Genomic regulation of senescence and innate immunity signaling in the retinal pigment epithelium

Abstract

The tumor suppressor p53 is a major regulator of genes important for cell cycle arrest, senescence, apoptosis, and innate immunity, and has recently been implicated in retinal aging. In this study we sought to identify the genetic networks that regulate p53 function in the retina using quantitative trait locus (QTL) analysis. First we examined age-associated changes in the activation and expression levels of p53; known p53 target proteins and markers of innate immune system activation in primary retinal pigment epithelial (RPE) cells that were harvested from young and aged human donors. We observed increased expression of p53, activated caspase-1, CDKN1A, CDKN2A (p16INK4a), TLR4, and IFNα in aged primary RPE cell lines. We used the Hamilton Eye Institute (HEI) retinal dataset ( www.genenetwork.org ) to identify genomic loci that modulate expression of genes in the p53 pathway in recombinant inbred BXD mouse strains using a QTL systems biology-based approach. We identified a significant trans-QTL on chromosome 1 (region 172-177 Mb) that regulates the expression of Cdkn1a. Many of the genes in this QTL locus are involved in innate immune responses, including Fc receptors, interferon-inducible family genes, and formin 2. Importantly, we found an age-related increase in FCGR3A and FMN2 and a decrease in IFI16 levels in RPE cultures. There is a complex multigenic innate immunity locus that controls expression of genes in the p53 pathway in the RPE, which may play an important role in modulating age-related changes in the retina.

Figure 1

Aging activates the p53 pathway in RPE cells. A) Primary cultures of RPE cells obtained from two young and two aged (29, 40, and 84, 86 years, respectively) human donor eyes and were grown to confluence. RPE cell lysates were analyzed by western blot for total-p53, CDKN1A, CDKN2A, TLR4, active caspase-1, IFNG, and IFNA2 using specific antibodies. Beta actin was used as an internal loading control. B) Densitometric values from young RPE cells were set at 100%. Data shows the mean +/− SE. *, significantly different compared with young RPE cells (P<0.05). N=3.

Figure 2

Identification of genomic networks that regulate expression of Cdkn1a, Cdkn2a or Cdkn2b. The data illustrates the likelihood ratio statistic (LRS) scores for expression of (A) Cdkn1a (B) Cdkn2b in the retina of BXD RI mice. QTLs with significant LRS scores are indicated by a horizontal solid black line and regions with suggestive LRS scores are indicated by a dashed black line (~17.2, and ~10.5, respectively). Arrowhead indicates the query gene locus. Insert (A) shows the trans-QTL on Chr 1 (Qrr1).

Figure 3

Graphic representation of genomic networks that regulate the expression of p53 pathway in the retina. The data illustrates the likelihood ratio statistic (LRS) scores for the combined expression of the Cdkn1a, Cdkn2a and Cdkn2b in the retina of BXD RI mice. A significant cis-QTL peak on mouse chromosome 4 (LRS=124) at the location of the Cdkn2b gene and a trans-QTL on chromosome 1 (LRS= 21) is seen. Horizontal line marks the threshold for significance (LRS=15).

Figure 4

Aging alters expression of genes in the Qrr1 locus in RPE cells. A) mRNA expression from primary cultures of young or aged RPE cells were analyzed by qRT-PCR for expression IFI16, FMN2, FCER1γ and GAPDH. Cycle threshold values for each transcript were normalized to GAPDH and young RPE samples using the delta-Ct method. N=12. *, denotes significantly different values (P<0.05). B). Cell lysates from primary cultures of young or aged RPE cells were analyzed by Western blot for expression of IFI16, FMN2, and FCGR3A. Beta actin was used as an internal loading control. C) Densitometry analysis of Western blot bands. Values from young RPE cells were set at 100%. Data shows the mean +/− SE. *, significantly different compared with young RPE cells (P<0.05). N=3

Figure 5

Schematic showing Qrr1 activation of Cdkn1a and p53 pathways that are important for immunity and aging. 1) Various exogenous and endogenous stimuli, like TLR4 and IFN signaling, DNA damage, oxidative stress and foreign dsDNA, activates proteins in Qrr1 locus and p53. 2) Qrr1 proteins (IFI family) can also increase or decrease p53 transcriptional activity. Importantly, p53 triggers expression of some of innate immunity genes in the Qrr1 locus on chromosome 1. 3) Once activated, p53 initiates expression of genes that play an important role in apoptosis, cell cycle arrest, and innate and adaptive immunity. 4) Misregulation of these pathways are thought to be important for aging and AMD. Cdkn1a, which was used for the QTL analysis, is in blue and underlined. Genes in red indicate a possible negative regulator of p53 activity.