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 6

MKRN3 RING finger mutants lose the ability to inhibit KISS1 and TAC3 promoters.

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MKRN3 RING finger mutants lose the ability to inhibit KISS1 and TAC3 pro...
Effect of missense MKRN3 mutations identified in patients with CPP on KISS1 (A) and TAC3 (B) promoter activity. The mutants located in the RING finger domain, p.C340G and p.R365S, lost the ability to inhibit the KISS1 and TAC3 promoters, similarly to WT MKRN3. The mutant located in the zinc finger domain, p.F417I, inhibited the KISS1 and TAC3 promoters at least as effectively as WT MKRN3, while p.H420Q, located in the same domain as F417, inhibited the TAC3 promoter similarly to WT MKRN3 but had compromised ability to inhibit the KISS1 promoter. Yellow bars represent the KISS1 or TAC3 promoter, blue bars represent the KISS1 or TAC3 promoter with cotransfection of MKRN3. Groups with different symbols (†, ‡, §, #, ¶) are significantly different (P < 0.05), as determined by 1-way ANOVA followed by Student–Newman–Keuls test. (C) Schematic representation of the makorin ring finger protein encoded by MKRN3 showing the zinc finger domains, which are RNA binding domains, the RING finger domain, responsible for E3 ubiquitin ligase activity, and the specific makorin-type domain. The arrows point to the locations of the missense mutations studied here. Zn, zinc finger; pink circles, cysteine residues in the zinc fingers; green circles, histidine residues in the zinc fingers; blue circles represent the amino acids in the protein. The numbers underneath the schematic indicate amino acid locations in the protein.