Sex Hormones Modulate the Length of Telomeres of Normal and Telomerase-Mutant Leukocytes through the Estrogen Receptor Pathway.
Authors: Calado Rodrigo T. , Yewdell William T. , Wilkerson Keisha L. , Regal Joshua A. , Kajigaya Sachiko , Young Neal S. 
Published: October 2019

Abstract
Telomeres are nucleoprotein complexes at chromosome ends that protect chromosomes from end-to-end fusion, damage, and recombination. Telomeres are eroded during cell division due to DNA polymerase’s inability to fully duplicate them. Immature cells, including hematopoietic stem cells, express telomerase to maintain telomere lengths. Mutations in genes encoding the telomerase complex (TERT, coding for the reverse transcriptase, and TERC, coding for the RNA component) are associated with constitutional and acquired aplastic anemia, causing low telomerase activity, short telomeres, and a reduced stem cell compartment. Bone marrow failure in telomerase-mutated patients often responds to androgen therapy, but the mechanism of action is unknown. We investigated whether sex steroids modulated telomerase expression and function in hematopoietic cells from healthy subjects and individuals carrying TERT mutations. Peripheral blood lymphocytes were cultured for two days in phenol-free RPMI 1640 supplemented with charcoal-treated 10% fetal bovine serum containing interleukin-2 and phycohemagglutinin in the presence or absence of methyltrienolone (R1881), 19-nortestosterone-17 decanoate (19-NT), 6-hydroxy-testosterone (6-HT), estradiol (E2), and/or tamoxifen, an estrogen antagonist, and/or letrozole, an aromatase inhibitor. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay, and TERT expression by Real Time PCR. Both androgens and E2 stimulated telomerase activity in lymphocytes from normal subjects in a dose-dependent fashion that correlated to higher TERT mRNA levels; tamoxifen abolished sex steroid effects on telomerase function, while letrozole abrogated androgen from up-regulating telomerase in lymphocytes, indicating that sex steroids stimulate TERT expression through the estrogen receptor, which may interact with the estrogen receptor elements present in the promoter region of the TERT gene. As the PI3-K/Akt pathway has been implicated in the sex steroid-dependent activation of telomerase, we tested if this pathway was also activated in lymphocytes. In both immunoblot and flow cytometry analysis, we failed to observe any Akt phosphorylation induced by sex hormones, making it unlikely that this pathway plays a role in androgen-mediated telomerase activation in lymphocytes. We also addressed the effects of androgens of normal bone marrow CD34+ cells, which were purified in immunomagnetic columns and cultured in long-term liquid media, and R1881 also stimulated telomerase in normal marrow CD34+ cells. Peripheral blood lymphocytes from three TERT mutation healthy carriers were also cultured in the presence of androgens. As expected, TERT mutant cells had low baseline telomerase activity, which was restored to normal levels by R1881. These results indicate that sex steroids can modulate TERT expression and telomerase activity in hematopoietic cells signaling through estrogen receptors, and they can partially restore telomerase activity in TERT mutation carriers in vitro. Our findings provide a mechanism for androgen therapy in patients with either acquired or constitutional marrow failure caused by defects in telomere repair. These results also have implications in the physiological variation of telomere shortening, since telomere length of women during fertile age is stable, and hormone replacement therapy decreases the rate of telomere shortening in postmenopausal women.