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.

Keywords: Endocrinology; Sex hormones.

Figure 1. MKRN3 mRNA levels in the mediobasal hypothalamus (MBH) decrease before puberty initiation in different species.

Expression of Mkrn3 mRNA in the hypothalamus of the female rat (A) or rhesus monkey (B) as determined by RT-qPCR. RNA expression data were normalized using peptidylprolyl isomerase A (Ppia) as the housekeeping gene and by dividing each individual value by the average of the P7 group for rats or the <6m group for monkeys. Bars represent mean ± SEM. In A, groups are as follows: 7 days old (P7; n = 8), 14 days old (P14; n = 7), 21 days old (P21; n = 4), 28 days old (P28; n = 5), and 32–34 days old (P32–34; n = 5). In B, groups are as follows: monkeys younger than 6 months of age (<6m; n = 4), 6 months to 1 year of age (6m–1y; n = 4), 1 to 2.5 years of age (1–2.5y; n = 7), 2.5 to 4 years of age (2.5–4y; n = 8). Mkrn3 mRNA levels decreased from P14 to P21 in the hypothalamus of rats (A), and from age 6m–1y to age 1.5–2y in the hypothalamus of monkeys (B). Groups with different symbols (†, ‡) are significantly different (A, P = 0.001; B, P = 0.046), as determined by 1-way ANOVA followed by the Student-Newman-Keuls test.

Figure 2. The prepubertal decrease in hypothalamic Mkrn3 expression is independent of gonadal activation.

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.

Figure 3. Mkrn3 expression is higher in the hypothalamus and cortex of mice compared with peripheral tissues before puberty onset.

(A) Representative photomicrographs showing Mkrn3 expression by in situ hybridization (ISH) in female mice in the caudal hypothalamus. A high, diffuse level of Mkrn3 expression was detected in the hypothalamus of P1 mice, with more specific localization of expression in the ARC and VMN at P10. Expression was reduced by P15 and low in the adult in both ovariectomized (OVX) and estradiol-replaced (E2-replaced) mice. A sense probe did not detect Mkrn3 mRNA. 3V, third ventricle; ARC, arcuate nucleus; VMN, ventromedial nucleus. (B) Schematic representation of the neuroanatomical distribution of Mkrn3 mRNA in the brain of female P10 mice, as assessed by ISH. Red dots indicate areas where Mkrn3 mRNA was detected, darker areas with higher concentrations, and lighter red with relatively low Mkrn3 expression. MnPo, median preoptic nucleus; VOLT, vascular organ of the lamina terminalis; H, hippocampus; DMH, dorsomedial nucleus of hypothalamus; PLCo, posterolateral cortical amygdaloid nucleus; BMP, accessory basal amygdaloid nucleus; PH, posterior hypothalamic area; PMV, premammillary nucleus, ventral part; PMCo, posteromedial cortical amygdaloid nucleus; LV, lateral ventricle. (C) Mkrn3 expression in tissues of P9 male mice. (D–G) Mkrn3 expression in the hypothalamus (D), cortex (E), liver (F), and testes (G) of mice at P9 (white bar) and P90 (blue bar). The bar graphs show the relative change in mRNA levels in different tissues, compared with Mkrn3 levels in the hypothalamus in C and with levels at P9 in D–G. Mean (±SEM) values are shown at each age (n = 3–4 mice per group, with each measurement performed in triplicate). In C, groups with different symbols (†, ‡) are significantly different (P = 0.039), as determined by 1-way ANOVA followed by Tukey’s multiple-comparisons test. In D–G, statistical analysis was performed by unpaired 2-tailed t test. Asterisks indicate P = 0.002 in D and P = 0.02 in E.

Figure 4. Mkrn3 is coexpressed in Kiss1 neurons in the MBH of mice.

(A and B) Double-labeled fluorescent in situ hybridization (FISH) for Mkrn3 (green) and Kiss1 (magenta) mRNA in the MBH of P12 and P30 female mice. Mkrn3 mRNA was detected in several neurons in the ARC of P12 mice (A), with a decrease in P30 mice (B). Cell nuclei identified by Hoechst staining (blue) compared with Mkrn3 expression of (C–E) P12 and (F–H) P30 mice in higher-magnification views. (I and J) Neurons of P12 mice coexpress Mkrn3 and Kiss1 mRNA. Scale bars: 100 μm (A and B) and 10 μm (I–J and C–H). (K) Representative gel showing Mkrn3 and Kiss1 expression by RT-PCR in individual Kiss1^CreGFP cells from the ARC in intact P13 females. The expected sizes for Mkrn3 and Kiss1 are 113 and 120 bp, respectively. RNA extracted from the MBH was used as positive (+, with reverse transcriptase) and negative (–, without reverse transcriptase) controls (TC, tissue control). (L) Bar graphs summarizing the percentage (mean ± SEM) of Kiss1^CreGFP neurons (73 neurons from 4 animals) that expressed Kiss1 and Mkrn3 mRNA.

Figure 5. MKRN3 inhibits KISS1 and TAC3 promoter activity.

Effect of MKRN3 on luciferase activity regulated by the human KISS1 (A), TAC3 (B), and PDYN (C) promoter (p). Effect of short truncated MKRN3 (sMKRN3) on KISS1 (D) and TAC3 (E) promoter activity. Neuro-2a cells were transfected with luciferase reporter constructs containing the 5′ flanking region of the indicated genes, in addition to an expression vector encoding WT MKRN3-HA or MKRN3 mutants. Forty-eight hours later, the cells were harvested and assayed for luciferase activity. Bars represent mean ± SEM (n = 4–6). (A–C) Although WT MRKN3 inhibited KISS1 and TAC3 (P = 0.0056) promoter activity, it did not inhibit PDYN promoter activity. (D and E) A truncated MKRN3 (sMKRN3) with 102 amino acids did not inhibit KISS1 or TAC3 promoter activity. Yellow bars represent the indicated gene promoters only, blue bars represent the indicated gene promoters and cotransfection with MKRN3, pink when both MKRN3 and sMKRN3 were cotransfected, and green when only sMKRN3 was cotransfected. Groups with different symbols (†, ‡, §, #, ¶) are significantly different (P < 0.05), as determined by 1-way ANOVA followed by the Student-Newman-Keuls test.

Figure 6. MKRN3 RING finger mutants lose the ability to inhibit KISS1 and TAC3 promoters.

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.

Figure 7. WT and mutant MKRN3 bind to KISS1 and TAC3 promoters.

Association of WT MKRN3 and mutant variants, identified in patients with CPP, with the 5′ promoter region of the KISS1 (A) and TAC3 (B) genes as determined by chromatin immunoprecipitation assay. HEK293T cells were transfected with MKRN3 constructs carrying a 3× hemagglutinin (HA) tag (WT MKRN3 and mutants C340G, R365S, P417I, and H420Q), and the negative control was transfected with a GFP construct devoid of MKRN3. Immunoprecipitation was carried out using anti-HA antibodies. Bars represent mean ± SEM (n = 3). Blue bars represent WT MRKN3. Groups with different symbols (†, ‡, §) are significantly different from one another (P < 0.001), as determined by 1-way ANOVA followed by the Student-Newman-Keuls test.

Figure 8. MKRN3 undergoes auto-ubiquitination and MKRN3 mutants are less ubiquitinated than WT MKRN3.

(A) HEK293T cells were transfected with a plasmid encoding WT MKRN3 (WT), or were untransfected (UT). The membrane was immunoblotted with anti-MKRN3, and with anti–β-actin as control. Whole-cell lysates revealed a smear, typical of the pattern shown by polyubiquitinated proteins. Coexpression of his-ubiquitin with MKRN3 resulted in a smear of larger complexes, corresponding to polyubiquitinated protein. (B and C) HEK293T cells were transfected with empty pcDNA, WT MKRN3, or mutant MKRN3. Immune complexes were precipitated using protein A anti-HA beads and probed with anti-ubiquitin (upper panel) or anti-HA (middle panel); lower panels correspond to whole-cell lysates probed with anti-HA. (B) Transfected cells treated with vehicle. Upper panel: An upper smear is present when immunoblotted with anti-ubiquitin; it is stronger in the lane loaded with protein from cells transfected with WT MKRN3 than with MKRN3 mutants. Middle panel: Immunoblotting with anti-HA shows a main band below 75 kDa in lanes with WT MKRN3 and MKRN3 mutants, likely corresponding to a mature form of MKRN3; a band at 50 kDa was also detected in lysates from both WT and mutant MRKN3, but was significantly weaker in intensity for C340G MKRN3 compared with WT MKRN3 and other mutants. (C) Transfected cells treated with MG132. Upper panel: Probing with anti-ubiquitin results in a smear suggestive of auto-ubiquitination that is stronger with WT MKRN3 compared with the MKRN3 mutants located in the zinc finger, F417I and H420G, and is weakest for the RING finger mutants, C340G and R365S. Middle panel: Probed with anti-HA, protein levels are similar for WT MKRN3 and all mutants. In all, anti–β-actin antibody (bottom panels) was a loading control. WT, WT MKRN3; UT, untransfected cells; MW, molecular weight.