Abstract
Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1−/− cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.
Highlights
Cellular mechano-sensitivity plays fundamental roles in cell viability and function, tissue development, and maintenance of organs [1]
Several inherited renal diseases are a consequence of dysfunctional primary cilia, with Polycystic Kidney Disease (PKD) as most prominent example [15]
Our results show fluid flow induced transforming growth factor β (TGF-β)/ALK5-mediated signaling, SMAD2/3 phosphorylation, and target gene expression in proximal tubular epithelial cells (PTEC)
Summary
Cellular mechano-sensitivity plays fundamental roles in cell viability and function, tissue development, and maintenance of organs [1]. The signaling modules responsible for the flow-sensing response involve a number of proteins located in the cell membrane, cilium and/or at the ciliary base, including polycystin-1 (PC-1) and the ion channel polycystin-2 (PC-2), encoded by the genes mutated in patients with autosomal dominant polycystic kidney disease (ADPKD) [4, 5]. Mutations or deletions of other ciliary proteins can cause renal cystic disease in mouse models and patients, indicating the role of cilia during cystogenesis [16, 17]. In the absence of polycystins, renal epithelial cells lack the capability to respond to signals needed to maintain the epithelium differentiated, resulting in cyst formation [15]
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