Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation

Abstract

Diabetic nephropathy, a devastating consequence of diabetes mellitus, is characterized by the accumulation of extracellular matrix (ECM) that disrupts the kidney’s filtration apparatus. Elevated glucose levels increase the deposition of a fibronectin (FN) matrix by mesangial cells, the primary matrix-producing cells of the kidney, and also increase acetyl-CoA leading to higher levels of lysine acetylation. Here, we investigated the connection between acetylation and the ECM and show that treatment of mesangial cells with deacetylase inhibitors increases both acetylation and FN matrix assembly compared to untreated cells. The matrix effects were linked to lysine 794 (K794) in the β1 integrin cytoplasmic domain based on studies of cells expressing acetylated (K794Q) and non-acetylated (K794R) mimetics. β1(K794Q) cells assembled significantly more FN matrix than wildtype β1 cells, while the non-acetylated β1(K794R) form was inactive. We show that mutation of K794 affects FN assembly by stimulating integrin-FN binding activity and cell contractility. Wildtype and β1(K794Q) cells but not β1(K794R) cells further increased their FN matrix when stimulated with deacetylase inhibitors indicating that increased acetylation on other proteins is required for maximum FN assembly. Thus, lysine acetylation provides a mechanism for glucose-induced fibrosis by up-regulation of FN matrix assembly.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

“…For these experiments, wild-type and Fn1 mEGFP/+ MEFs were sparsely-plated in Ibidi 8-well glass-bottom chambers overnight, allowing to observe the structure of thin fibrils. Cells were then fixed and stained with antibodies recognizing different Fn1 epitopes: polyclonal rabbit antibodies raised to recognize the first six type III repeats of Fn1 (Fn1 III1-6)(Vega et al, 2020) (Fig. 5 column 1), a monoclonal antibody recognizing an epitope within the central region of Fn1(Fig.…”

Elevated glucose alters global gene expression and tenascin-C alternative splicing in mesangial cells

“…Conditioned medium was collected from cells before cell lysis and stored with addition of 10 mM EDTA, 2 mM PMSF at −20 °C. Cells were lysed in deoxycholate (DOC) buffer, DOC-soluble cell lysate was separated from DOC-insoluble matrix by centrifugation, and the insoluble pellet was solubilized in SDS buffer with boiling [ [29] , [30] , [31] ]. Total protein concentration of the DOC-soluble fraction was measured using BCA assay (Pierce).…”

“…For conditioned medium, 10 μl for each condition were separated by SDS-PAGE on a 5% gel. Gels were transferred to a nitrocellulose membrane and immunoblotted as described [ 29 ]. The following antibodies were used: for TN-C - R759 rabbit polyclonal antiserum at 1:10,000 and for GAPDH - Cell Signaling (14C10) rabbit monoclonal antibody at 1:5000.…”

“…Differentiated mesangial cells in either 5 mM or 30 mM glucose medium with 10 μg/ml human plasma FN were fixed with 3.7% formaldehyde in PBS and incubated with anti-TN-C (R759 rabbit polyclonal antiserum diluted 1:100) or anti-human FN (mouse monoclonal concentrated supernatant HFN7.1 diluted 1:100, DSHB, Iowa) followed by Alexa-Fluor-488- or 568-conjugated goat anti-rabbit or goat anti-mouse IgG (Invitrogen) antibody (1:600) [ 8 ]. Samples were mounted in ProLong Gold antifade reagent (LifeTechnologies).…”

“…Excess amounts of the ECM proteins fibronectin (FN) and type IV collagen can be detected in renal biopsies of patients with diabetic nephropathy [ 5 ], and cultured mesangial cells, the major ECM-producing cells in the glomerulus, increased assembly of FN and collagen fibrils when exposed to elevated glucose levels [ 6 ]. Using a mouse mesangial cell model, we have previously described several glucose-dependent mechanisms for excessive FN matrix assembly including increases in activity of integrin receptors for FN [ 6 ], cell interactions with advanced glycation end products [ 7 ], and increased lysine acetylation [ 8 ]. In addition to ECM-specific effects of glucose, others have identified roles for epigenetic changes in various diabetic animal and cell models of diabetic nephropathy [ 9 , 10 ].…”