (Wolf and Ziyadeh, 1999; Sartorelli and Fulco, 2004; Neubauer, 2007). How glucose induces increased cell size is usually poorly comprehended. Increased Akt activity has been shown to stimulate transport and metabolism of glucose and triggers TOR-dependent increases in protein translation (Plas and Thompson, 2005; Manning and Cantley, 2007). Several observations correlate hyperglycemia to increased activity of transforming growth factor- (TGF-). In diabetic patients and rodent models of diabetes, continuous exposure of cells to high glucose has been linked to hypertrophy of proximal tubular and mesangial cells, and accumulation of extracellular Kaempferol matrix proteins and fibrosis (Ziyadeh, 2004). Consistent with the induction of extracellular matrix protein expression by TGF- and with TGF-s role in fibrosis (Zavadil and Bottinger, 2005), TGF-1 levels were increased in the glomerular and tubular compartments of the kidney in rodent models of diabetes, and Smad3 activation was observed in these cells (Kolm-Litty et al., 1998; Hong et al., 2001; Isono et al., 2002). High glucose was also shown to induce TGF- expression, leading to production of extracellular matrix proteins (Ziyadeh et al., 1994), and exposure of cells to high glucose can increase the Kaempferol expression of TGF-1 and/or the TRII receptor (Hong et al., 2001; Iglesias-de la Cruz et al., 2002). These observations suggest a functional linkage of glucose-stimulated increase of protein synthesis, in particular of extracellular matrix proteins, with increased TGF- signaling. However, a direct role of TGF- signaling in the glucose-stimulated increase in cell size has not been revealed. TGF-, the prototype of a 33-member TGF- family, functions through cell surface receptor complexes of two type I and two type II receptors, i.e. TRI and TRII. Following ligand binding, the TRII receptors phosphorylate and activate the Kaempferol TRI receptors, which C-terminally phosphorylate and thereby activate Smad2 and Smad3. These then form a complex with Smad4, translocate into the nucleus, and regulate the transcription of TGF- responsive genes (Shi and Massague, 2003; Feng and Derynck, 2005). Smad signaling does not account for other TGF- responses and, accordingly, non-Smad mechanisms that relay TGF- signals have been characterized (Derynck and Zhang, 2003; Moustakas and Heldin, 2005). Recent findings revealed that TGF- can activate PI3K, leading to activation of the PI3KCAkt-TOR-S6 kinase pathway in response.These observations have relevance for the physiology of hyperglycemia-induced pathologies that are associated with tissue hypertrophy, including cancer. Results Glucose increases cell size and protein content To evaluate the effect of glucose, we analyzed cells by circulation cytometry using forward light scatter as parameter indicative of cell size. induced a rapid increase in cell surface levels of the TRI and TRII receptors, and a rapid activation of TGF- ligand by matrix metalloproteinases, including MMP-2 and MMP-9. The consequent autocrine TGF- signaling in response to glucose led to Akt-TOR pathway activation. Accordingly, preventing MMP-2/MMP-9 or TGF–induced TOR activation inhibited high glucose-induced cell hypertrophy. Introduction Cell size is usually highly controlled and its deregulation has been implicated in obesity, diabetes and malignancy. Cell growth is usually defined as increase in cell mass, often associated with increased protein synthesis (Mamane et al., 2006). The best characterized signaling pathway that regulates cell size is usually defined by the sequential activation of phosphatidylinositol 3-kinase (PI3K), Akt, TOR and S6 kinase. Growth factors that take action through tyrosine kinase receptors, such as insulin and IGF-1, activate PI3K, thus enhancing the phosphorylation of Akt. Consequent activation of mTOR results in phosphorylation of S6 kinase and 4ECBP1, leading to enhanced translation (Hay and Sonenberg, 2004; Manning and Cantley, 2007). The contribution of additional signaling pathways that control cell size during homeostasis remains poorly understood. Glucose is an essential nutrient for cells and provides energy for cell growth. After being transported into the cell by glucose transporters, glucose undergoes a metabolic process known as glycolysis, which generates ATP and NADPH as energy source and regulates the activity of TOR, protein synthesis and cell size (Herman and Kahn, 2006). High glucose induces increased protein synthesis and cell size, and promotes cell hypertrophy in various tissues and organs, including muscle mass, kidney and heart Elevated levels of blood glucose, i.e. hyperglycemia, consequently increase the risk and complications of diseases such as obesity, diabetes and heart disease (Wolf and Ziyadeh, 1999; Sartorelli and Fulco, 2004; Neubauer, 2007). How glucose induces increased cell size is usually poorly understood. Increased Akt activity has been shown to stimulate transport and metabolism of glucose and triggers TOR-dependent increases in protein translation (Plas and Thompson, 2005; Manning and Cantley, 2007). Several observations correlate hyperglycemia to increased activity of transforming growth factor- (TGF-). In diabetic patients and rodent models of diabetes, continuous exposure of cells to high glucose has been linked to hypertrophy of proximal tubular and mesangial cells, and accumulation of extracellular matrix proteins and fibrosis (Ziyadeh, 2004). Consistent with the induction of extracellular matrix protein expression by TGF- and with TGF-s role in fibrosis (Zavadil and Bottinger, 2005), TGF-1 levels were increased in the glomerular and tubular compartments of the kidney in rodent models of diabetes, and Smad3 activation was observed in these cells (Kolm-Litty et al., 1998; Hong et al., 2001; Isono et al., 2002). High glucose was also shown to induce TGF- expression, leading to production of extracellular matrix proteins (Ziyadeh et al., 1994), and exposure of cells to high glucose can increase the expression of TGF-1 and/or the TRII receptor (Hong et al., Kaempferol 2001; Iglesias-de la Cruz et al., 2002). These observations suggest a functional linkage of glucose-stimulated increase of protein synthesis, in particular of extracellular matrix proteins, with increased TGF- signaling. However, a direct role of TGF- signaling in the glucose-stimulated increase in cell Kaempferol size has not been revealed. TGF-, the prototype of a 33-member TGF- family, acts through cell surface receptor complexes of two type I and two type II receptors, i.e. TRI and TRII. Following ligand binding, the TRII receptors phosphorylate and activate the TRI receptors, which C-terminally phosphorylate and thereby activate Smad2 and Smad3. These then form a complex with Smad4, translocate into the nucleus, and regulate the transcription of TGF- responsive genes (Shi and Massague, 2003; Feng and Derynck, 2005). Smad signaling does not account for other TGF- responses and, accordingly, non-Smad mechanisms that relay TGF- signals have been characterized (Derynck and Zhang, 2003; Moustakas and Heldin, 2005). Recent findings revealed that TGF- can activate PI3K, leading to activation of the PI3KCAkt-TOR-S6 kinase pathway in response to TGF-. Activation of this pathway by Rabbit Polyclonal to KRT37/38 TGF- was observed in cells undergoing epithelial to mesenchymal transition, and allows TGF- to directly regulate translation, complementing the Smad-mediated.