Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components

Abstract

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.

Matrix-Stiffness–Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis

“…12 In addition to serving transcriptional repressor for cyclin D1, Klf4 also functions as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. Soft matrixpreserved Klf4 should thus be emphasized to maintain phenotype differentiation and growth arrest.…”

“…12 All procedures were reviewed and approved through the Institute of Animal Care and Use Committee at the Medical College of National Cheng Kung University (Tainan, Taiwan).…”

“…12 The Young's moduli of these matrices were measured with atomic force microscopy (AFM). Briefly, the Young's modulus of the MG is approximately 66.0 AE 0.3 Pa, and both the dish and MG-coated dish are approximately giga Pa.…”

“…12 Briefly, mPTECs were cultured on the indicated conditions for 3 days and incubated with EdU for 7.5 hours before analysis. For some experiments, cells were incubated with primary rabbit antibody against Klf5 (Novus) at 4 C overnight.…”

“…EMT induced by transformation growth factor-b1, a key mediator in renal fibrosis, 11 is also inhibited by soft matrix. 12 In the present work, we studied the detailed mechanisms underlying the matrix stiffness-modulated behavior of mPTECs, particularly in relation to proliferation.…”