Background/Purpose

The immunomodulating roles of estrogens are not completely understood. Although 17β estradiol (E2) has been shown to promote systemic autoimmunity, it has also been shown to inhibit pro-inflammatory cytokine secretion. We showed previously that treatment of male mice with E2 led to improved survival in nephrotoxic serum induced nephritis (NTN). In this study we aimed at determining the effect of E2 on intrinsic renal cells and to understand the role of E2 in regulating inflammation in vivo in the kidney during nephritis. Vascular Cell Adhesion Molecule, VCAM-1, is an adhesion molecule for leukocytes that is upregulated in vivo in mice with autoimmune nephritis, and in vitro in tubular epithelial cells and mesangial cells (MCs) upon stimulation with tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ). In this study we determined whether E2 regulates the extent of renal inflammation by regulating adhesion molecule expression by MCs.

Methods

We used nephrotoxic serum induced nephritis as a model of autoimmune nephritis. Mice were treated with E2 pellets prior to induction of nephritis. We determined the molecular mechanisms of VCAM-1 regulation by E2 using cell and molecular biology techniques such as flow cytometry, immunofluorescence, Chromatin immunoprecipitation (ChIP) assays, and quantitative PCRs  in TNFα stimulated MCs. 

Results

We show that E2 treatment inhibited VCAM-1 upregulation in kidneys in vivo during NTN in both male and female mice. E2 also inhibited upregulation of VCAM-1 in MC upon TNFα stimulation. The VCAM-1 upregulation in MCs was regulated by the transcription factor NFκB, since inhibition of NFκB inhibited the upregulation. We further determined the molecular mechanism of regulation of VCAM-1 by E2. We show that E2 does not regulate the nuclear translocation of p65 subunit. ChIP assays showed that although E2 does not inhibit p65 binding to the VCAM-1 promoter, it inhibits the recruitment of RNA polymerase II to the promoter, suggesting that E2 may inhibit the formation of pre-initiation complex at the promoter. We showed previously that absence of Poly (ADP-Ribose) Polymerase-1 (PARP-1) inhibited TNFα stimulated VCAM-1 upregulation in mouse MCs. PARP-1 has been shown to interact with estrogen receptor and we showed that E2 inhibits PARP-1 activity in macrophages. PARP-1 has been also proposed as a co-factor for NFkB activation. We therefore determined whether E2 regulates VCAM-1 upregulation through PARP-1. Indeed our data show that PARP-1 interacts with p65 upon TNFα stimulation and this interaction is inhibited in the presence of E2. 

Conclusion

Our data show that E2 inhibits upregulation of VCAM-1 in nephritic kidneys. Using mesangial cells we further showed that E2 inhibits VCAM-1 upregulation by inhibiting the formation of pre-initiation complex at the VCAM-1 promoter. E2 inhibits PARP-1 recruitment to p65, further inhibiting the recruitment of RNA polymerase II and transcription at the VCAM-1 promoter. We propose that E2 plays an important role in regulating renal inflammation locally, which may explain why nephritis in systemic autoimmunity tends to be worse in males.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/17%ce%b2-estradiol-regulates-vcam-1-expression-during-glomerulonephritis/