Notch4 activation aggravates NF-κB-mediated inflammation in HIV-1-associated nephropathy

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Notch pathway activation plays a central role in the pathogenesis of many glomerular diseases. We have previously shown that Notch4 expression was upregulated in various renal cells in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) patients and rodent models of HIVAN. In this study, we examined whether the Notch pathway can be distinctly activated by HIV-1 gene products and whether Notch4, in particular, can influence disease progression. Using luciferase reporter assays, we did not observe activation of the NOTCH4 promoter with the HIV protein Nef in podocytes. Further, we observed upregulated expression of a gamma secretase complex protein, presenilin 1, but not Notch4, in podocytes infected with an HIV-1 expression construct. To assess the effects of Notch4 on HIVAN disease progression, we engineered Tg26 mice with global deletion of the Notch4 intracellular domain (Notch4dl), which is required for signaling function. These mice (Notch4d1/Tg26+) showed a significant improvement in renal function and a significant decrease in mortality compared to Tg26 mice. Histological examination of kidneys showed that Notch4d1/Tg26+ mice had overall glomerular, tubulointerstitial injury and a marked decrease in interstitial inflammation. A significant decrease in the proliferating cells was observed in the tubulointerstitial compartments of Notch4d1/Tg26+ mice. Consistent with the diminished inflammation, kidneys from Notch4d1/Tg26+ mice also showed a significant decrease in expression of the inflammatory cytokine transcripts Il-6 and Ccl2, as well as the master inflammatory transcription factor NF-κB (Nfkb1 transcripts and p65 protein). These data identify Notch4 as an important mediator of tubulointerstitial injury and inflammation in HIVAN and a potential therapeutic target.National Institutes of Health (R01DK108433 awarded to M.S.

Secretion Stimulates Intramembrane Proteolysis of a Secretory Granule Membrane Enzyme*

Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH2 termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus

Thermodynamic and structural insights into CSL-DNA complexes

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo