Transforming growth factor-β₁differentially mediates fibronectin and inflammatory cytokine expression in kidney tubular cells

Abstract

Transforming growth factor-beta(1) (TGF-beta(1)) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-beta(1) induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1), and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1, and fibronectin on TGF-beta(1) expression is unknown. The TGF-beta(1) gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased (P < 0.05) in TGF-beta(1)-silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF-beta(2) (P < 0.05), TGF-beta(3) (P < 0.05) isoforms and pSmad2 (P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF-beta antibody reversed the observed increase in fibronectin expression, suggesting that TGF-beta(2) and TGF-beta(3) isoforms mediate the increased fibronectin expression in TGF-beta(1)-silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF-beta(1)-silenced cells. In contrast, nuclear factor-kappaB (NF-kappaB) DNA binding activity was significantly decreased (P < 0.0005). These studies demonstrate that TGF-beta(1) is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF-beta isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-beta(1) reduces chemokine production in association with reduced NF-kappaB DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-beta(1). Conversely, decreased expression of TGF-beta(1) results in increased TGF-beta(2), TGF-beta(3), AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin.

High Glucose-Induced Thioredoxin-Interacting Protein in Renal Proximal Tubule Cells Is Independent of Transforming Growth Factor-β1

“…We have previously demonstrated the increased levels of TGF-␤2 and -3 in TGF-␤1-silenced cells result in overexpression of fibronectin. 16 High-glucose exposure for 72 hours further increased Txnip expression by 3.5-fold in TGF-␤1-silenced cells compared with wild-type cells (P Ͻ 0.0005) ( Figure 5D), which is consistent with Txnip protein expression by Western blotting ( Figure 5, E and F). These data suggest that high glucose induced Txnip expression through a TGF-␤1-independent pathway.…”

“…We have recently demonstrated that increased TGF-␤2 and TGF-␤3 expression in TGF-␤1-silenced cells results in a paradoxical increase in fibronectin expression. 16 We hypothesize that overexpression of Txnip in TGF-␤1-silenced cells is caused by the previously confirmed increase in expression of TGF-␤2 and TGF-␤3, 16 which in turn increases Txnip expression. Indeed, we have confirmed this hypothesis by the addition of pan-specific TGF-␤-neutralizing antibody to TGF-␤1-silenced cells and increased level of Txnip returned to control level.…”

“…TGF-␤1 gene was effectively silenced in HK-2 cells using siRNA technology as previously described. 16 Txnip mRNA expression significantly increased by 21-fold in TGF-␤1-silenced cells (P Ͻ 0.0005) ( Figure 5A). However, the increased level of Txnip returned to control (nonspecific siRNA) level after the addition of pan-specific TGF-␤-neutralizing antibody in TGF-␤1-silenced cells ( Figure 5, B and C), suggesting TGF-␤2 or -3 might increase in TGF-␤1-silenced cells and these two isoforms subsequently increased Txnip expression.…”

TGF-β induces miR-30d down-regulation and podocyte injury through Smad2/3 and HDAC3-associated transcriptional repression

“…Smad signaling can cause both transcription activation and repression, in which Smad recruits histone deacetylases (HDAC) and transcription repressors to negatively regulate the downstream targets [22]. To explore the possible HDAC involvement in the repression, we first examined the effect of HDAC inhibition on the miR-30d promoter transcription activity.…”

Transforming Growth Factor-Beta in Kidney Transplantation: A Double-Edged Sword

“…A variety of biological activities of TGF-beta have been demonstrated in different experimental systems, including stimulation of cellular proliferation and cellular differentiation, or oppositely induction of cell apoptosis and anti-proliferation (Siegel and Massague, 2003), suggesting that TGF-beta, particularly TGF-beta 1, is a key regulatory factor for tissue homeostasis. In cultured renal cells, these three TGF-beta isoforms have similar activities (Yu et al, 2003;Qi et al, 2006), but the activities of TGF-beta 2 and TGF-beta 3 may be partially mediated by TGF-beta 1 (Yu et al, 2003). Kidney transplantation is the best therapy for individuals who unfortunately have end-stage kidney disease; individuals with kidney transplants live longer with a better quality of life compared to those on dialysis (Port et al, 1993;Laupacis et al, 1996;Schnuelle et al, 1998).…”