Abstract
Interaction of transforming growth factor-β (TGFβ)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFβ receptor involved in the noncanonical signaling is unknown. Here, we show that TGFβ increased the catalytic loop phosphorylation of platelet-derived growth factor receptor β (PDGFRβ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFβ increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRβ, thus activating Akt. Inhibition of PDGFRβ using a pharmacological inhibitor and siRNAs blocked TGFβ-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRβ-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRβ in mesangial cells. Additionally, we demonstrate that PDGFRβ-initiated phosphorylation of PRAS40 is required for TGFβ-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRβ is also associated with enhanced TGFβ expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFβ noncanonical signaling, we identified how TGFβ receptor I achieves mTORC1 activation through PDGFRβ-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRβ in TGFβ-driven renal fibrosis.
Highlights
Chronic kidney disease affects 10% of the world’s population, including 20 million Americans, and causes increased risk of cardiovascular diseases and loss of renal function, leading to end-stage renal disease with significant public health costs
Activation of TGFbRI stimulates plateletderived growth factor receptor b (PDGFRb) autophosphorylation in mesangial cells Previously, we demonstrated that transforming growth factor-b (TGFb) increases the tyrosine phosphorylation of several proteins, including a protein at 190 kDa to induce noncanonical signaling in mesangial cells [15]
To characterize the tyrosine kinase involved in the action of TGFb, we considered PDGFRb because it is a receptor tyrosine kinase, it is one of most abundant receptors expressed in mesangial cells, and it elicits pathology in glomerulosclerosis [5, 19, 27]
Summary
Chronic kidney disease affects 10% of the world’s population, including 20 million Americans, and causes increased risk of cardiovascular diseases and loss of renal function, leading to end-stage renal disease with significant public health costs PRAS40, which results in the increased mTORC1 activity necessary for mesangial cell hypertrophy and expression of matrix proteins fibronectin and collagen I (a2). These results conclusively demonstrate that Akt-mediated phosphorylationdependent inactivation of PRAS40 downstream of PDGFRb regulates TGFb-induced mTORC1 activation in mesangial cells.
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