Transforming growth factor (TGF)- has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-1-increased collagen I expression. TGF-1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-1 treatment. The PI3K inhibitor LY294002 decreases TGF-1-stimulated ␣1(I) and ␣2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF- receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This crosstalk between the Smad and PI3K pathways likely contributes to TGF-1 induction of glomerular scarring.
Selected flavonoids were tested for their ability to inhibit the catalytic activity of DNA topoisomerase (topo) I and II. Myricetin, quercetin, fisetin, and morin were found to inhibit both enzymes, while phloretin, kaempferol, and 4',6,7-trihydroxyisoflavone inhibited topo II without inhibiting topo I. Flavonoids demonstrating potent topo I and II inhibition required hydroxyl group substitution at the C-3, C-7, C-3', and C-4' positions and also required a keto group at C-4. Additional B-ring hydroxylation enhanced flavonoid topo I inhibitory action. A C-2, C-3 double bond was also required, but when the A ring is opened, the requirement for the double bond was eliminated. Genistein has been previously reported to stabilize the covalent topo II-DNA cleavage complex and thus function as a topo II poison. All flavonoids were tested for their ability to stabilize the cleavage complex between topo I or topo II and DNA. None of the agents stabilized the topo I-DNA cleavage complex, but prunetin, quercetin, kaempferol, and apigenin stabilized the topo II DNA-complex. Competition experiments have shown that genistein-induced topo II-mediated DNA cleavage can be inhibited by myricetin, suggesting that both types of inhibitors (antagonists and poisons) interact with the same functional domain of their target enzyme. These results are of use for the selection of flavonoids that can inhibit specific topoisomerases at specific stages of the topoisomerization reaction.
Basu RK, Hubchak S, Hayashida T, Runyan CE, Schumacker PT, Schnaper HW. Interdependence of HIF-1␣ and TGF-/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.
Transforming growth factor (TGF)-beta is a central stimulus of the events leading to chronic progressive kidney disease, having been implicated in the regulation of cell proliferation, hypertrophy, apoptosis and fibrogenesis. The fact that it mediates these varied events suggests that multiple mechanisms play a role in determining the outcome of TGF-beta signaling. Regulation begins with the availability and activation of TGF-beta and continues through receptor expression and localization, control of the TGF-beta family-specific Smad signaling proteins, and interaction of the Smads with multiple signaling pathways extending into the nucleus. Studies of these mechanisms in kidney cells and in whole-animal experimental models, reviewed here, are beginning to provide insight into the role of TGF-beta in the pathogenesis of renal dysfunction and its potential treatment.
Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging.
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