RasGrf1: genomic imprinting, VSELs, and aging

Abstract

Increase in life span in RasGrf1-deficient mice revealed that RasGrf1 deficiency promotes longevity. Interestingly, RasGrf1 is one of parentally imprinted genes transcribed from paternally-derived chromosome. Erasure of its imprinting results in RasGrf1 downregulation and has been demonstrated in a population of pluripotent adult tissues-derived very small embryonic like stem cells (VSELs), stem cells involved in tissue organ rejuvenation. Furthermore, based on recent observation that RasGrf1 signaling molecule is located downstream from insulin (Ins) and insulin like growth factor-1 (Igf-1) receptors, the extended life-span of RasGrf1-/- mice may support beneficial effect of reduced Ins/Igf-1 signaling on longevity. Similarly, downregulation of RasGrf1 in VSELs renders them resistant to chronic Ins/Igf-1 signaling and protects from premature depletion from adult tissues. Thus, the studies in RasGrf1-/- mice indicate that some of the imprinted genes may play a role in ontogenetic longevity and suggest that there are sex differences in life span that originate at the genome level. All this in toto supports a concept that the sperm genome may have a detrimental effect on longevity in mammals. We will discuss a role of RasGrf1 on life span in context of genomic imprinting and VSELs.

Figure 1. RasGrf1 phosporylation in murine embryonic ES-D3 cells.

Panel A. RasGrf1 protein phophorylation at Ser929 was detected by western blot after stimulation for 5 min with LIF (50 ng/mL), insulin (10 ng/mL), IGF-1 (100 ng/mL), and IGF-2 (100 ng/ml). Panel B. The total level of Ras-Grf1 protein. The experiment was repeated twice with similar results.

Figure 2. Insulin/IGF-1 and -2 signaling and imprinted genes.

In mammals, there are three insulin factors (insulin, IGF-1, and IGF-2) that bind to two tyrosine kinase receptors, insulin receptor (InsR), and IGF-1 receptor (IGF-1R). Igf2R is a non-signaling mannose-type sink receptor for IGF-2. Depending on cell type, activation of InsR and IGF-1R lead to metabolic and proliferative responses. RasGrf1 is a small GTP exchange factor (GEF) and is involved in signaling from InsR and IGF-1R. VSELs, due to changes in the epigenetic state of parentally imprinted genes, show a decrease in Igf-2 and RasGrf1 expression (green) and Igf2R overexpression (red). These epigenetic changes in genes regulating insulin/Igf signaling keep VSELs quiescent in adult tissues. We hypothesize that chronic exposure to Ins/Igf accelerates premature depletion of VSELs, while RasGrf1 deficiency has the opposite effect.

Figure 3. The change of genomic imprinting during VSEL ageing

Panel A. RQ-PCR analysis of RasGrf1, Igf2, and H19 expression in VSELs isolated from 4-week-old and 18-month-old C57BL/6 mice. The relative expression level was represented as the percentage of β2 microglobin (β2mg) expression and shown as the mean ± SD. Please note reciprocal expression in Igf-2 (that promotes proliferation) and H19 (that encodes for mRNA giving rise to several miRNA negatively affecting cell proliferation). Both of these genes (Igf2-H19) are regulated in tandem by a common DMR that affects their expression - when one is silenced another one is upregulated. Panel B. Combined bisulfate-restriction analysis (COBRA) assay of Igf2-H19 DMR1 (upper panel) and RasGrf1 DMR (lower panel) by BstUI restriction enzyme in 6-month-old control wild type (WT) and 6-month-old transgenic bovine GH-overexpressing mice (bGH-TG). The COBRA assay was performed on VSELs, hematopoietic stem cells (HSC), and bone marrow mononuclear cells (BMMNC). “-” indicates bisulfate modification was not performed on the DNA (control); “+” indicates that DNA was subjected to bisulfate modification. The unmethylated DNA (uMe) was not cleaved, in contrast to methylated DNA (Me), because of a sequence change at the site recognized by a restriction enzyme after bisulfite reaction.