Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro

Abstract

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

TGF-β signaling in the kidney: profibrotic and protective effects

“…Further evidence indicates that loss of Smad4 represses Smad7 transcription, which leads to decreased nuclear factor of light polypeptide gene enhancer in B-cells inhibitor ␣ (IB␣) expression but enhanced NF-B activation in the tubulointerstitium after UUO, thereby promoting renal inflammation (60). Moreover, Smad7 knockout mice display enhanced renal inflammation with increased NF-B/p65 phosphorylation and increased macrophage infiltration in models of UUO and STZinduced diabetes (8,14).…”

“…Similarly, disruption of T␤RII in cultured renal tubular epithelial cells and fibroblasts resulted in impairment of the anti-inflammatory effect of TGF-␤1 (59). Furthermore, conditional Smad4 knockout mice in which Smad4 was specifically deleted from kidney tubular epithelial cells displayed significantly enhanced renal inflammation as evidenced by increased infiltration of CD45 ϩ leukocytes and F4/80 ϩ macrophages and upregulation of proinflammatory cytokines IL-1␤, TNF-␣, monocyte chemoattractant protein-1 (MCP-1), as well as intercellular adhesion molecule-1 (ICAM-1) in the UUO kidney (60). Thus inhibition of TGF-␤ signaling through disruption of either T␤RII or Smad4 resulted in enhanced renal inflammation, suggesting that TGF-␤ possesses anti-inflammatory effects in the kidney.…”

Delivery of microRNA-146a with polyethylenimine nanoparticles inhibits renal fibrosis in vivo

“…[4][5][6] Smad4 knockout in the kidney was found to enhance inflammation, as evidenced by leukocyte and macrophage infiltration. 6 Unresolved inflammation characterized by macrophage infiltration has also been reported to promote progressive renal fibrosis. [7][8][9] Infiltration by macrophages is invariably observed at sites of renal fibrosis, with the degree of infiltration correlated with the extent of fibrosis.…”

“…[4][5][6] TGF-β 1 receptor-activated Smad3 combines with a common Smad, Smad4, and this complex is translocated to the nucleus, where it binds to DNA elements to promote the transcription of various pro-fibrotic genes. [4][5][6] Smad4 knockout in the kidney was found to enhance inflammation, as evidenced by leukocyte and macrophage infiltration. 6 Unresolved inflammation characterized by macrophage infiltration has also been reported to promote progressive renal fibrosis.…”

Nano-sized carriers in gene therapy for renal fibrosisin vivo

“…Numerous studies have demonstrated that transforming growth factor (TGF)-β 1 and downstream intracellular proteins, known as small mothers against decapentaplegic (Smad), play a central role as a pro-fibrotic pathway in renal fibrosis (Figure 1) [16–22]. TGF-β 1 receptor-activated Smad3 combines with a common Smad, called Smad4, and this complex is translocated into the nucleus where it binds to DNA elements to promote the transcription of various pro-fibrotic genes (Figure 1) [16–19].…”