The role of Memo in premature aging and FGFR signaling

Memo was recently identified in a screen for proteins required for ErbB receptor induced cell migration. Encoded by a single-copy gene present in all branches of life, it is ubiquitously expressed during all stages of embryogenesis and in adult tissues. This study aimed to investigate the physiological role of Memo. Initially, we generated a conventional knock out of Memo, which was embryonic lethal between E12.5 to E14.5. We subsequently generated a mouse strain to allow temporal control of Memo deletion by crossing a mouse strain harboring a homozygous floxed Memo allele (Memofl/fl) with pCX-CreERTM transgenics, where the ubiquitous actin promoter drives the Cre recombinase. Following tamoxifen treatment, Memo was deleted in all organs (Memo null mice). Knock out of Memo in mice at the age of 2 to 14 weeks revealed a severe premature aging phenotype accompanied by alterations in insulin and glucose metabolism. The phenotype of the Memo null mice, especially the metabolic phenotype, revealed an interesting similarity to the Klotho and FGF23 mutant mice. In particular, insulin and glucose metabolism was very similar to the phenotype shown by both Klotho mutant and FGF23 knock-out mice. To investigate this phenotype further, and as Klotho is mainly expressed in the renal tubular cells, we generated a mouse model, which lacked Memo specifically in the kidney. We chose a Pax8 promoter system to delete Memo in the renal tubular cells (1). Kidney specific Memo knock-out mice were generated by crossing Memo floxed (Memofl/fl) mice with a mouse line expressing the tetracycline-sensitive transactivator rtTA under control of the Pax8 promoter, and a mouse line harboring Cre recombinase under a promoter that contains a tetracycline-responsive promoter element. In these mice doxycycline treatment induced loss of Memo in renal tubular epithelial cells.
By investigation of the Memo null and kidney-specific Memo KO mice we found that only the Memo null mice showed premature aging symptoms. However, both KO models showed deregulation of the FGF23-Klotho axis measuring expression of enzymes involved in vitamin D metabolism and phosphate reabsorption. Moreover, blood analysis of both models showed renal insufficiency and hypercalcemia. For the kidney-specific Memo KO animals we also performed analysis of the urine and found hypercaliuria. Surprisingly, for both models there was no difference in serum phosphate levels, which has earlier been shown by others to be causative for the premature aging syndrome.
In conclusion, we found that deletion of Memo in the full body or specifically in the kidney induces renal insufficiency and hypercalcemia. Furthermore, Memo deletion in the full body results in severe premature aging symptoms that cannot be explained by elevated serum phosphate levels. To date, it is not clear how hypercalcemia and hypercalciuria affects Memo mice and what specifically induces the premature aging in Memo null animals.
To investigate the function of Memo in vitro we isolated and immortalized mouse embryonic fibroblasts (MEFs) from Memofl/fl embryos. These cells were then infected with a Cre recombinase containing vector, which following 4-hydroxytamoxifen treatment ablated Memo expression (KO MEFs). Our studies showed that signaling downstream of FGF2 was reduced in activity and duration in Memo KO MEFs and furthermore that Memo was associated with the FGFR signaling complex (FGFR-FRS2-GRB2-GAB1). In addition, we tested mammary carcinoma cells (4T1) for sensitivity to FGFR inhibition and revealed lower sensitivity to FGFR inhibition in Memo downregulated cells. To investigate the role of Memo in the metabolic signaling we used HEK293 cells that are stably transfected with Klotho or ßKlotho and therefore responsive to FGF19, 21 and 23. We found that in Memo downregulated HEK293-Klotho and –ßKlotho cells, FGFR signaling activity after stimulation with FGF23 and FGF19 was affected.
In summary, this study provides evidence for a physiological role of Memo downstream of the FGFR pathway. We show that Memo is part of the FGFR signaling complex. Loss of Memo affects the intensity and duration of the FGFR signaling and modulates sensitivity to FGFR inhibition and to oxidative stress. Furthermore, we uncovered an important role of Memo in renal physiology that contributed to a premature aging phenotype, which is similar to that observed in Klotho mutant or FGF23 knock out animals.