MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons
Ana Paula Abreu, Carlos A. Toro, Yong Bhum Song, Victor M. Navarro, Martha A. Bosch, Aysegul Eren, Joy N. Liang, Rona S. Carroll, Ana Claudia Latronico, Oline K. Rønnekleiv, Carlos F. Aylwin, Alejandro Lomniczi, Sergio Ojeda, Ursula B. Kaiser
Ana Paula Abreu, Carlos A. Toro, Yong Bhum Song, Victor M. Navarro, Martha A. Bosch, Aysegul Eren, Joy N. Liang, Rona S. Carroll, Ana Claudia Latronico, Oline K. Rønnekleiv, Carlos F. Aylwin, Alejandro Lomniczi, Sergio Ojeda, Ursula B. Kaiser
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Research Article Endocrinology

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons

Abstract

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Authors

Ana Paula Abreu, Carlos A. Toro, Yong Bhum Song, Victor M. Navarro, Martha A. Bosch, Aysegul Eren, Joy N. Liang, Rona S. Carroll, Ana Claudia Latronico, Oline K. Rønnekleiv, Carlos F. Aylwin, Alejandro Lomniczi, Sergio Ojeda, Ursula B. Kaiser

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Figure 2

The prepubertal decrease in hypothalamic Mkrn3 expression is independent of gonadal activation.

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The prepubertal decrease in hypothalamic Mkrn3 expression is independent...
Mkrn3 expression in the ARC (left) and AVPV (right) of intact WT (white bars) and hpg (blue bars) mice across postnatal development as determined by RT-qPCR. The bar graphs show the relative change in mRNA levels in female (A) and male (B) mice, compared with levels at P10, normalized to levels of endogenous ribosomal protein L19 (RpL19) mRNA. Mean (±SEM) values are shown at each age (n = 3–4 mice per group, with each measurement performed in triplicate). Statistical analysis of effects of age and genotype were compared by 2-way ANOVA with a post hoc Tukey’s multiple-comparisons test. Mkrn3 mRNA levels decreased from P10 to P45 in the ARC and AVPV of WT mice, similarly to hpg mice. There was no difference in Mkrn3 expression between WT and hpg female or male mice across pubertal maturation in either the ARC or AVPV. Groups with different symbols (†, ‡, §) are significantly different (P = 0.0002). (C–F) Expression of Mkrn3 (C and E) and Kiss1 (D and F), quantified by RT-qPCR, in the MBH (C and D) and POA (E and F) of WT female mice treated with 2 μg of estradiol benzoate (EB)or vehicle at age P11. All mice were sacrificed 24 hours after treatment. The bar graphs show relative mRNA levels in female EB-treated (green bars) mice compared with vehicle-treated (white bars) mice, normalized to levels of endogenous RpL19 mRNA. Mean (±SEM) values are shown for 4–6 mice in each group, with each measurement performed in triplicate. Statistical analysis of effects of EB treatment was by unpaired 2-tailed t test. Asterisks indicate P < 0.0001 for D and P = 0.014 for F.